European Journal of Pharmacology (v.650, #1)

Recent advances in the study on capsaicinoids and capsinoids by Xiu-Ju Luo; Jun Peng; Yuan-Jian Li (1-7).
Chili peppers are the major source of nature capsaicinoids, which consist of capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homodihydrocapsaicin, and homocapsaicin, etc. Capsaicinoids are found to exert multiple pharmacological and physiological effects including the activities of analgesia, anticancer, anti-inflammation, antioxidant and anti-obesity. Therefore, capsaicinoids may have the potential value in clinic for pain relief, cancer prevention and weight loss. In addition, capsaicinoids also display the benefits on cardiovascular and gastrointestinal system. It has been shown that capsaicinoids are potential agonists of capsaicin receptor or transient receptor potential vanilloid subfamily member 1 (TRPV1). They could exert the effects not only through the receptor-dependent pathway but also through the receptor-independent one. CH-19 Sweet peppers are the source of nature capsinoids, which share similar structure with capsaicinoids and consist of capsiate, dihydrocapsiate, and nordihydrocapsiate, etc, Comparing with capsaicinoids, capsinoids are less pungent and easily broken down in the normal aqueous conditions. So far, it has been found that capsinoids possess the biological properties of antitumor, antioxidant and anti-obesity. Since capsinoids are less toxic than capsaicinoids, therefore, capsinoids may have the advantages over capsaicinoids in clinical applications such as cancer prevention and weight loss.
Keywords: Capsaicinoid; Capsinoid; Capsaicin; Capsiate; TRPV1;

Extracellular signal-regulated kinases in pain of peripheral origin by John P.M. White; Mario Cibelli; Antonio Rei Fidalgo; Istvan Nagy (8-17).
Activation of members of the family of enzymes known as extracellular signal-regulated kinases (ERKs) is now known to be involved in the development and/or maintenance of the pain associated with many inflammatory conditions, such as herniated spinal disc pain, chronic inflammatory articular pain, and the pain associated with bladder inflammation. Moreover, ERKs are implicated in the development of neuropathic pain signs in animals which are subjected to the lumbar 5 spinal nerve ligation model and the chronic constriction injury model of neuropathic pain. The position has now been reached where all scientists working on pain subjects ought to be aware of the importance of ERKs, if only because certain of these enzymes are increasingly employed as experimental markers of nociceptive processing. Here, we introduce the reader, first, to the intracellular context in which these enzymes function. Thereafter, we consider the involvement of ERKs in mediating nociceptive signalling to the brain resulting from noxious stimuli at the periphery which will be interpreted by the brain as pain of peripheral origin.
Keywords: MAPK; ERK; Pain; Inflammation; Neuropathy; Intracellular signalling;

Involvement of notch signaling pathway in amyloid precursor protein induced glial differentiation by Young-Don Kwak; Amelia Marutle; Elise Dantuma; Stephanie Merchant; Sergey Bushnev; Kiminobu Sugaya (18-27).
The amyloid precursor protein (APP) has been mainly studied in its role in the production of amyloid β peptides (Aβ), because Aβ deposition is a hallmark of Alzheimer's disease. Although several studies suggest APP has physiological functions, it is still controversial. We previously reported that APP increased glial differentiation of neural progenitor cells (NPCs). In the current study, NPCs transplanted into APP23 transgenic mice primarily differentiated into glial cells. In vitro treatment with secreted APP (sAPP) dose-dependently increased glial fibrillary acidic protein (GFAP) immuno-positive cells in NPCs and over expression of APP caused most NPCs to differentiate into GFAP immuno-positive cells. Treatment with sAPP also dose-dependently increased expression levels of GFAP in NT-2/D1 cells along with the generation of Notch intracellular domain (NICD) and expression of Hairy and enhancer of split 1 (Hes1). Treatment with γ-secretase inhibitor suppressed the generation of NICD and reduced Hes1 and GFAP expressions. Treatment with the N-terminal domain of APP (APP 1–205) was enough to induce up regulation of GFAP and Hes1 expressions, and application of 22 C11 antibodies recognizing N-terminal APP suppressed these changes by sAPP. These results indicate APP induces glial differentiation of NPCs through Notch signaling.
Keywords: Alzheimer's disease; Amyloid precursor protein; Notch; Glial differentiation; Neural progenitor cells;

Intracellular cAMP assay and Eu-GTP-γS binding studies of chimeric opioid peptide YFa by Krishan Kumar; Sambuddha Kumar; Raj Kumar Kurupati; Mahesh Kumar Seth; Anita Mohan; M Ejaz Hussain; Santosh Pasha (28-33).
In our previous studies chimeric peptide of Met-enkephalin and FMRFa, YGGFMKKKFMRFamide (YFa), demonstrated concentration dependent κ– and μ–opioid receptor mediated antinociception without tolerance development. To gain further insight of the observed behavior of YFa, the present study was undertaken. The effect of chimeric peptide on forskolin-stimulated cAMP formation under acute and chronic treatment and stimulation of Eu-GTP-γS binding in CHO cells stably expressing κ– and μ–opioid receptors was assessed. YFa showed concentration dependent inhibition of forskolin-stimulated cAMP in both hKOR and hMOR-CHO cells; however, the inhibition at 1 nM was significantly higher in hKOR cells and comparable to DynA (1–13) than that shown at 20 nM in hMOR cells. Chronic treatment of YFa, similar to DynA (1–13), did not show significant change in forskolin-stimulated cAMP level in both hKOR and hMOR cells. However, chronic treatment of morphine and DAMGO showed an increase in forskolin-stimulated cAMP level in hMOR-CHO cells indicating superactivation of adenylyl cyclase. Eu-GTP-γS binding studies of YFa showed a concentration dependent adherent binding with κ– and μ–opioid receptors; however, the latter demonstrated significant binding at higher concentration. Thus the study indicates the chimeric opioid peptide YFa as a potent κ- receptor specific antinociceptive moiety, showing no tolerance and hence may serve as a lead in understanding the mechanism of tolerance development, antinociception and its modulation.
Keywords: Cyclic adenosine monophosphate; Antinociception; Adenylyl cyclase; Tolerance;

Muscarinic acetylcholine receptors present in human osteoblast and bone tissue by Pei-Shan Liu; Yi-Yin Chen; Chi-Kuang Feng; Yi-Hsuan Lin; Tien-Chi Yu (34-40).
Acetylcholine is the predominant neurotransmitter in the neuromuscular junction, and a role in bone has been postulated. The expression of nicotinic receptors has been reported in osteoblasts, but the expression and function of muscarinic receptor in bone remain obscure. In this study, we investigated the expression and functional activities of muscarinic receptor subtypes in human osteoblast cell lines and animal and human bone tissue. The mRNA levels of muscarinic receptor subtypes were detected by reverse-transcription polymerase chain reaction. We found that muscarinic subtypes m1, m2, m3, m4, and m5 were expressed at different levels in human osteosarcoma HOS cells, rat femur, and human rib bone tissue; m1, m4, m5 were in cultured mouse femur bone cells and cultured mouse calvarial bone cells; m2, m3, m4 were in bovine bone. The mRNA of neuronal markers, light-, medium- and heavy-neurofilament, was not found in human bone tissues to exclude the possible contamination from neuronal tissue. Methacholine induced an elevation in cytosolic calcium concentration and proliferation in HOS cells. Both effects were blocked by atropine. We conclude that muscarinic receptor is present in bone tissue to evoke calcium signaling and modulate cell proliferation. Different muscarinic receptor subtypes are distributed in various parts of the animal skeletal system including the different species and bone portions. Bone remodeling involving osteoblast proliferation leads the possibilities that muscarinic receptor may play roles in bone remodeling.
Keywords: Muscarinic acetylcholine receptor; Human osteosarcoma HOS cell; Human bone tissue; Calcium signaling; Proliferation;

Curcumin is a phytochemicals which is able to inhibit carcinogenesis in a variety of cell lines. However little is known about its effect on the cell-surface and the interaction between cell-surface and the reacting drug. In this study, we found that curcumin could inhibit the growth of human hepatocellular carcinoma cell line (HepG2), change the cell-surface morphology and trigger the pro-apoptotic factor to promote cell apoptosis. Cell counting kit results indicated that the cell viability had a dose-dependent relationship with the curcumin concentration in 24 h. The 50% inhibiting concentration (IC50) was 17.5 ± 3.2 μM. It was clear that curcumin could lead to apoptosis, and the apoptosis increased as the reacting concentration goes up. Moreover, curcumin could also affect the disruption of mitochondrial membrane potential and the disturbance of intracellular free Ca2+ concentration. All these alterations changed the cell morphology and cell-surface ultrastructure with atomic force microscopy (AFM) detecting at nanoscale level. AFM results indicated that cells in control group clearly revealed a typical long spindle-shaped morphology. Cell tails was wide and unrolled. The ultrastructure showed that cell membrane was made up of many nanoparticles. After being treated with curcumin, cell tail was narrowed. The size of membrane nanoparticles became small. These results can improve our understanding of curcumin which can be potentially developed as a new agent for treatment of hepatocellular carcinoma since it has been reported to have a low cytotoxic effect on healthy cell. AFM can be used as a powerful tool for detecting ultrastructures.
Keywords: Curcumin; Apoptosis; Atomic force microscope; Intracellular free Ca2+; Mitochondrial membrane potential;

1-Methyl-4-phenylpyridinium ion (MPP+), a neurotoxin selective to dopaminergic neurons and an inhibitor of mitochondrial complex I, has been widely used as an etiologic model of Parkinson's disease. In this study, we investigated the protective effects of a novel synthetic compound, 8-Phenyl-6a,7,8,9,9a,10-hexahydro-6H-isoindolo[5,6-g]quinoxaline-7,9-dione (PHID), on MPP+-induced cytotoxicity in SH-SY5Y cells. MPP+ induced apoptosis characterized by generation of reactive oxygen species, caspase-3 activation, poly ADP ribose polymerase proteolysis and increase in Bax/Bcl-2 ratio were blocked by PHID in a dose-dependent fashion. Furthermore, MPP+-mediated activation of stress-activated protein kinase/c-Jun N-terminal kinase (JNK) was also inhibited by PHID in a dose-dependent manner. The results indicate that PHID protects against MPP+-induced apoptosis by blocking reactive oxygen species stimulation and JNK signaling pathways in SH-SY5Y cells, implicating the novel compound in the prevention of progressive neurodegenerative diseases such as Parkinson's disease.
Keywords: PHID; MPP+; Reactive oxygen species; Apoptosis; Parkinson's disease; Cytotoxicity;

Pharmacological profile of AS1670542, a novel orally-active human thrombopoietin receptor agonist by Masaki Abe; Ken-ichi Suzuki; Chinatsu Sakata; Keizo Sugasawa; Fukushi Hirayama; Yuji Koga; Tomihisa Kawasaki; Shin Naganuma; Hiroyuki Itoh (58-63).
Eltrombopag, an orally-active small molecule thrombopoietin (TPO) receptor agonist, was used for the first time in 2008 to treat patients with chronic idiopathic thrombocytopenic purpura. Here, we investigated the pharmacological effect of a new orally-active small molecule TPO receptor agonist which may be effective in treating these patients. 50% effective concentration values for cell proliferation with AS1670542 or eltrombopag were 1.9 and 13 nM, respectively, while those for megakaryocyte colony formation from human cord blood CD34+ cells with AS1670542 or eltrombopag were 260 and 950 nM, respectively. On Day 14 after the start of administration, AS1670542 significantly increased the number of human platelets in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice with transplanted human hematopoietic stem cells at 0.3 (P  < 0.05); in contrast, while administration of eltrombopag also increased the numbers of these platelets at 30 mg/kg/day (P  = 0.058), no statistical significance was noted in the increase. Here, we identified AS1670542, a novel orally-active TPO receptor agonist which mimics the biological activity of TPO and may demonstrate greater in vitro and in vivo pharmacologically efficacy than eltrombopag.
Keywords: Thrombopoietin; TPO receptor; Megakaryocyte; NOD/SCID mice; AS1670542; Eltrombopag;

Novel hexahydrocannabinol analogs as potential anti-cancer agents inhibit cell proliferation and tumor angiogenesis by Dinesh Thapa; Jong Suk Lee; Se-Woong Heo; Yong Rok Lee; Keon Wook Kang; Mi-Kyoung Kwak; Han Gon Choi; Jung-Ae Kim (64-71).
Both natural and synthetic cannabinoids have been shown to suppress the growth of tumor cells in culture and in animal models by affecting key signaling pathways including angiogenesis, a pivotal step in tumor growth, invasion, and metastasis. In our search for cannabinoid-like anticancer agents devoid of psychoactive side effects, we synthesized and evaluated the anti-angiogenic effects of a novel series of hexahydrocannabinol analogs. Among these, two analogs LYR-7 [(9S)-3,6,6,9-tetramethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol] and LYR-8 [(1-((9S)-1-hydroxy-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-2-yl)ethanone)] were selected based on their anti-angiogenic activity and lack of binding affinity for cannabinoid receptors. Both LYR-7 and LYR-8 inhibited VEGF-induced proliferation, migration, and capillary-like tube formation of HUVECs in a concentration-dependent manner. The inhibitory effect of the compounds on cell proliferation was more selective in endothelial cells than in breast cancer cells (MCF-7 and tamoxifen-resistant MCF-7). We also noted effective inhibition of VEGF-induced new blood vessel formation by the compounds in the in vivo chick chorioallantoic membrane (CAM) assay. Furthermore, both LYR analogs potently inhibited VEGF production and NF-κB transcriptional activity in cancer cells. Additionally, LYR-7 or LYR-8 strongly inhibited breast cancer cell-induced angiogenesis and tumor growth. Together, these results suggest that novel synthetic hexahydrocannabinol analogs, LYR-7 and LYR-8, inhibit tumor growth by targeting VEGF-mediated angiogenesis signaling in endothelial cells and suppressing VEGF production and cancer cell growth.
Keywords: Synthetic hexahydrocannabinol; Anti-angiogenesis; Cell proliferation; NF-κB; Tamoxifen-resistant MCF-7 breast cancer;

Oxidative stress-induced apoptosis in lens epithelial cells plays an important role in cataract formation, and its prevention may be of therapeutic interest. This study was performed to investigate the protective effect and mechanisms of honokiol on H2O2-induced apoptosis in human lens epithelial (HLE) cells. HLE cells (SRA01-04) were pretreated with honokiol at concentrations of 5 μM, 10 μM and 20 μM before 50 μM H2O2 treatment. The results demonstrated that pretreatment of honokiol inhibited the activation of caspase-3 and caspase-9 and downregulated the expression of Bcl-2. Mechanistically, honokiol suppressed H2O2-induced phosphorylation of ERK1/2, p38 mitogen-activated protein kinase (MAPK), JNK and Akt. Honokiol also inhibited H2O2-induced nuclear factor-κB (NF-κB)/p65 phosphorylation and translocation in HLE cells. These results demonstrate that honokiol suppresses H2O2-induced HLE cell apoptosis via interference with the MAPKs, Akt and NF-κB signaling, suggesting that honokiol might have a potential effect against cataract formation.
Keywords: Honokiol; Apoptosis; Human lens epithelial cells; Mitogen-activated protein kinase; Akt;

Effects of quercetin on α9α10 nicotinic acetylcholine receptor-mediated ion currents by Byung-Hwan Lee; Sun-Hye Choi; Tae-Joon Shin; Mi Kyung Pyo; Sung-Hee Hwang; Sang-Mok Lee; Hyun-Dong Paik; Hyoung-Chun Kim; Seung-Yeol Nah (79-85).
Quercetin, one of the flavonoids, is a low molecular weight substance found in fruits and vegetables. Quercetin, like other flavonoids, has a wide range of neuropharmacological actions and antioxidant effects. The α9α10 nicotinic acetylcholine receptor is one of the numerous nicotinic acetylcholine receptors that exist as a heteropentameric form between efferent olivocochlear fibers and hair cells of the cochlea. In this study, we report the effects of quercetin on rat α9α10 nicotinic acetylcholine receptor-mediated ion currents using the two-electrode voltage-clamp technique. Treatment with acetylcholine evoked inward currents (I ACh) in oocytes heterologously expressing the α9α10 nicotinic acetylcholine receptor. Quercetin blocked I ACh in concentration-dependent and reversible manners, and the blocking effect on I ACh was stronger with pre-application than co-application of quercetin. The half maximal inhibitory concentration (IC50) of quercetin was 45.4 ± 10.1 μM. Quercetin-mediated I ACh inhibition was not affected by acetylcholine concentration and was independent of membrane-holding potential. Although the inhibitory effect of quercetin was significantly attenuated in the absence of extracellular Ca2+, the action of quercetin was independent of extracellular Ca2+ concentration, indicating that the presence of extracellular Ca2+ might be needed for quercetin-related effects and might play an important role in quercetin-mediated regulation of the α9α10 nicotinic acetylcholine receptor. These results indicate that quercetin-mediated regulation of the α9α10 nicotinic acetylcholine receptor could provide a molecular basis for quercetin actions at the cellular level.
Keywords: Flavonoid; Quercetin; α9α10 nicotinic acetylcholine receptor; Xenopus oocyte;

Pharmacological activation and genetic manipulation of cystathionine beta-synthase alter circulating levels of homocysteine and hydrogen sulfide in mice by Kristian K. Jensen; Neil S. Geoghagen; Lan Jin; Tom G. Holt; Qi Luo; Lorraine Malkowitz; Weihua Ni; Shuo Quan; M. Gerard Waters; Aiwu Zhang; Heather H. Zhou; Kang Cheng; Ming-juan Luo (86-93).
Hydrogen sulfide (H2S) is a recently discovered gasotransmitter found in mammalian tissues and blood. Treatment with H2S donor molecules has shown promising results in preclinical models of inflammatory and cardiovascular diseases. Augmentation of H2S levels thus holds promise as a novel therapeutic approach for treatment of disease in man. Cystathionine β-synthase (CBS) has been shown to catalyze H2S production in vitro. CBS enzyme activity is allosterically regulated by the endogenous activator S-adenosyl methionine. This mode of regulation suggests the possibility for designing a small molecule activator of CBS to enhance H2S production. This hypothesis, however, has not been directly tested in vivo. We show here that CBS contributes significantly to endogenous H2S production in mice: adenovirus mediated over expression of CBS in the liver significantly increased circulating levels of H2S, whereas CBS deficiency resulted in reduced levels. We demonstrate that CBS enzyme from endogenous sources can be activated by S-adenosyl methionine to a greater extent compared to recombinant enzyme, suggesting greater potential for activation than previously anticipated. Importantly, we show that circulating H2S levels are increased by pharmacological activation of CBS in vivo; i.e. in the presence of the endogenous activator. Together, our data demonstrate that CBS activity partially regulates endogenous H2S in mice, and suggest that pharmacological activation of CBS is a promising approach for enhancing endogenous production of H2S for the treatment of cardiovascular and other diseases.
Keywords: Hydrogen sulfide; Cystathionine β-synthase; Homocysteine;

Cerebellar and cerebrocortical A-type γ-aminobutyric acid (GABAA) receptors were examined in mice and rats. In wild-type mouse cerebellum, the agonists GABA and gaboxadol exerted heterogeneous displacement of [3H]ethynylbicycloorthobenzoate (EBOB) binding with nanomolar and submicromolar affinities. In mouse cerebella lacking α6 subunits (α6KO), nanomolar displacement by GABA agonists was absent, while micromolar displacement was potentiated to 12-fold by 0.3 μM 5α-tetrahydrodeoxycorticosterone (5α-THDOC). In α6KO cerebellum, 60% of [3H]EBOB binding was neurosteroid-insensitive, while 5α-THDOC elicited enhancement with EC50  = 150 nM instead of nanomolar displacement. In conclusion, nanomolar displacement of cerebellar [3H]EBOB binding by GABA agonists and neurosteroids can be attributed to GABAA receptors containing α6 and δ subunits. In contrast, [3H]EBOB binding to rat cerebral cortex was affected by allopregnanolone and 5α-THDOC in bidirectional manner with nanomolar enhancement (EC50 ~ 80 nM) and micromolar displacement. Nonequilibrium binding conditions with decreased incubation time tripled the maximal enhancement of [3H]EBOB binding by 5α-THDOC. 5ß-THDOC enhanced the cortical [3H]EBOB binding with EC50 ~ 0.5 μM and it attenuated bidirectional modulation by 5α-THDOC. Allopregnanolone and 5α-THDOC produced biphasic enhancements of chloride currents elicited by 1 μM GABA in cerebellar granule cells, for 5α-THDOC with EC50,1 ~ 16 nM and EC50,2 ~ 1.3 μM. Differences in peak current enhancements in the absence minus presence of 0.1 mM furosemide corresponding to α6ßδ GABAA receptors were augmented only by micromolar 5α-THDOC while the difference curve for allopregnanolone was polyphasic as without furosemide. Consequently, these neurosteroids differentially affected the binding and function of various GABAA receptor populations.
Keywords: α6ßδ GABAA receptor; [3H]EBOB binding; Gaboxadol; Allopregnanolone; 5α/ß-THDOC; α6KO mouse;

Psoralidin has been reported to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production, but the mechanisms of the action remain unclear. Thus, the impact of psoralidin on signaling pathways known to be implicated in NO synthesis was explored in LPS-activated RAW264.7 macrophages by using RT-PCR and Western blotting. Consistent with NO inhibition, psoralidin suppressed LPS-induced expression of inducible NO synthase (iNOS) by abolishing IκB kinase (IKK) phosphorylation, IκB degradation and nuclear factor κB (NF-κB) nuclear translocation without effecting mitogen-activated protein kinases (MAPKs) phosphorylation. Exposure to wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression, suggesting activation of such a signal pathway might also be phosphoinositide-3-kinase (PI3K) dependent. By using Src inhibitor PP2, Janus kinase 2 (JAK-2) inhibitor AG490, Bruton's tyrosine kinase (Btk) inhibitor LFM-A13 and spleen tyrosine kinase (Syk) inhibitor piceatannol, the results showed that piceatannol clearly repressed NO production more potently than the other inhibitors. Furthermore, piceatannol significantly repressed LPS-induced PI3K/Akt phosphorylation and the downstream IKK/IκB activation, suggesting that Syk is an upstream key regulator in the activation of PI3K/Akt-mediated signaling. In fact, transfection with siRNA targeting Syk obviously reduced iNOS expression. Interestingly, LPS-induced phosphorylations of Syk and PI3K-p85 were both significantly blunted by psoralidin treatment. The present results show that interfering with Syk-mediated PI3K phosphorylation might contribute to the NO inhibitory effect of psoralidin via blocking IKK/IκB signaling propagation in LPS-stimulated RAW 264.7 macrophages.
Keywords: Psoralidin; LPS; Syk; PI3K; IKK; NF-κB; iNOS;

Crocetin prevents retinal degeneration induced by oxidative and endoplasmic reticulum stresses via inhibition of caspase activity by Mika Yamauchi; Kazuhiro Tsuruma; Shunsuke Imai; Tomohiro Nakanishi; Naofumi Umigai; Masamitsu Shimazawa; Hideaki Hara (110-119).
Crocetin is a carotenoid that is the aglicone of crocin, which are found in saffron stigmas (Crocus sativus L.) and gardenia fruit (Gardenia jasminoides Ellis). In this study, we investigated the effects of crocetin on retinal damage. To examine whether crocetin affects stress pathways, we investigated intracellular oxidation induced by reactive oxygen species, expression of endoplasmic reticulum (ER) stress-related proteins, disruption of the mitochondrial membrane potential (ΔΨm), and caspases activation. In vitro, we employed cultured retinal ganglion cells (RGC-5, a mouse ganglion cell-line transformed using E1A virus). Cell damage was induced by tunicamycin or hydrogen peroxide (H2O2) exposure. Crocetin at a concentration of 3 μM showed the inhibitory effect of 50–60% against tunicamycin- and H2O2-induced cell death and inhibited increase in caspase-3 and -9 activity. Moreover, crocetin inhibited the enzymatic activity of caspase-9 in a cell-free system. In vivo, retinal damage in mice was induced by exposure to white light at 8000 lx for 3 h after dark adaptation. Photoreceptor damage was evaluated by measuring the outer nuclear layer thickness at 5 days after light exposure and recording the electroretinogram (ERG). Retinal cell damage was also detected with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining at 48 h after light exposure. Crocetin at 100 mg/kg, p.o. significantly inhibited photoreceptor degeneration and retinal dysfunction and halved the expression of TUNEL-positive cells. These results indicate that crocetin has protective effects against retinal damage in vitro and in vivo, suggesting that the mechanism may inhibit increase in caspase-3 and -9 activities after retinal damage.
Keywords: Crocetin; Retinal damage; Apoptosis; Light exposure; Caspase;

Activation of p38 MAPK by damnacanthal mediates apoptosis in SKHep 1 cells through the DR5/TRAIL and TNFR1/TNF-α and p53 pathways by Feng-Lang Lin; Jue-Liang Hsu; Chang-Hung Chou; Wen-Jun Wu; Chi-I Chang; Hung-Jen Liu (120-129).
The effect of the natural compound damnacanthal from Morinda citrifolia on SKHep 1 cell growth regulation was investigated. Treatment of SKHep 1 cells with damnacanthal for 24 h indicated a dose-dependent antiproliferative activity. Damnacanthal seems to be selective for tumor cell lines, since there is only minimal toxicity against normal hepatocyte cells (FL83B). This is first demonstration that damnacanthal-mediated apoptosis involves the sustained activation of the p38 MAPK pathway, leading to the transcription of the death receptor family genes encoding DR5/TRAIL and TNF-R1/TNF-α genes as well as the p53-regulated Bax gene. The damnacanthal-mediated expression of DR5/TRAIL and TNF-R1/TNF-α results in caspase 8 activation, leading to Bid cleavage. In turn, activated Bid, acting with p53-regulated Bax, leads to cytochrome c released from mitochondria into the cytoplasm. Combined activation of the death receptors and mitochondrial pathways results in activation of the downstream effecter caspase 3, leading to cleavage of PARP. TRAIL- and TNF-α-mediated damnacanthal-induced apoptosis could be suppressed by treatment with caspase inhibitors as well as soluble death receptors Fc:DR5 and Fc:TNF-R1 chimera. Taken together, this study provided first evidence demonstrating that TRAIL-, TNF-α-, and p53-mediated damnacanthal-induced apoptosis require the activation of p38 MAPK and mitochondrion-mediated caspase-dependent pathways.
Keywords: Damnacanthal; Morinda citrifolia; p38 MAPK; TRAIL; TNF-α; p53;

Resveratrol protects human keratinocytes HaCaT cells from UVA-induced oxidative stress damage by downregulating Keap1 expression by Yong Liu; Fangxiao Chan; Haimei Sun; Jihong Yan; Dongying Fan; Dongzhi Zhao; Jing An; Deshan Zhou (130-137).
Ultraviolet radiation A (UVA)-induced oxidative stress is recognized as an important factor in the development of skin carcinogenesis. Resveratrol is demonstrated to possess remarkable antioxidant activity in the organism. The aim of this study was to investigate the protective role of resveratrol in human keratinocytes (HaCaT) against UVA-induced oxidative damage and the possible mechanism of the translocation of NF-E2-related factor-2 (Nrf2) into the nucleus. The HaCaT cells were UVA-irradiated and the effects of resveratrol on cell viability, reactive oxygen species generation and membrane-lipid peroxidation were measured. The proteins and mRNA of Nrf2 and Kelch-like-ECH-associated protein 1 (Keap1) were determined by immunofluorescence staining, Western blot and quantitative PCR, respectively. UVA exposure led to a decrease in viability and an increase in reactive oxygen species generation in HaCaT cells. Resveratrol could effectively increase the viability of HaCaT cells after UVA exposure and protect them from UVA-induced oxidative stress. Moreover, resveratrol increased the level of Nrf2 protein and facilitated Nrf2 accumulation in the nucleus; as a result, the activity of antioxidant enzymes was also upregulated. The main finding was that Keap1 protein, a repressor of Nrf2 in the cytoplasm, was clearly decreased by resveratrol treatment 12 h and beyond though the level of Keap1 mRNA still increased. Our results suggest that resveratrol can degrade Keap1 protein and facilitate Nrf2 accumulation in the nucleus, thereby protecting HaCaT cells from UVA-induced oxidative stress. Resveratrol could be a more useful natural medicine for the protection of epidermal cells from UVA-induced damage.
Keywords: Resveratrol; HaCaT cell; Oxidative stress; Nrf2; Keap1;

Fluconazole inhibits hERG K+ channel by direct block and disruption of protein trafficking by Shengna Han; Yu Zhang; Qiu Chen; Yanyan Duan; Tenghao Zheng; Xiangjie Hu; Zhao Zhang; Lirong Zhang (138-144).
Fluconazole, a commonly used azole antifungal drug, can induce QT prolongation, which may lead to Torsades de Pointes and sudden death. To investigate the arrhythmogenic side effects of fluconazole, we studied the effect of fluconazole on human ether-a-go-go-related gene (hERG) K+ channels (wild type, Y652A and F656C) expressed in human embryonic kidney (HEK293) cells using a whole-cell patch clamp technique, Western blot analysis and confocal microscopy. Fluconazole inhibited wild type hERG currents in a concentration-dependent manner, with a half-maximum block concentration (IC50) of 48.2 ± 9.4 μM. Fluconazole did not change other channel kinetics (activation and steady-state inactivation) of hERG channel. Mutations in drug- binding sites (Y652A or F656C) of the hERG channel significantly attenuated the hERG current blockade by fluconazole. In addition, fluconazole inhibited the trafficking of hERG protein by Western blot analysis and confocal microscopy, respectively. These findings indicate that fluconazole may cause acquired long QT syndrome (LQTS) via a direct inhibition of hERG current and by disrupting hERG protein trafficking, and the mutations Y652 and F656 may be obligatory determinants in inhibition of hERG current for fluconazole.
Keywords: Fluconazole; hERG channel; Long QT syndrome; hERG protein trafficking; Human embryonic kidney cell;

Rho-kinase inhibitor upregulates migration by altering focal adhesion formation via the Akt pathway in colon cancer cells by Seiji Adachi; Ichiro Yasuda; Masanori Nakashima; Takahiro Yamauchi; Takashi Yoshioka; Yukio Okano; Hisataka Moriwaki; Osamu Kozawa (145-150).
Although Rho-kinase is reportedly implicated in carcinogenesis and the progression of human cancers, its precise mechanism has not been fully elucidated. We recently reported that Rho-kinase negatively regulates epidermal growth factor (EGF)-stimulated cancer progression in SW480 colon cancer cells. In the present study, we investigated the effect of Rho-kinase on the migration of SW480 colon cancer cells and the mechanism underlying the involvement of Rho-kinase. Interestingly, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y27632), a specific inhibitor of Rho-kinase, dose-dependently enhanced cell migration. SW480 cells spontaneously release vascular endothelial growth factor (VEGF), however, Y27632 had little effect on its release. While Rho-kinase, which is generally phosphorylated in unstimulated cells, was clearly suppressed by Y27632, exogenous VEGF did not affect its phosphorylation. Immunofluorescence microscopy revealed that Y27632 caused a dramatic change in the localization of focal adhesion components, vinculin, phosphorylated caveolin-1 and tyrosine-phosphorylated proteins in SW480 cells. Furthermore, Akt inhibitor restored the loss of vinculin-stained focal adhesion formation induced by Y27632. We also observed similar effects for Y27632 on the migration and localization of focal adhesion components such as vinculin in another colon cancer cell line, HT29. Taken together, these results strongly suggest that Rho-kinase negatively regulates the migration of colon cancer cells by altering focal adhesion formation via the Akt pathway.
Keywords: Rho-kinase; Cell migration; Colon cancer; Focal adhesion; Akt;

Interaction of ethyl pyruvate in vitro with NF-κB subunits, RelA and p50 by Ayako Mizutani; Noriko Maeda; Seikichi Toku; Sayomi Higa-Nakamine; Yoichiro Isohama; Hajime Sunakawa; Kazuhiro Sugahara; Hideyuki Yamamoto (151-156).
Ethyl pyruvate, an aliphatic ester derived from pyruvate, reportedly has anti-inflammatory actions through inhibition of the transcription mediated by nuclear factor-kappa B (NF-κB). It was suggested that ethyl pyruvate inhibited NF-κB/DNA-binding activity through the covalent modification of RelA. However, the interaction of ethyl pyruvate with RelA in vitro has not been reported. In the present study, we confirmed that treatment of cultured alveolar epithelial cells, A549 cells, with tumor necrosis factor α (TNFα) increased the NF-κB/DNA-binding activity. When the nuclear extract of the cells was incubated with ethyl pyruvate, the NF-κB/DNA-binding activity was strongly inhibited. Because we previously found that the NF-κB/DNA complex included RelA and p50, we bacterially expressed a deletion mutant of RelA, RelA (1-220), and a full-length form of p50. Incubation of RelA (1-220) or p50 with ethyl pyruvate induced dramatic changes in mobility in two types of nondenaturing gel electrophoresis. Electrophoretic mobility shift assays revealed that incubation of RelA (1-220) or p50 with ethyl pyruvate inhibited the DNA-binding activity. Furthermore, immunostaining of A549 cells revealed that ethyl pyruvate inhibited the nuclear association of RelA after TNFα treatment. These results suggest that ethyl pyruvate interacts with RelA and p50 to inhibit their functions at multiple points.
Keywords: A549 cells; Ethyl pyruvate; NF-κB; p50; RelA;

Tempol attenuates cocaine-induced death of PC12 cells through decreased oxidative damage by Ran Numa; Meggie Baron; Ron Kohen; Rami Yaka (157-162).
The association between cocaine administration and induction of oxidative stress in different brain regions suggests that oxidative damage is an important factor participating in cocaine disruption of normal central nervous system functions. In order to deal with this topic, brain penetrating exogenous antioxidants were suggested as a tool to prevent cocaine-induced oxidative damage and behavioral changes. Lately, we have shown that Tempol, a stable nitroxide radical reduced oxidative damage and attenuated the development and expression of cocaine psychomotor sensitization. To examine whether nitroxides, represented by Tempol, can exhibit protective effects against cocaine-induced cell death and to elucidate the molecular mechanism of cocaine-induced oxidative damage, we used the well established PC12 cell line model. The results showed that (1) cocaine induced cell death in a dose-dependent manner (2) and that it was reduced significantly by the stable nitroxide radical Tempol. Furthermore, (3) Tempol significantly inhibited oxidative damage induced by cocaine as reflected by mitochondrial superoxide radical and peroxide enhancement. Finally, (4) Tempol restored the total scavenging capacity which was reduced by cocaine in PC12 cells. Cumulatively, these results suggest that nitroxides such as Tempol can attenuate oxidative damage and cell death induced by cocaine and that PC12 cells can be used as an in vitro model to further investigate the precise molecular mechanism of these compounds.
Keywords: PC12; Cocaine; Oxidative stress; Tempol; Antioxidant; Superoxide radical;

Neferine inhibits cultured hepatic stellate cell activation and facilitates apoptosis: A possible molecular mechanism by Hui Ding; Jinghong Shi; Ying Wang; Jia Guo; Juhui Zhao; Lei Dong (163-169).
Neferine is a major alkaloid component of “Lian Zi Xin”, embryos of the seeds of Nelumbo nucifera Gaertner, Nymphaeaceae. Previous studies have shown that neferine has an inhibitory effect on pulmonary fibrosis through its anti-inflammatory and anti-oxidative activities and inhibition of cytokines and NF-κB. However, it is unknown whether neferine also has an inhibitory effect on liver fibrosis through inhibition of TGF-β1 and collagen I and facilitation of apoptosis of hepatic stellate cells. This study examined the effects of neferine on cultured hepatic stellate (HSC-T6) cells and explored its possible action mechanisms by means of MTT assay, enzyme-linked immunosorbent assay, flow-cytometric annexin V–PI assay and Hoechst 33258 staining, as well as real-time PCR and western blotting. The results showed that neferine administration (2, 4, 6, 8 and 10 μmol/l) significantly decreased the TGF-β1 and collagen I produced in HSC-T6 cells, and increased the HSC-T6 cell apoptosis in a dose-dependent manner. Neferine treatment for 48 h at concentrations of 6 and 10 μmol/l significantly increased Bax and caspase 3 mRNAs and proteins, and reduced Bcl2 and alpha-smooth muscle actin (α-SMA) mRNAs and proteins. Our data indicate that neferine efficiently inhibits cultured HSC-T6 cell activation and induces apoptosis by increasing Bax and caspase 3 expression via the mitochondrial pathway.
Keywords: Neferine; Apoptosis; Hepatic stellate cell; Hepatic fibrosis;

Pancreatic cancer is one of the most aggressive human malignancies with an increasing incidence worldwide. Despite an increase in the number of systemic treatments available for pancreatic cancer, the impact of therapy on the clinical course of the disease has been modest, underscoring an urgent need for new therapeutic options. Although selective cyclooxygenase-2 inhibitors have been demonstrated to have cancer-preventive effects, the mechanism of their effects is not clearly known. Moreover, there have been no unbiased studies to identify novel molecular targets of NS-398 regarding pancreatic cancer. Here we undertook a gene expression profiling study to identify novel molecular targets modulating the growth inhibitory effects of NS-398 on pancreatic cancer cell lines. Our mRNA-based gene expression results showed that the growth inhibitory effect of NS-398 was accompanied with an activation of G1/S and G2/M cell cycle regulation, P53 signalling, apoptotic, aryl hydrocarbon receptor and death receptor signalling pathways. Moreover, we reported, for the first time, that the growth inhibitory effect of NS-398 is mediated by down-regulation of RRM2, CTGF, MCM2 and PCNA and up-regulation of NAG-1 in all cell lines.
Keywords: Pancreatic cancer; Microarray; Ingenuity; NS-398;

Pharmacological characterization of the ghrelin receptor antagonist, GSK1614343 in rat RC-4B/C cells natively expressing GHS type 1a receptors by Elisabetta Perdonà; Federico Faggioni; Alberto Buson; Fabio Maria Sabbatini; Corrado Corti; Mauro Corsi (178-183).
A novel growth hormone secretagogues type 1a (GHS1a) receptors antagonist (2R)-N'-[3,5- bis(trifluoromethyl)phenyl]-2-[(8aR)-hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl]-2-(3- pyridinyl)ethanohydrazide (GSK1614343) was functionally characterised in rat pituitary adenoma cell line, RC-4B/C endogenously expressing GHS1a receptors. The antagonism profile of GSK1614343 was compared with that of 6-[(4-fluorophenyl)oxy]-2-methyl-3-{[(3S)-1-(1-methylethyl)-3- piperidinyl]methyl}-4(3H)-quinazolinone (YIL-781) another ghrelin receptor antagonist recently published. The activity of both compounds was also evaluated at rat recombinant GHS1a receptors. The characterization of the two antagonists was performed by intracellular calcium mobilization measurements by using fluorometric imaging plate reader (FLIPR) technology and inositol phosphate (IP) turnover measurements by [3H]-IP accumulation assay. RC-4B/C and U2-OS cells transiently transduced with rat GHS1a receptors virus were used. In RC-4B/C cells, GSK1614343 and YIL-781, depressed the ghrelin maximal response in FLIPR assay as result of hemi-equilibria phenomenon. When using the [3H]-IP accumulation assay both compounds behaved as competitive antagonist with pKB values of 8.03 for GSK1614343 and 7.54 for YIL-781. In rat recombinant receptor, GSK1614343 and YIL-781 inhibited the calcium response induced by ghrelin with pIC50 values of 7.90 and 8.27, respectively. GSK1614343 and YIL-781 did not show intrinsic activity in both endogenously expressed and recombinant rat GHS1a receptors. The new ghrelin receptor antagonist GSK1614343 is a potent competitive antagonist in rat pituitary RC-4B/C cells endogenously expressing GHS1a receptors when equilibrium conditions between ligand and receptor are reached in the test assay. GSK1614343 represents a useful tool to investigate the physiological relevance of the ghrelin system in rat models.
Keywords: Ghrelin; RC-4B/C cells; GHS1a receptor; GSK1614343; YIL-781;

The accumulation of malondialdehyde (MDA), a lipid peroxidation by-product that has been used as an indicator of cellular oxidation status, is significantly increased in many neurological diseases such as brain ischemia/reperfusion, Alzheimer's disease and Parkinson's disease in vivo. In the present study, we found that MDA treatment in vitro reduced cortical neuronal viability in a time- and dose-dependent manner and induced cellular apoptosis as well as necrosis simultaneously. Furthermore, exposure to MDA led to accumulation of intracellular reactive oxygen species, dysfunction of mitochondria (denoted by the loss of mitochondrial transmembrane potential (Δψm)) and activation of JNK and ERK. Carnosine exhibited better protection against MDA-induced cell injury than antioxidant N-acetyl-cysteine (NAC) with its multi-potency, which alleviated MDA-induced protein cross-linking, Δψm decrease, reactive oxygen species burst, JNK and ERK activation. In conclusion, our results suggest that MDA induced cell injury in vitro via protein cross-linking and successive mitochondrial dysfunction, and the activation of reactive oxygen species-dependent MAPK signaling pathway. Carnosine alleviated all these alterations induced by MDA, but NAC merely inhibited Bcl-2 family-related activation of JNK and ERK. These results prompt the possibility that carnosine, but not other conventional antioxidants, can protect neurons against MDA-induced injury through decomposition of protein cross-linking toxicity and may serve as a novel agent in the treatment of neurodegenerative diseases.
Keywords: MDA; Carnosine; Lipid peroxidation; Protein cross-linking; Oxidative stress; N-acetyl-cysteine;

The dipeptidyl peptidase IV (CD26, EC inhibitor vildagliptin is a potent antihyperalgesic in rats by promoting endomorphin-2 generation in the spinal cord by Kornél Király; Anne-Marie Lambeir; Judit Szalai; Apolka Szentirmay; Walter Luyten; István Barna; Zita Puskár; Márk Kozsurek; András Z. Rónai (195-199).
We have reported previously that the dipeptidyl peptidase IV inhibitor Ile-Pro-Ile had an antihyperalgesic action in rats when given intrathecally in the carrageenan-induced hyperalgesia, as detected by the Randall–Selitto test. Vildagliptin, a non-peptide inhibitor of the same enzyme, which is already on the market as an “euglycemic” agent in diabetics, has a slightly more potent and more sustained antihyperalgesic effect in the same test when given by the same route. The action of 3 nmol/rat vildagliptin could be antagonized by subcutaneous naltrexone (0.5 mg/kg) pretreatment, or by intrathecally co-administered specific antiserum to endomorphin-2. Thus, the antihyperalgesia by vildagliptin, similarly to Ile-Pro-Ile, was opioid receptor-mediated and could be attributed to the promotion of endomorphin-2 generation in rat spinal cord dorsal horn. Furthermore, vildagliptin (1 mg/kg) is a potent antihyperalgesic also when given subcutaneously.
Keywords: Vildagliptin; Antihyperalgesic effect; Dipeptidyl peptidase IV; Endomorphin-2 generation; Carrageenan-induced hyperalgesia;

Agmatine modulates neuroadaptations of glutamate transmission in the nucleus accumbens of repeated morphine-treated rats by Xiao-Fei Wang; Ning Wu; Rui-Bin Su; Xin-Qiang Lu; Yin Liu; Jin Li (200-205).
It has been proved that agmatine inhibits opioid dependence, yet the neural mechanism remains unclear. In the present study, the effect of agmatine on the neuroadaptation of glutamate neurotransmission induced by morphine dependence, including changes of the extracellular glutamate level and glutamate receptors in the nucleus accumbens was investigated.We found that agmatine (2.5–20 mg/kg, s.c.) inhibited development of morphine dependence, which was consistent with our previous report. In rats repeatedly treated with morphine, the glutamate level in the nucleus accumbens dialysate was markedly increased after naloxone-precipitated withdrawal. When agmatine (20 mg/kg, s.c.) was co-pretreated with morphine or was applied before naloxone-precipitated withdrawal, this elevation of the extracellular glutamate level was inhibited. In the synaptosome model, repeated morphine treatment and naloxone precipitation induced an increase in glutamate release, while agmatine (20 mg/kg, s.c.) co-pretreated with morphine reversed the increase of glutamate release. However, neither morphine or agmatine treatment alone nor morphine and agmatine co-administration had any influence on [3H]-glutamate uptake. It indicated that the elevation of the glutamate level in the nucleus accumbens might be caused by the increase of glutamate release of synaptosome in the withdrawal conditions of morphine-dependent rat. Furthermore, agmatine concomitant treatment with morphine entirely abolished the up-regulation of the NR1 subunit of N-methyl- d -aspartate (NMDA) receptors in the nucleus accumbens in repeated morphine-treated rats.Taken together, the present study demonstrated that agmatine could modulate the neuroadaptations of glutamate transmission in the nucleus accumbens in the case of morphine dependence, including modulating extracellular glutamate concentration and NMDA receptor expression.
Keywords: Agmatine; Opioid dependence; Extracellular glutamate; NMDA receptors;

Tetrahydroxystilbene glucoside, a plant-derived cognitive enhancer, promotes hippocampal synaptic plasticity by Ting Wang; Yuan-Jian Yang; Peng-Fei Wu; Wei Wang; Zhuang-Li Hu; Li-Hong Long; Na Xie; Hui Fu; Fang Wang; Jian-Guo Chen (206-214).
Plant or food derived polyphenols have received a great deal of attention due to their biological properties including anti-oxidative effects, neuroprotection and memory enhancement. Here, we investigated the roles of 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), an active component of the rhizome extracted from Polygonum multiflorum, in the regulation of hippocampal synaptic plasticity in normal mice as well as the underlying mechanisms. It was shown that TSG promoted the differentiation of PC12 cells and increased the intracellular calcium level in hippocampal neurons. TSG facilitated high-frequency stimulation (HFS)-induced hippocampal long-term potentiation (LTP) in a bell-shaped manner. The facilitation of LTP induction by TSG required calcium/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) activation. Taken together, our data demonstrate that TSG promotes LTP induction and this effect may contribute to the enhancement of learning and memory seen in animal models.
Keywords: 2,3,5,4′-Tetrahydroxystilbene-2-O-β-d-glucoside; Synaptic plasticity; Calcium/calmodulin-dependent protein kinase II; Extracellular signal-regulated kinase;

The present study investigated whether clonidine – an α2-adrenoceptor agonist known to relieve pain – is able to suppress itch-related behavior in mice. An intraplantar injection of serotonin induced biting (an itch-related response), which was inhibited by intraperitoneal and intrathecal, but not intraplantar or intracisternal, clonidine injections. The effect of intrathecal clonidine was inhibited by intrathecal injections of phentolamine (a non-selective α-adrenoceptor antagonist) and yohimbine (a selective α2-adrenoceptor antagonist), but not by prazosin (a selective α1-adrenoceptor antagonist). The effect of intraperitoneal clonidine was also inhibited by intrathecal yohimbine. These results suggest that clonidine is an effective antipruritic agent and that the effect is mainly mediated by the stimulation of α2-adrenoceptors in the dorsal horn.
Keywords: Clonidine; Itch-related response; Biting; Pruritus; Intrathecal injection; α2-Adrenoceptor;

Co-administration of caffeine profoundly enhances the acute toxicity of 3,4 methylenedioxymethamphetamine (MDMA) in rats. The aim of this study was to determine the ability of caffeine to impact upon MDMA-induced dopamine release in superfused brain tissue slices as a contributing factor to this drug interaction. MDMA (100 and 300 μM) induced a dose-dependent increase in dopamine release in striatal and hypothalamic tissue slices preloaded with [3H] dopamine (1 μM). Caffeine (100 μM) also induced dopamine release in the striatum and hypothalamus, albeit to a much lesser extent than MDMA. When striatal tissue slices were superfused with MDMA (30 μM) in combination with caffeine (30 μM), caffeine enhanced MDMA-induced dopamine release, provoking a greater response than that obtained following either caffeine or MDMA applications alone. The synergistic effects in the striatum were not observed in hypothalamic slices. As adenosine A1 receptors are, one of the main pharmacological targets of caffeine, which are known to play an important role in the regulation of dopamine release, their role in the modulation of MDMA-induced dopamine release was investigated. 1 μM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a specific A1 antagonist, like caffeine, enhanced MDMA-induced dopamine release from striatal slices while 1 μM 2,chloro-N(6)-cyclopentyladenosine (CCPA), a selective adenosine A1 receptor agonist, attenuated this. Treatment with either SCH 58261, a selective A2A receptor antagonist, or rolipram, a selective PDE-4 inhibitor, failed to reproduce a caffeine-like effect on MDMA-induced dopamine release. These results suggest that caffeine regulates MDMA-induced dopamine release in striatal tissue slices, via inhibition of adenosine A1 receptors.
Keywords: MDMA; Caffeine; Superfusion; Adenosine A1 receptor; Dopamine; (Rat);

Effects of adolescent social isolation on the expression of brain-derived neurotrophic factors in the forebrain by Qingxuan Meng; Nanxin Li; Xiao Han; Feng Shao; Weiwen Wang (229-232).
Adolescence is a critical period for the development of neural plasticity. Social isolation is an important animal model for various neurodevelopmental disorders. Brain derived neurotrophic factor (BDNF) mediates many important brain functions such as neural plasticity, and aberrations in its expression have been implicated in many brain disorders. However, to date there have been few reports of effects of adolescent social isolation on BDNF expression in adult animals. In the present study, we subjected weaning Sprague Dawley rats to a four-week early adolescence social isolation procedure followed by four-week standard housing until adulthood for molecular assays. BDNF protein levels in several key forebrain regions relevant to brain development were investigated using immunohistochemistry, including frontal and cingulate cortex as well as hippocampus. Our results show that adolescent social isolation significantly increased BDNF protein expression in the medial prefrontal cortex and all three sub-fields of the hippocampus, including CA1, CA2/3 and dentate gyrus. This study advances the use of adolescent social isolation as an animal model for studying neurobiological underpinnings of various neurodevelopmental disorders.
Keywords: Adolescent; BDNF; Frontal cortex; Hippocampus; Social isolation;

Mu and delta opioid receptors modulate the reinforcing effects of ethanol, however, their role in the subjective effects of ethanol is not well understood. This study evaluated the contribution of mu and delta opioid receptors to the subjective effects of ethanol using drug discrimination procedures. Monkeys were trained to discriminate ethanol from saline under a schedule of food delivery. In tests, ethanol engendered increases in drug-lever responding, reaching a maximum of > 80%. The mu opioid receptor agonists fentanyl and buprenorphine and the delta opioid receptor agonists SNC 80 and SNC 162 did not substitute for the discriminative stimulus effects of ethanol. As pretreatments, the full agonists fentanyl and SNC 80 enhanced the effects of low doses of ethanol and fentanyl attenuated the effects of the ethanol training dose. Although the possibility of pharmacological antagonism of the effects of ethanol cannot be ruled out, a more likely alternative is that the diminished effects of ethanol were due to perceptual masking of the ethanol stimulus. In contrast, the partial agonists buprenorphine and SNC 162 did not alter ethanol's effects. Finally, the discriminative stimulus effects of ethanol were attenuated following administration of presumably mu-selective doses of the antagonist naltrexone, but not after administration of the delta opioid receptor antagonist naltrindole. The ability of naltrexone to block the discriminative stimulus effects of ethanol likely reflects its capacity to attenuate ethanol-induced increases in endogenous opioids, in particular beta-endorphin, because attenuation of the ethanol stimulus was not accompanied by significant suppression of response rate.
Keywords: Mu opioid receptor; Delta opioid receptor; Drug discrimination; Beta-endorphin; Perceptual masking; (Monkey);

Suppression of nitric oxide synthesis by L-NAME reverses the beneficial effects of pioglitazone on scopolamine-induced memory impairment in mice by Nika Allami; Mehrak Javadi-Paydar; Farhoud Rayatnia; Kourosh Sehhat; Reza Rahimian; Abbas Norouzi; Ahmad Reza Dehpour (240-248).
Pioglitazone, an agonist of peroxisome proliferator-activated receptor gamma (PPARγ), which is widely used in treatment of type 2 diabetes, has shown some therapeutic effect in Alzheimer's disease. In this study, effects of acute pioglitazone on acquisition, consolidation and retrieval of memory, and also the involvement of nitric oxide (NO) in the effects of pioglitazone on spatial recognition memory has been investigated in a two-trial recognition Y-maze test and passive avoidance in mice. Memory impairment was induced by scopolamine (1 mg/kg, i.p.). Pioglitazone (10 and 20 mg/kg, p.o.) was administrated prior to either acquisition, consolidation or retention trials, while L-NAME (N-nitro-l-arginine methyl ester), a non-specific NO synthase inhibitor, was administered (10 mg/kg, i.p.) 30 min before each trial. Results: 1) pioglitazone improved the acquisition of recognition spatial memory-impaired by scopolamine; L-NAME dramatically reversed improving effects of pioglitazone on memory acquisition; 2) pioglitazone did not change the consolidation of spatial memory, impaired by scopolamine; 3) pioglitazone improved the retrieval of spatial memory and L-NAME did not alter the beneficial effect of pioglitazone; 4) pioglitazone did not affect scopolamine-induced cognitive impairments in the passive avoidance test.The present study demonstrates the beneficial effect of acute pioglitazone administration on acquisition and retrieval of scopolamine-induced cognitive deficits. This effect was reversed only in acquisition phase by nitric oxide synthase inhibitor, L-NAME, therefore, it could be concluded that NO might be involved in the pioglitazone beneficial effect of spatial memory acquisition.
Keywords: Pioglitazone; Spatial recognition memory; Nitric oxide; L-NAME; (Mice);

The glucagon-like peptide 1 receptor agonist exendin-4 improves reference memory performance and decreases immobility in the forced swim test by Ruben Isacson; Elisabet Nielsen; Karin Dannaeus; Göran Bertilsson; Cesare Patrone; Olof Zachrisson; Lilian Wikström (249-255).
We have earlier shown that the glucagon-like peptide 1 receptor agonist exendin-4 stimulates neurogenesis in the subventricular zone and excerts anti-parkinsonian behavior. The aim of this study was to assess the effects of exendin-4 treatment on hippocampus-associated cognitive and mood-related behavior in adult rodents. To investigate potential effects of exendin-4 on hippocampal function, radial maze and forced swim test were employed. The time necessary to solve a radial maze task and the duration of immobility in the forced swim test were significantly reduced compared to respective vehicle groups if the animals had received exendin-4 during 1–2 weeks before testing. In contrast to the positive control imipramine, single administration of exendin-4 1 h before the challenge in the forced swim test had no effect. Immunohistochemical analysis showed that the incorporation of bromodeoxyuridine, a marker for DNA synthesis, as well as doublecortin expression was increased in the hippocampal dentate gyrus following chronic treatment with exendin-4 compared to vehicle-treated controls. The neurogenic effect of exendin-4 on hippocampus was confirmed by quantitative PCR showing an upregulation of mRNA expression for Ki-67, doublecortin and Mash-1. Since exendin-4 significantly improves hippocampus-associated behavior in adult rodents, it may be a candidate for alleviation of mood and cognitive disorders.
Keywords: Hippocampus; Glucagon-like peptide 1 receptor; Exendin-4; Depression; Cognition;

Histamine H1 antagonist treatment with pyrilamine reduces nicotine self-administration in rats by Edward D. Levin; Susan Slade; Corinne Wells; Margaret Pruitt; Vanessa Cousins; Marty Cauley; Ann Petro; Dawn Hampton; Jed Rose (256-260).
Nicotine has been definitively shown to be critically involved in the neural bases of tobacco addiction. However, nicotine releases a wide variety of neurotransmitters. Nicotine-induced dopamine release has been shown to play a key role in facilitating nicotine self-administration. Other transmitter systems may also play important roles in the pharmacological effects of nicotine and may provide important leads for combating nicotine self-administration. Clozapine, an antipsychotic drug, which blocks a variety of different transmitter receptors including serotonin 5HT2 and histamine H1 receptors, has been found to decrease smoking. Previously we found that the serotonin 5HT2 antagonist, ketanserin, significantly reduced nicotine self-administration. In the current study, we assessed histamine H1 receptor interaction with nicotine self-administration. Young adult female Sprague–Dawley rats were fitted with IV catheters and trained to self-administer nicotine (0.03 mg/kg/infusion). Acute doses of 40 mg/kg of pyrilamine, a histamine H1 antagonist, significantly reduced nicotine self-administration. We also found that repeated injections (20 mg/kg) or chronic infusion via osmotic minipumps (50 mg/kg/day) of pyrilamine also significantly decreased nicotine self-administration. The peripherally restricted H1 antagonist ebastine was ineffective in reducing nicotine self-administration, pointing to central H1 receptor blockade as key for the effectiveness of pyrilamine. H1 antagonists may be a promising avenue to explore for new treatments to aid smoking cessation.
Keywords: Nicotine; Self-administration; Histamine; H1; Pyrilamine; Ebastine; (Rat);

The aim of the present study was to assess the effect of chronic naltrexone treatment on daily patterns of food intake in food-deprived and free-feeding rats. In experiment 1, Wistar male rats had continuous access to food and water, while in experiment 2 they were deprived of food for 12 h/day. Animals in both experiments were studied as follows: a baseline period (7 days), followed by a treatment period (14 days) with either saline or naltrexone at 10 mg/kg/day. Finally, a post-treatment period (7 days) was assessed. Food and water consumption were measured every 2 h after the naltrexone or saline injection for 12 h and once more 12 h later. Experiment 1: Food intake was higher in the naltrexone group 10 h after injection. Total food intake and body weight gain were higher in the naltrexone group than in the saline group in the second week of treatment and in the post-treatment period. Experiment 2: The overeating observed in the saline group in the hours following the 12 h of the food deprivation period was suppressed by naltrexone, though total daily food intake was not affected. Body weight gain was initially reduced by naltrexone, but a rebound effect was observed during the post-treatment period in the naltrexone group. Naltrexone produced a differential effect on food intake and body weight that depended on the rats' food deprivation status. These results could be explained in terms of opioid receptor up-regulation that enhances the rewarding effects of food or by naltrexone-produced changes in palatability.
Keywords: Naltrexone; Food intake; Body weight; Water consumption; Opioid antagonist; Alimentary behaviour;

Caffeine/nutrition interaction in the rat brain: Influence on latent inhibition and cortical spreading depression by Márlison José Lima de Aguiar; Cilene Rejane Ramos Alves de Aguiar; Rubem Carlos Araújo Guedes (268-274).
Caffeine, like malnutrition, can produce behavioral and electrophysiological alterations. However, the interaction of both factors remains unclear. Here this interaction has been studied in male Wistar rats previously malnourished during the lactation period by feeding their dams the “regional basic diet” of Northeast Brazil, containing about 8% protein, predominantly from vegetable sources (RBD8). At 70–75 days of life, a subset of the pups was treated intraperitoneally with 30 mg/kg caffeine for 4 days while being tested according to the behavioral model of latent inhibition. Another group was subjected to an electrophysiological recording of the phenomenon known as cortical spreading depression, and the effects of caffeine injected during the recording session were evaluated. Caffeine did not affect cortical spreading depression, but antagonized latent inhibition in both the RBD8-malnourished rats and in the well-nourished control group fed a chow diet with 22% protein. This effect of caffeine was not seen in malnourished rats fed a protein-supplemented RBD (protein increased to 22% by increasing the proportion of foodstuffs from vegetable origin; RBD22 group), suggesting that the amino acid imbalance of this diet may modulate the caffeine effects on latent inhibition. The results indicate a differential effect of caffeine in the latent inhibition behavioral model, as compared to the cortical spreading depression phenomenon, and this effect is influenced by the early nutritional status of the animal. We suggest that caffeine may modulate dopaminergic subcortical receptors participating in attention processes, but does not interact at the cortical level, in a way that would affect cortical spreading depression.
Keywords: Protein malnutrition; Caffeine; Attention behavior; Latent inhibition; Brain electrophysiology; Cortical spreading depression;

Tanshinone IIA attenuates atherosclerosis in ApoE−/− mice through down-regulation of scavenger receptor expression by Fu-Tian Tang; Yuan Cao; Tie-Qiao Wang; Li-Jing Wang; Jiao Guo; Xiao-Shi Zhou; Suo-wen Xu; Wei-Hua Liu; Pei-Qing Liu; He-Qing Huang (275-284).
This study is designed to investigate the protection of tanshinone IIA (TSIIA) against atherosclerosis in apolipoprotein E deficient (ApoE−/−) mice and to explore the mechanisms by focusing on the expressions of scavenger receptors, scavenger receptor-A (SR-A) and CD36. The in vivo study demonstrated that TSIIA (10–90 mg/kg) inhibited the atherosclerotic lesions, down-regulated the CD68 protein expression in lesion and decreased the contents of cholesterol in aortas of ApoE−/− mice. In addition, TSIIA reduced the serum levels of oxidized LDL (oxLDL) and down-regulated the mRNA expression of CD36, SR-A and peroxisome proliferator-activated receptor gamma (PPARγ) in aortas. The in vitro study showed that TSIIA (0.1–10 μM) decreased cholesterol level and DiI-oxLDL uptake in mouse peritoneal macrophages treated with oxLDL (50 μg/ml). In addition, TSIIA down-regulated the mRNA and protein expression of CD36 but not that of SR-A in oxLDL treated macrophages. TSIIA also down-regulated the mRNA expression of PPARγ in oxLDL treated macrophages. Furthermore, TSIIA reduced the mRNA expression of CD36 in macrophages treated with PPARγ agonist 15d-PGJ2 (2 μM) or troglitazone (50 μM), whereas both 15d-PGJ2 (0.5–1.5 μM) and troglitazone (5–20 μM) dose-dependently abolished the down-regulation of CD36 expression by TSIIA in oxLDL treated macrophages. These results suggest that TSIIA attenuates the atherosclerotic lesion in ApoE−/− mice, which might be attributed to the properties of both anti-oxidation and down-regulation of scavenger receptors. Furthermore, antagonism of PPARγ might be involved in the down-regulation of CD36 by TSIIA.
Keywords: Atherosclerosis; Tanshinone IIA; Oxidative stress; Scavenger receptor; PPARγ antagonism;

Magnesium lithospermate B decreases [Ca2+]i in endothelial cells by inhibiting K+ currents by Haifei Zhang; Jie Zhang; Ruopeng Zha; Miaomiao Hu; Yiping Wang (285-289).
Magnesium lithospermate B (MLB) is a hydrophilic active component of Salviae miltiorrhizae Radix. Studies have shown that MLB affected intracellular calcium ([Ca2+]i), but the underlying mechanism was unclear yet. The present work was aimed to investigate the underlying mechanism of MLB affecting [Ca2+]i in endothelial cells (ECs). Isolated mesentery arteries were employed to test the involvement of L-Ca2+ channel. [Ca2+]i was measured in ECs loaded with Fluo-3. Membrane potential and membrane currents were recorded in ECs using patch-clamp techniques. Results showed that MLB did not inhibit Ca2+ influx via L-Ca2+ channel in isolated mesenteric arteries. However, MLB decreased [Ca2+]i in a concentration-dependent manner in ECs. MLB depolarized the membrane potential of ECs and inhibited K+ currents. These results demonstrated that MLB decreased [Ca2+]i by inhibiting K+ currents and depolarizing membrane potential in ECs.
Keywords: Endothelial cell; Magnesium lithospermate B; K+ current; Membrane potential;

Protective effects of novel single compound, Hirsutine on hypoxic neonatal rat cardiomyocytes by Li Xin Wu; Xian Feng Gu; Yi Chun Zhu; Yi Zhun Zhu (290-297).
Uncaria rhynchophylla is a traditional Chinese herb that has been applied in China for treatment of ailments of the cardiovascular system, but little is known about its active constituents and effect in cardiomyocytes. In present study, we investigated the cardioprotective effect of 0.1 μΜ, 1 μΜ and 10 μΜ Hirsutine isolated from the methanolic extracts of Uncaria rhynchophylla by high performance liquid chromatography (HPLC) on neonatal rat cardiomyocytes treated with hypoxia to determine the mechanism underlying the protective effect with regard to cardiac anti-oxidant enzymes and apoptosis genes. Hirsutine significantly increased the viability of cardiomyocytes injured by hypoxia. Gene expression levels of proapoptotic genes (Bax, Fas and caspase-3) were significantly downregulated compared with the hypoxic control group (P < 0.05), whereas the expression level of Bcl-2 was upregulated following Hirsutine treatment (P < 0.05). Correspondingly, Hirsutine treatment increased Bcl-2 protein level and decreased Bax protein level. Assay investigating cardiac anti-oxidant enzymes provided further evidence for the protective effect of Hirsutine, as indicated by the induction of the anti-oxidant enzymes superoxide dismutase. The results of present study suggest that the mechanism of action of Hirsutine in hypoxic neonatal rat cardiomyocytes may be related to its anti-oxidant and anti-apoptotic properties. This may open an avenue for developing novel candidate compounds with cardioprotective effect from unique Chinese plant.
Keywords: Hirsutine; Cardiomyocytes; Hypoxia; Apoptosis; Superoxide dismutase;

PTEN inhibitors cause a negative inotropic and chronotropic effect in mice by Lingyun Zu; Zhenyun Shen; Jacob Wesley; Zheqing P. Cai (298-302).
Inactivation of phosphatase and tensin homologue deleted on chromosome ten (PTEN) decreases cardiac contractility under basal conditions and induces cardioprotection against ischemia–reperfusion injury. However, the pharmacological effect of PTEN inhibitors on cardiac contractility has not been studied before. In the present study, we investigated the hypothesis that PTEN inhibition decreases cardiac contractility in mice. We first exposed isolated mouse hearts to the PTEN inhibitor bpV(phen) (40 μM), the phosphoinositide-3 kinase inhibitor wortmannin (1 μM), and the PTEN-resistant PIP3 analog 3-phosphorothioate-PtdIns(3,4,5)P3 (3-PT-PTP, 0.5 μM) for 10 min. Left ventricular pressure was measured by a Mikro-tip pressure catheter. We then inhibited PTEN in mice by intra-peritoneal injection of VO-OHpic (10 μg/kg) 30 min before ischemia and then exposed them to 30 min of ischemia and 120 min of reperfusion. At the end of the experiments, hearts were isolated for measurement of myocardial infarct size by 1.5% triphenyltetrazolium chloride. Left ventricular systolic pressure and heart rate were significantly decreased by bpV(phen). Consistent with the result, the maximal rate of left ventricular pressure increase or decrease was significantly decreased by bpV(phen). 3-PT-PIP3 mimicked the effect of bpV(phen), and the opposite effect on cardiac contractility was seen with wortmannin. Moreover, inhibition of PTEN in vivo by VO-OHpic decreased left ventricular systolic pressure and heart rate before ischemia, but resulted in an increase in cardiac functional recovery and a decrease in myocardial infarct size after ischemia–reperfusion. In conclusion, PTEN inhibition causes a negative inotropic and chronotropic effect while inducing cardioprotection against ischemia–reperfusion injury.
Keywords: PTEN; PI3K; Cardiac contractility; Reperfusion injury; Myocardial infarction;

Effects of β-adrenoceptor antagonists on anaphylactic hypotension in conscious rats by Wei Zhang; Toshishige Shibamoto; Yasutaka Kurata; Hiroyuki Kohno (303-308).
Anaphylactic shock is sometimes fatal or resistant to therapy in patients treated with propranolol, a nonselective β-adrenoceptor antagonist, against cardiovascular diseases. However, it remains unknown which subtype of β-adrenoceptors, β1- or β2-adrenoceptor, is primarily responsible for the detrimental effects of propranolol on anaphylactic hypotension. Effects of β1- and β2-adrenoceptor antagonists were therefore determined on the survival rate and systemic hypotension in conscious Sprague–Dawley rats that suffered from anaphylactic shock. Mean arterial pressure and portal venous pressure were simultaneously measured. The control rats showed a decrease in mean arterial pressure and an increase in portal venous pressure, but did not die within 48 h after an injection of ovalbumin antigen. The survival rate of the rats pretreated with propranolol (1 mg/kg; n  = 7), the selective β2-adrenoceptor antagonist ICI 118,551 (0.5 mg/kg; n  = 7), or adrenalectomy (n  = 7) was significantly smaller than that with the selective β1-adrenoceptor antagonist atenolol (2 mg/kg; n  = 7). However, the changes in mean arterial pressure and portal venous pressure were similar for 10 min after antigen among any groups, although propranolol and atenolol attenuated the antigen-induced increase in heart rate. Furthermore, bolus injections of epinephrine (3 μg/kg) at 3 and 5 min after antigen prevented the death of the atenolol-pretreated rats, but only marginally prolonged the survival rates for the ICI 118,551- or propranolol-pretreated and adrenalectomized rats. In conclusion, in rat anaphylactic shock, inhibition of β2-adrenoceptor causes more detrimental effects than that of the β1-adrenoceptor. These β-adrenoceptor antagonists may exert detrimental effects on rat systemic anaphylaxis via inhibiting beneficial actions of catecholamines endogenously released from the adrenal gland.
Keywords: Anaphylactic shock; β-Adrenoceptor antagonist; Propranolol; Atenolol; ICI 118,551; Portal venous pressure; Conscious rat;

Nicorandil normalizes prolonged repolarisation in the first transgenic rabbit model with Long-QT syndrome 1 both in vitro and in vivo by Jürgen Biermann; Kezhong Wu; Katja E. Odening; Stefan Asbach; Gideon Koren; Xuwen Peng; Manfred Zehender; Christoph Bode; Michael Brunner (309-316).
Transgenic rabbits expressing loss-of-function pore mutants of the human gene KCNQ1 (KvLQT1-Y315S) have a Long QT-Syndrome 1 (LQT1) phenotype. We evaluated for the first time the effect of nicorandil, an opener of ATP-sensitive potassium channels, and of isoproterenol on cardiac action potential duration and heart rate dependent dispersion of repolarisation in transgenic LQT1 rabbits. In vivo LQT1 and littermate control were subjected to transvenous electrophysiological studies; in vitro monophasic action potentials were recorded from explanted Langendorff-perfused hearts. In vivo ventricular effective refractory periods (VERP) at the right ventricular base were significantly prolonged in LQT1 as compared to littermate control, resulting in a more pronounced VERP dispersion in LQT1. This difference in VERP dispersion between LQT1 and littermate control disappeared after infusion of nicorandil. In vitro, mean action potential durations (APD75 and APD90) of LQT1 were significantly prolonged compared to littermate control at baseline. Nicorandil decreased APD75 and APD90 in LQT1 and littermate control at all stimulated heart rates. After adding nicorandil, the APD90 at all hearts rates and the APD75 at high heart rates were no longer different. Dispersion of repolarisation (∆APD75 and ∆APD90) was heart rate dependently decreased after nicorandil at all tested stimulation cycle lengths only in LQT1. We demonstrated phenotypic differences of LQT1 and littermate control in vivo and in vitro. Nicorandil 20 μmol/l improved repolarisation abnormalities and heterogeneities in transgenic LQT1 rabbits.
Keywords: Long QT-Syndrome; Polymorphic ventricular tachycardia; Transgenic rabbit model; Electrophysiological studies; Nicorandil; Dispersion of repolarisation;

We previously reported that chronic ethanol lowers blood pressure in female rats. In this study, hemodynamic, biochemical, and immunoblot analyses were performed to investigate: (i) the roles of cardiac contractility and autonomic activity in the hypotensive action of ethanol, and (ii) whether endotoxemia-induced upregulation of cardiac and/or vascular nitric oxide synthase (NOS) isoforms underlies the hypotensive and cardiac effects of ethanol. Telemetric monitoring of blood pressure, heart rate, and myocardial contractility (dP/dt max) was performed in female rats receiving liquid diet with or without ethanol (5% w/v, 13 weeks). Autonomic control was assessed by frequency domain analysis of interbeat intervals (IBI) and systolic blood pressure (SBP). Compared with pair-fed controls, ethanol caused sustained reductions in blood pressure, heart rate, and + dP/dt max. Ethanol feeding increased the spectral power of high-frequency band (IBIHF, 0.75–3 Hz) and decreased the low-frequency band (IBILF, 0.25–0.75 Hz) and IBILF/HF ratio, suggesting increased cardiac parasympathetic dominance. In contrast, vascular tone was not affected by ethanol because SBP spectral bands and plasma norepinephrine remained unchanged. Myocardial expressions of eNOS and its upstream regulators, phosphatidylinositol 3-kinase (PI3K) and Akt, and plasma endotoxin and nitrite/nitrate were increased by ethanol. Myocardial iNOS was also increased by ethanol whereas nNOS remained unchanged and aortic levels of all NOS isoforms were not altered by ethanol. These findings suggest that facilitation of myocardial PI3K/Akt/eNOS and iNOS pathways, due possibly to ethanol-induced endotoxemia and/or increased cardiac parasympathetic dominance, might constitute a cellular mechanism for the reduced myocardial contractility and hypotension caused by ethanol in female rats.
Keywords: Ethanol; Blood pressure; Myocardial contractility; Female rats; Nitric oxide synthase; Hemodynamic variability;

Na+–H+ exchanger activation on resuscitation following hemorrhagic shock has been shown to result in myocardial injury and dysfunction. Amiloride, an inhibitor of the Na+–H+ exchanger has been shown to protect the myocardium against reperfusion injury in the ischemic hearts. However, the mechanism of protection remains unclear. Na+–H+ exchanger blockers have been implicated in the regulation of inflammatory responses and chemokine production. The present study investigated the therapeutic anti-inflammatory value of amiloride on myocardial contractile function in post-resuscitation following hemorrhagic shock in rats. Male Sprague–Dawley rats were assigned into 3 groups: 1) hemorrhage, 2) hemorrhage treated with amiloride, and 3) sham hemorrhage (n = 6 per group). Rats were hemorrhaged over 60 min to reach a mean arterial blood pressure of 40 mm Hg. After 60 min of hemorrhagic shock, rats were treated or not by injection of 1 ml of 100 μM amiloride (0.027 mg/ml) intra-arterially. Resuscitation was made in vivo by reinfusion of the shed blood to restore norm tension for 30 min. Left ventricular contractile function was measured in the isolated hearts following hemorrhage and in vivo resuscitation using the Langendorff apparatus. Arterial blood samples were collected from all groups at the end of the experimental period (90 min) for cytokine measurements (TNF-α). Amiloride decreased the inflammatory response to hemorrhagic shock and resuscitation by lowering the levels of TNF. These results indicate that amiloride protects the myocardium by down regulating the inflammatory response to hemorrhagic shock and resuscitation.
Keywords: Amiloride; Hemorrhagic shock; TNF; Inflammation;

Tourniquet-induced acute ischemia–reperfusion injury in mouse skeletal muscles: Involvement of superoxide by Thai P. Tran; Huiyin Tu; Iraklis I. Pipinos; Robert L. Muelleman; Hassan Albadawi; Yu-Long Li (328-334).
Although arterial limb tourniquet is one of the first-line treatments to prevent exsanguinating hemorrhage in both civilian pre-hospital and battlefield casualty care, prolonged application of a limb tourniquet can lead to serious ischemia–reperfusion injury. However, the underlying pathomechanisms of tourniquet-induced ischemia–reperfusion injury are still poorly understood. Using a murine model of acute limb ischemia–reperfusion, we investigated if acute limb ischemia–reperfusion injury is mediated by superoxide overproduction and mitochondrial dysfunction. Hind limbs of C57/BL6 mice were subjected to 3 h ischemia and 4 h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Approximately 40% of the gastrocnemius muscle suffered infarction in this model. Activities of mitochondrial electron transport chain complexes including complex I, II, III, and IV in the gastrocnemius muscle were decreased in the ischemia–reperfusion group compared to sham. Superoxide production was increased while activity of manganese superoxide dismutase (MnSOD, the mitochondria-targeted SOD isoform) was decreased in the ischemia–reperfusion group compared to the sham group. Pretreatment with tempol (a SOD mimetic, 50 mg/kg) or co-enzyme Q10 (50 mg/kg) not only decreased the superoxide production, but also reduced the infarct size and normalized mitochondrial dysfunction in the gastrocnemius muscle. Our results suggest that tourniquet-induced skeletal muscle ischemia–reperfusion injuries including infarct size and mitochondrial dysfunction may be mediated via superoxide overproduction and reduced antioxidant activity. In the future, this murine ischemia–reperfusion model can be adapted to mechanistically evaluate anti-ischemic molecules in tourniquet-induced skeletal muscle injury.
Keywords: Infarct size; Ischemia–reperfusion injury; Mitochondria; Superoxide; Tourniquet;

Cisplatin-induced cardiotoxicity: Mechanisms and cardioprotective strategies by El-Sayed E. El-Awady; Yasser M. Moustafa; Dina M. Abo-Elmatty; Asmaa Radwan (335-341).
Increased oxidative stress and apoptosis have been implicated in the cardiotoxicity that limits the clinical use of cisplatin as an anti-tumoral drug. Our study was conducted to evaluate the protective potential of acetyl-l-carnitine, DL-α-lipoic acid and silymarin against cisplatin-induced myocardial injury. Eighty male albino rats were divided into eight groups. The first four groups were treated with normal saline, acetyl-l-carnitine (500 mg/kg, i.p.), DL-α-lipoic acid (100 mg/kg, p.o.) and silymarin (100 mg/kg, p.o.) respectively, for 10 successive days. The remaining groups were treated with the same doses of normal saline, acetyl-l-carnitine, DL-α-lipoic acid and silymarin, respectively, for 5 successive days before and after a single dose of cisplatin (10 mg/kg, i.p.). Serum activities of lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase isoenzyme MB (CK-MB) and plasma cardiac troponin I (cTnI) concentration were estimated. Malondialdehyde (MDA), reduced glutathione (GSH) contents, superoxide dismutase activity (SOD) and protein content in cardiac tissues were measured. Moreover, integrity of both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) was also examined. Cisplatin-treated rats experienced a significant elevation of serum activities of LDH, CK, CK-MB and cTnI plasma concentration. These effects were accompanied by a significant increase in MDA level. On the other hand, a significant decrease in GSH content, SOD activity and total protein content was observed. In addition, both mtDNA and nDNA were heavily damaged. However, acetyl-l-carnitine, DL-α-lipoic acid and silymarin significantly attenuated the cisplatin-evoked disturbances in the above-mentioned parameters. In conclusion, the former drugs were proven to be potential candidates to ameliorate cisplatin-induced cardiotoxicity.
Keywords: Cisplatin-induced cardiotoxicity; Acetyl-l-carnitine; DL-α-lipoic acid; Silymarin; Mitochondrial DNA; Apoptosis;

Inhibitory effect of fenofibrate on neointima hyperplasia via G0/G1 arrest of cell proliferation by Jung-Jin Lee; Ji-Yeon Yu; Wei-Yun Zhang; Tack-Joong Kim; Yong Lim; Jin-Sook Kwon; Dong-Woon Kim; Chang-Seon Myung; Yeo-Pyo Yun (342-349).
We have previously reported that fenofibrate displayed a potent antithrombotic effect by the inhibition of platelet aggregation. The present study was designed to investigate the effects of fenofibrate on the neointimal hyperplasia and its possible molecular mechanism. Neointimal hyperplasia was measured in balloon-inflated–induced vascular injury model of male Sprague–Dawley rats and cell proliferation was measured in primary cultured rat aortic vascular smooth muscle cells (VSMCs). Fenofibrate-treated group showed a significant reduction in neointimal formation (0.07 ± 0.04 mm2) from the control (0.13 ± 0.04 mm2). Fenofibrate significantly inhibited platelet-derived growth factor (PDGF)-BB-induced cell counting and [3H]-thymidine incorporation into DNA. Fenofibrate suppressed the PDGF-BB-inducible progression through G0/G1 to S phase of cell cycle. Moreover, fenofibrate inhibited not only phosphorylation of retinoblastoma (Rb) protein and expression of cyclin D/E, CDK 2/4 and proliferating cell nuclear antigen (PCNA) proteins but also mitogen-activated protein kinase (MAPK) signaling pathways such as ERK 1/2, p38 and JNK phosphorylation. In conclusion, the present study demonstrates that fenofibrate significantly inhibits neointimal formation via G0/G1 arrest of PDGF-BB-induced cell proliferation in association with the inhibition of MAPK, which resulted in the downregulation of expressions of cyclin D/E, CDK 2/4 and PCNA proteins, suggesting that fenofibrate may be useful for individuals with a high risk of thrombotic or cardiovascular diseases.
Keywords: Neointima hyperplasia; Fenofibrate; G0/G1 arrest; Cardiovascular disease;

Can guanine-based purines be considered modulators of intestinal motility in rodents? by Maria Grazia Zizzo; Flavia Mulè; Mariangela Mastropaolo; Daniele F. Condorelli; Natale Belluardo; Rosa Serio (350-355).
Adenine-based purines play a pivotal role in the control of gastrointestinal motility in rodents. Recently, guanine-based purines have been also shown to exert extracellular effects in the central nervous system raising the possibility of the existence of distinct receptors for guanine-based purines. Thus, it seems likely to speculate that also guanine-based purines may play a role in the modulation of the intestinal contractility. Spontaneous and neurally-evoked mechanical activity was recorded in vitro as changes in isometric tension in circular muscle strips from mouse distal colon. Guanosine up to 3 mM or guanine up to 1 mM failed to affect the spontaneous mechanical activity, but reduced the amplitude of the electrical field stimulation (EFS)-induced cholinergic contractions, without affecting the early nitrergic relaxation. Both compounds failed to affect the direct contractile responses evoked by carbachol. No desensitization of the response was observed. Guanine-based purine effects were not altered by theophylline, P1 purinoceptor antagonist, by PPADS or suramin, P2 purinoceptor antagonists, by ODQ, guanilyl cyclase inhibitor, or by DDA, adenylyl cyclase inhibitor. Nucleoside uptake inhibitors, dipyridamole or 6-[(4-Nitrobenzyl)thio]-9-β-D-ribofuranosylpurine (NBTI), antagonized the inhibitory effects induced by guanosine without interfering with guanine. On the contrary, adenine, a competitive inhibitor of nucleobase uptake, antagonized guanine-induced effects. In conclusion, our data indicate that guanosine and guanine are able to modulate negatively the excitatory cholinergic neurotransmission in the circular muscle layer of mouse colon. Guanine-based purines appear to interfere with prejunctional acethylcoline release. Their effects are dependent by their cellular uptake, and independent by adenine-based purine receptors.
Keywords: Colon; Circular muscle; Guanosine; Guanine; Cholinergic contraction; (Mouse);

Botulinum neurotoxin-type A (BoNTA) is an emerging therapeutic option for the treatment of benign prostatic hyperplasia. Recent reports indicate increased incidence of benign prostatic hyperplasia in the insulin-resistant individuals. Insulin-resistance is associated with the compensatory rise in the plasma insulin, which is known to have growth-promoting effects. The present study investigated the effect of insulin-resistance on the effectiveness of BoNTA in inducing prostatic atrophy in rats. Sprague-Dawley rats (200–220 g), maintained on normal-pellet or high-fat diet, were injected in the ventral prostate with 200 μl of saline or the same volume containing 5U BoNTA at the end of 9 weeks and were sacrificed 3 weeks later. Ventral prostate was carefully isolated, weighed, fixed and stained to examine the cellular morphology, cell death and proliferation. High-fat diet produced insulin-resistance, hyperinsulinemia and prostatic enlargement in rats. BoNTA caused prostatic atrophy and apoptosis in both insulin-resistant and insulin-sensitive rats. However, the effect of BoNTA was more prominent in insulin-sensitive rats (apoptosis-2 fold, prostatic atrophy-3 fold) as compared to the insulin-resistant rats. Significant increase in the phosphorylation of ERK-1/2 and expression of the proliferating cell nuclear antigen was observed in the prostate of insulin-resistant rats. In the present investigation we report that diet-induced insulin-resistance activates mitogenic signaling of insulin, increases cellular proliferation and reduces BoNTA-induced prostatic atrophy and apoptosis in rats. Results of the present study indicate that the insulin-resistance can affect the therapeutic outcome of BoNTA.
Keywords: Insulin-resistance; Botulinum neurotoxin-type A; Prostate; Apoptosis; Cell-proliferation;

Hepatoprotective role of naringin on nickel-induced toxicity in male Wistar rats by Leelavinothan Pari; Kasinathan Amudha (364-370).
Aim of the present study was planned to determine the protective role of naringin in attenuating the toxicity induced by nickel sulfate in rat liver. In this investigation nickel sulfate (20 mg/kg body weight) was administered intraperitoneally for 20 days to induce toxicity. Naringin was administered orally (20, 40 and 80 mg/kg body weight) for 20 days with intraperitoneal administration of nickel sulfate. Liver injury was measured by the increased activities of serum hepatic enzymes namely aspartate transaminase, alanine transaminase, alkaline phosphatase, gamma glutamyl transferase, lactate dehydrogenase and total bilirubin along with increased elevation of lipid peroxidation markers, thiobarbituric reactive acid substances, lipid hydroperoxides, protein carbonyl content and conjugated dienes. The toxic effect of nickel was also indicated by significantly decreased activities of enzymatic antioxidants like superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase and glucose-6-phosphate dehydrogenase and non-enzymatic antioxidants like reduced glutathione, total sulfhydryl groups, vitamin C and vitamin E levels were significantly decreased. Naringin administered at a dose of 80 mg/kg body weight significantly reversed the activities of hepatic marker enzymes, decreasing lipid peroxidative markers, increasing the antioxidant cascade and decreasing the nickel concentration in the liver. The effect at a dose of 80 mg/kg body weight was more pronounced than that of other two doses (20 and 40 mg/kg body weight). All these changes were supported by histopathological observations. These results clearly demonstrate that naringin has the potential in alleviating the toxic effects of nickel in rat liver.
Keywords: Naringin; Nickel; Hepatotoxicity; Oxidative stress; Antioxidant; Lipid peroxidation;

Tetrahydrobiopterin analogues with NO-dependent pulmonary vasodilator properties by Suma P. Kunuthur; Philip H. Milliken; Colin L. Gibson; Colin J. Suckling; Roger M. Wadsworth (371-377).
Reduced NO levels due to the deficiency of tetrahydrobiopterin (BH4) contribute to impaired vasodilation in pulmonary hypertension. Due to the chemically unstable nature of BH4, it was hypothesised that oxidatively stable analogues of BH4 would be able to support NO synthesis to improve endothelial dysfunction in pulmonary hypertension. Two analogues of BH4, namely 6-hydroxymethyl pterin (HMP) and 6-acetyl-7,7-dimethyl-7,8-dihydropterin (ADDP), were evaluated for vasodilator activity on precontracted rat pulmonary artery rings. ADDP was administered to pulmonary hypertensive rats, followed by measurement of pulmonary vascular resistance in perfused lungs and eNOS expression by immunohistochemistry. ADDP and HMP caused significant relaxation in vitro in rat pulmonary arteries depleted of BH4 with a maximum relaxation at 0.3 μM (both P < 0.05). Vasodilator activity of ADDP and HMP was completely abolished following preincubation with the NO synthase inhibitor, L-NAME. ADDP and HMP did not alter relaxation induced by carbachol or spermine NONOate. BH4 itself did not produce relaxation. In rats receiving ADDP 14.1 mg/kg/day, pulmonary vasodilation induced by calcium ionophore A23187 was augmented and eNOS immunoreactivity was increased. In conclusion, ADDP and HMP are two analogues of BH4, which can act as oxidatively stable alternatives to BH4 in causing NO-mediated vasorelaxation. Chronic treatment with ADDP resulted in improvement of NO-mediated pulmonary artery dilation and enhanced expression of eNOS in the pulmonary vascular endothelium. Chemically stable analogues of BH4 may be able to limit endothelial dysfunction in the pulmonary vasculature.
Keywords: Tetrahydrobiopterin; Pulmonary artery; Nitric oxide; Pulmonary hypertension; Nitric oxide synthase;

Diazoxide attenuates indomethacin-induced small intestinal damage in the rat by Alessandro Menozzi; Cristina Pozzoli; Enzo Poli; Benedetta Passeri; Paola Gianelli; Simone Bertini (378-383).
ATP-sensitive potassium (KATP) channel openers have been shown to protect against cellular damage in neurons, cardiac muscle, and kidney and to effectively reduce nonsteroidal anti-inflammatory drug (NSAID)-induced gastric damage in rats. We investigated the effects of KATP channel opener diazoxide on small intestinal injury induced in rats by indomethacin administration. The effect of glibenclamide, a KATP channel blocker, was also evaluated. Diazoxide (15, 45 and 135 mg/kg) or glibenclamide (18 mg/kg), were given by oral gavage 1 h before and 6 h after indomethacin treatment (20 mg/kg p.o.). After 24 h, macroscopic and histologic lesions, myeloperoxidase (MPO) activity and lipid peroxidation levels were evaluated. Diazoxide at 15 mg/kg was ineffective, while at doses of 45 mg/kg and 135 mg/kg was able to significantly improve all damage parameters. Glibenclamide administration enhanced intestinal injury. These results show for the first time a beneficial effect of diazoxide in indomethacin-induced enteritis in the rat. Several mechanisms, such as oxidative phosphorylation uncoupling and hypermotility seem particularly important in NSAID-induced intestinal injury. Such events lead to increased mucosal permeability and to penetration of noxious lumen components, which ignite the inflammatory response. Since KATP channel openers were shown to protect against mitochondrial damage, to reduce intercellular permeability and to relax smooth muscle, we suggest that diazoxide could exert its beneficial effects by one or more of these actions.
Keywords: Diazoxide; Indomethacin; Small intestine; Rat; KATP channel;

Although nonalcoholic steatohepatitis (NASH) is associated with insulin resistance partly due to reduced levels of circulating adiponectin, the role of adiponectin receptors including adiponectin receptor 1 and adiponectin receptor 2 in adipose tissues in NASH remains controversial. The present study showed that there was a marked decline in adiponectin receptor 1 and adiponectin receptor 2 expressions in liver and visceral fat, and these expressions were elevated in muscle of NASH rats 12 weeks after oral administration of a high-fat diet. An 8-week continuous treatment with rosiglitazone, a peroxisome proliferator-activated receptors γ (PPAR γ) agonist improved the histological lesions markedly in liver of NASH rats, and concurrently increased mRNA and protein expressions of adiponectin receptor 1 and adiponectin receptor 2 in liver and visceral fat, with down-regulation of the two receptors in muscle. There was a negative correlation between the ratio of adiponectin receptors/β-actin protein and serum TNF-α in the liver and visceral fat, and a positive correlation in muscle. Additionally, rosiglitazone increased circulating adiponectin, which was negatively correlated with serum TNF-α. These results indicated that rosiglitazone improved NASH by directly modulating adiponectin receptor 1 and adiponectin receptor 2 in various adipose tissues, or indirectly possibly via decreasing serum TNF-α.
Keywords: Nonalcoholic steatohepatitis; Rosiglitazone; Adiponectin; Receptor; Rats;

Therapeutic effects of SMND-309, a new metabolite of salvianolic acid B, on experimental liver fibrosis by Jian Hou; Jingwei Tian; Wanglin Jiang; Yubai Gao; Fenghua Fu (390-395).
(2E)-2-{6-[(E)-2-carboxylvinyl]-2,3-dihydroxyphenyl}-3-(3,4-dihydroxyphenyl) propenoic acid, a novel compound designated SMND-309, is a new metabolite of salvianolic acid B. The present study was carried out to investigate the effects of SMND-309 on experimental liver fibrosis in rats induced by subcutaneous injection of carbon tetrachloride and explore its possible mechanisms on the basis of biochemical, histopathologic and immunohistochemical studies. The results showed that intragastrical treatment with SMND-309 ameliorated liver function and decreased the elevation of serum hyaluronic acid, laminin, procollagen type III levels and hydroxyproline content in liver tissue. It also decreased the elevation in the malondialdehyde level and restored the decrease in superoxide dismutase and glutathione peroxidase activities. Upon histopathologic examination, the SMND-309-treated rats reduced the liver damage and the liver fibrosis grade. Moreover, the results of immunohistochemical examination showed that SMND-309 powerfully down-regulated the expression of connective tissue growth factor (CTGF) rather than transforming growth factor-beta1 (TGF-β1) in serum and liver. Meanwhile, SMND-309 exhibits significantly higher potency compared with salvianolic acid B (Sal B) at the same dose. The antifibrotic mechanisms of SMND-309 might be associated with its ability to suppress the expression of CTGF as well as scavenge lipid peroxidation products and increase endogenous antioxidant enzyme activity.
Keywords: (2E)-2-{6-[(E)-2-carboxylvinyl]-2,3-dihydroxyphenyl}-3-(3,4-dihydroxyphenyl) propenoic acid (SMND-309); Liver fibrosis; Carbon tetrachloride; Lipid peroxidation; Transforming growth factor-beta1 (TGF-β1); Connective tissue growth factor (CTGF);

Prostaglandin E2 receptor EP4-selective agonist (ONO-4819) increases bone formation by modulating mesenchymal cell differentiation by Tadashi Ninomiya; Akihiro Hosoya; Toru Hiraga; Masanori Koide; Kojiro Yamaguchi; Hiroji Oida; Yoshinori Arai; Noriyuki Sahara; Hiroaki Nakamura; Hidehiro Ozawa (396-402).
Prostaglandin E2 (PGE2) positively regulates bone resorption and formation mainly mediated through the EP4 receptor, a subtype of PGE2 receptors. ONO-4819, an EP4 receptor-selective agonist, has been shown to increase bone volume, density, and strength; however, the mechanism of these effects has yet to be fully elucidated. To explore this matter, ONO-4819 (10 μg/kg) was injected into intact rats twice a day for 5 weeks, and their bones were then analyzed by morphological techniques. The effects of ONO-4819 on the differentiation of bone cells were also examined in vitro. Bone morphometric analysis showed that osteoblast number, bone volume, mineral apposition rate, and bone formation rate were significantly increased by ONO-4819, whereas osteoclast number was not affected. Immunohistochemical examination demonstrated that ONO-4819 increased the number of Runx2- and Osterix-positive osteoblasts in rats. In vitro studies using the multipotent mesenchymal cell line C3H10T1/2 showed that ONO-4819 induced alkaline phosphatase (ALPase) activity and up-regulated the mRNA expression of ALPase and Osterix. In contrast, ONO-4819 reduced the mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) and inhibited adipocyte differentiation of C3H10T1/2 cells, which findings are consistent with the observation that the age-dependent increase in adipocyte number in the bone marrow was significantly suppressed in the ONO-4819-treated animals. ONO-4819 also dose-dependently increased osteoclast-like cell formation in vitro, but the required concentrations were much higher than those to induce osteoblast differentiation. These results collectively suggest that ONO-4819 increased bone formation by stimulating osteoblast differentiation and function, possibly through modulating mesenchymal cell differentiation in the bone.
Keywords: EP4 receptor; ONO-4819; Mesenchymal cells; Osteoblast differentiation; Adipocyte differentiation;

Serotonin (5-HT) enhances the neurogenic contractile response induced by electrical field stimulation (EFS) in the rat isolated urinary bladder. The aim of this study was to functionally characterize the receptors involved in this effect by using a range of 5-HT receptor subtype selective agonists and antagonists. 5-HT produced a concentration-dependent potentiation of contractile responses to EFS with a pEC50 value of 6.86 ± 0.24. SB-269970 (0.01, 0.1 and 1 μM), a selective 5-HT7 receptor antagonist, caused a concentration-dependent rightward shift of the 5-HT-induced response. The pA2 value was 8.16 with a slope of 0.46 ± 0.08. Neither ketanserine nor SB-204741, 5-HT2A and 5-HT2B receptors antagonists, respectively, affected the concentration–response curve to 5-HT. However, 5-HT response was antagonized by the selective 5-HT2C receptor antagonist SB-242084 (0.1 and 1 μM). In the presence of 1 μM of both antagonists SB-269970 and SB-242084, 5-HT response was almost fully inhibited. 5-CT, a 5-HT7 receptor agonist, induced a biphasic concentration-dependent potentiation of neurogenic contractions. SB-269970 concentration-dependently antagonized the first phase of 5-CT response with a pA2 value of 8.77 and a slope not significantly different from unity (0.91 ± 0.11) that suggests a competitive antagonism. WAY-161503, a 5-HT2C receptor agonist (0.01–10 μM), induced a concentration-dependent potentiation of contractile response to EFS while DOI (a selective 5-HT2A agonist) had no effect. SB-242084 (0.1 and 1 μM) antagonized the effect of WAY-161503 in a concentration-dependent manner. The current results demonstrate that 5-HT potentiates neurogenic contractions of rat isolated detrusor muscle through both 5-HT7 and 5-HT2c receptors.
Keywords: Serotonin; 5-HT7 receptor; 5-HT2 receptor; Urinary bladder; Electrical field stimulation; Rat;

Activation of α7 nicotinic acetylcholine receptors ameliorates indomethacin-induced small intestinal ulceration in mice by Ryoji Kawahara; Masashi Yasuda; Hiroshi Hashimura; Kikuko Amagase; Shinichi Kato; Koji Takeuchi (411-417).
Cholinergic anti-inflammatory actions have been shown to result mainly from the activation of α7 nicotinic acetylcholine receptors. Here, we investigated the possible role of α7 nicotinic acetylcholine receptors in the pathogenesis of indomethacin-induced small intestinal ulceration in mice. Male C57BL/6 mice were given indomethacin (10 mg/kg, s.c.), and sacrificed 24 h later. Nicotine (0.3–3 mg/kg) and PNU-282987 (a selective agonist of α7 nicotinic acetylcholine receptors; 1–10 mg/kg) were administered i.p. twice, at 0.5 h before and 8 h after indomethacin treatment, while methyllycaconitine (a selective antagonist of α7 nicotinic acetylcholine receptors; 10 mg/kg was administered twice, at 0.5 h before each nicotine treatment. Indomethacin caused severe hemorrhagic lesions in the small intestine with marked increases in myeloperoxidase (MPO) activity and inducible nitric oxide synthase (iNOS) expression in the mucosa. Pretreatment with nicotine reduced the severity of intestinal lesions in a dose-dependent manner. The protective effect of nicotine was mimicked by PNU-282987 and significantly attenuated by methyllycaconitine. The increases in MPO activity and iNOS expression induced by indomethacin were also significantly suppressed by nicotine and PNU-282987. Immunohistochemical study showed that the expression of α7 nicotinic acetylcholine receptors was clearly enhanced in the submucosa of the damaged area following indomethacin treatment. These results suggest that the activation of α7 nicotinic acetylcholine receptors ameliorates indomethacin-induced small intestinal ulceration, and that this effect may result from the inhibition of iNOS expression and neutrophil migration.
Keywords: α7 Nicotinic acetylcholine receptor; Nicotine; Indomethacin; Small intestinal ulceration; Neutrophil; Inducible nitric oxide synthase (iNOS);

Doxycycline attenuates acrolein-induced mucin production, in part by inhibiting MMP-9 by Shuang Ren; Ling-Li Guo; Jie Yang; Dai-Shun Liu; Tao Wang; Lei Chen; Ya-Juan Chen; Dan Xu; Yu-Lin Feng; Fu-Qiang Wen (418-423).
Matrix metalloproteinases (MMPs), especially MMP-9, have been found to increase the expression of epidermal growth factor (EGF) receptor, a possible regulator of acrolein-induced mucin expression in the airway epithelium. The aim of this study was to investigate whether doxycycline, a tetracycline antibiotic that inhibits MMPs, attenuates mucus production and synthesis of mucin MUC5AC in acrolein-exposed rats. Sprague–Dawley rats were exposed to acrolein aerosol [3.0 parts/million (ppm), 6 h/day, 12 days] and they received 20 mg/kg doxycycline daily by gavage, beginning two days before exposure to acrolein until the end of the experiment. The production of mucin glycoproteins and expression of the MMP-9 and MUC5AC genes were measured in rat trachea. The increase in levels of MMP-9 mRNA and protein in airway epithelium after acrolein exposure was accompanied by an increase in MUC5AC mRNA expression. Doxycycline significantly prevented these increases in acrolein-induced expression of MMP-9 and MUC5AC and attenuated mucus production in tracheal epithelium. These results indicate that doxycycline attenuated acrolein-induced mucin synthesis, in part by inhibiting expression of MMP-9. Thus doxycycline may have a prophylactic effect in the treatment of smoking-induced mucus hypersecretion.
Keywords: Acrolein; Airway mucus production; Doxycycline; MMP-9; MUC5AC;

Cysteinyl-leukotrienes are potent mediators involved in various inflammatory diseases and lung disorders such as asthma. However, their precise role in the pathogenesis of pulmonary fibrosis is unknown. In the present study, we investigated the effect of montelukast, a cysteinyl-leukotriene type 1 receptor antagonist, on bleomycin-induced pulmonary fibrosis in mice. Montelukast (10 mg/kg/day) was orally administered to the bleomycin-induced pulmonary fibrosis mice for 3 days before and 14 days after intratracheal instillation of bleomycin. We evaluated the effects of montelukast on the development of pulmonary fibrosis in these mice and investigated the expression of various cytokines and two cysteinyl-leukotriene receptors. Treatment with montelukast significantly attenuated the increased fibrotic area and hydroxyproline content in the fibrotic lungs of bleomycin-instilled mice. Montelukast treatment also decreased mRNA levels of IL-6, IL-10, IL-13, and TGF-β1, all of which were elevated in fibrotic lungs. In fibrotic lungs, TNF-α and IL-1β mRNA levels were increased and IFN-γ mRNA levels were decreased, but montelukast did not affect these mRNA levels. Furthermore, cysteinyl-leukotriene type 1 receptor mRNA levels were increased, whereas cysteinyl-leukotriene type 2 receptor mRNA levels were decreased in fibrotic lungs. Montelukast treatment induced the recovery of cysteinyl-leukotriene type 2 receptor mRNA levels to normal control levels but did not change cysteinyl-leukotriene type 1 receptor mRNA levels. These results suggest that montelukast exhibits its beneficial effects by inhibiting the overexpression of IL-6, IL-10, IL-13, and TGF-β1 and by modulating the homeostatic balance between the cysteinyl-leukotriene type 1 and type 2 receptors.
Keywords: Cysteinyl-leukotriene type 1 receptor; IL-6; IL-10; IL-13; TGF-β1;

Procaterol inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells by Mutsuo Yamaya; Hidekazu Nishimura; Yukimasa Hatachi; Motoki Yoshida; Hidenori Fujiwara; Masanori Asada; Katsutoshi Nakayama; Hiroyasu Yasuda; Xue Deng; Takahiko Sasaki; Hiroshi Kubo; Ryoichi Nagatomi (431-444).
β2 agonists reduce the frequency of exacerbations in patients with bronchial asthma and chronic obstructive pulmonary disease caused by respiratory virus infection. β2 agonists reduce the production of pro-inflammatory cytokines. However, the inhibitory effects of β2 agonists on the infection of rhinovirus, the major cause of exacerbations, have not been well studied. To examine the effects of a β2 agonist, procaterol, on rhinovirus infection and rhinovirus infection-induced airway inflammation, human tracheal epithelial cells were infected with a major group rhinovirus, type 14 rhinovirus. Rhinovirus infection increased viral titers and the content of pro-inflammatory cytokines, including interleukin-1β and interlukin-6, in supernatant fluids and rhinovirus RNA in the cells. Procaterol reduced rhinovirus titers and RNA, cytokine concentrations, and susceptibility to rhinovirus infection. Procaterol reduced the expression of intercellular adhesion molecule-1 (ICAM-1), the receptor for type 14 rhinovirus, and the number of acidic endosomes in the cells from which rhinovirus RNA enters into the cytoplasm. Procaterol inhibited the activation of nuclear factor kappa-B (NF-κB) proteins including p50 and p65 in the nuclear extracts, while it increased the cytosolic amount of the inhibitory kappa B-α and intracellular cyclic AMP (cAMP) levels. A selective β2-adrenergic receptor antagonist ICI 118551 [erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol] reversed the inhibitory effects of procaterol on rhinovirus titers and RNA, susceptibility to rhinovirus infection, pro-inflammatory cytokines production, ICAM-1 expression, acidic endosomes, and NF-κB. ICI 118551 also reversed the effects of procaterol on cAMP levels. Procaterol may inhibit rhinovirus infection by reducing ICAM-1 and acidic endosomes as well as modulate airway inflammation in rhinovirus infection.
Keywords: Procaterol; Human tracheal epithelial cell; Rhinovirus; Intercellular adhesion molecule; Acidic endosome; Pro-inflammatory cytokine;

Dipyridamole contributes to its beneficial effects on inflammatory responses in many cell types. The anti-inflammatory mechanisms of dipyridamole on glomerular mesangial cells are mostly uncharacterized. In this study, we monitored the influence of dipyridamole on the expression levels of cyclooxygenase-2 (COX-2) and monocyte chemoattractant protein-1 (MCP-1) in rat mesangial cells stimulated with lipopolysaccharide. Dipyridamole was found to inhibit lipopolysaccharide-induced COX-2 and MCP-1 expression, and reduced lipopolysaccharide-induced reactive oxygen species generation in rat mesangial cells. This inhibitory effect of dipyridamole is independent on cyclic AMP and cyclic GMP increase. Tin protoporphyrin IX (SnPP), a heme oxygenase-1(HO-1) inhibitor, blocked the inhibitory effect of dipyridamole on lipopolysaccharide-induced COX-2 and MCP-1 expression. By applying specific inhibitors in rat mesangial cells, ERK1/2 and p38 MAPK signaling pathways were demonstrated to be involved in the lipopolysaccharide-induced inflammatory responses, and were inhibited by SnPP and N-acetylcysteine treatment. Additionally, dipyridamole was also found to upregulate HO-1 in rat mesangial cells. Therefore, our data suggest that dipyridamole inhibits the expression of COX-2 and MCP-1 in lipopolysaccharide-treated rat mesangial cells via HO-1-mediated reactive oxygen species reduction.
Keywords: Dipyridamole; Lipopolysaccharide; Cyclooxygenase-2 (COX-2); Monocyte chemoattractant protein-1 (MCP-1); Heme oxygenase-1 (HO-1);

Downregulation of B lymphocyte stimulator expression by curcumin in B lymphocyte via suppressing nuclear translocation of NF-κB by Gang Huang; Yanchun Yang; Zhizhen Xu; Peng Zhou; Wei Gong; Yuan Li; Jishan Fan; Fengtian He (451-457).
Overexpression of B lymphocyte stimulator (BLyS) is closely involved in the pathogenesis and progression of some autoimmune diseases. Curcumin, a pharmacologically safe agent, has been shown to possess potent anti-inflammatory properties. However, it is not clear whether curcumin affects the expression of BLyS. In this study, we report that curcumin inhibits the expression of BLyS and that a DNA-binding site for the transcriptional factor NF-κB in the BLyS promoter region is required for this regulation. Moreover, we find that curcumin reduces the DNA-binding activity of NF-κB to the BLyS promoter region and suppresses nuclear translocation of p65, suggesting that curcumin may suppress BLyS expression via negatively interfering with NF-κB signaling. These results suggest that curcumin may serve as a novel therapeutic agent in the treatment of autoimmune diseases by targeting BLyS.
Keywords: B lymphocyte stimulator; Curcumin; NF-κB; Autoimmune disease;

In the present study, the efficacy of β-sitosterol isolated from an n-butanol extract of the seeds of the plant Moringa oleifera (Moringaceae) was examined against ovalbumin-induced airway inflammation in guinea pigs. All animals (except group I) were sensitized subcutaneously and challenged with aerosolized 0.5% ovalbumin. The test drugs, β-sitosterol (2.5 mg/kg) or dexamethasone (2.5 mg/kg), were administered to the animals (p.o.) prior to challenge with ovalbumin. During the experimental period (on days 18, 21, 24 and 29), a bronchoconstriction test (0.25% acetylcholine for 30 s) was performed and lung function parameters (tidal volume and respiration rate) were measured for each animal. On day 30, blood and bronchoalveolar lavaged fluid were collected to assess cellular content, and serum was collected for cytokine assays. Lung tissue was utilized for a histamine assay and for histopathology. β-sitosterol significantly increased the tidal volume (Vt) and decreased the respiration rate (f) of sensitized and challenged guinea pigs to the level of non-sensitized control guinea pigs and lowered both the total and differential cell counts, particularly eosinophils and neutrophils, in blood and bronchoalveolar lavaged fluid. Furthermore, β-sitosterol treatment suppressed the increase in cytokine levels (TNFα, IL-4 and IL-5), with the exception of IL-6, in serum and in bronchoalveolar lavaged fluid detected in model control animals. Moreover, treatment with β-sitosterol protected against airway inflammation in lung tissue histopathology. β-sitosterol possesses anti-asthmatic actions that might be mediated by inhibiting the cellular responses and subsequent release/synthesis of Th2 cytokines. This compound may have therapeutic potential in allergic asthma.
Keywords: Asthma; β-sitosterol; Cytokines; Moringa oleifera; Ovalbumin;

Antihyperglycaemic, antilipid peroxidative and antioxidant effects of gallic acid on streptozotocin induced diabetic Wistar rats by Vilapakkam Ranganathan Punithavathi; Ponnian Stanely Mainzen Prince; Ramesh Kumar; Jemmi Selvakumari (465-471).
The present study aims to evaluate the antihyperglycaemic, antilipid peroxidative and antioxidant effects of gallic acid on streptozotocin induced diabetic male Wistar rats. To induce diabetes mellitus, rats were injected with streptozotocin intraperitoneally at a single dose of 40 mg/kg. Streptozotocin induced diabetic rats showed significant (P  < 0.05) increase in the levels of blood glucose, glycosylated haemoglobin and significant (P  < 0.05) decrease in the levels of plasma insulin, body weight and total haemoglobin. Diabetic rats also showed significant (P  < 0.05) decrease in the activity of hepatic hexokinase and significant (P  < 0.05) increase in the activities of glucose-6-phosphatase and fructose-1, 6-bisphosphatase. The pancreatic thiobarbituric acid reactive substances and lipid hydroperoxides were significantly (P  < 0.05) increased and the activities of pancreatic superoxide dismutase, catalase and glutathione peroxidase were significantly (P  < 0.05) decreased in diabetic rats. Oral treatment with gallic acid (10 and 20 mg/kg) daily for a period of 21 days showed significant (P  < 0.05) protective effects on all the biochemical parameters studied. Histopathology of pancreas confirmed the protective effects of gallic acid in diabetic rats. In vitro study also revealed the potent antioxidant effect of gallic acid. Thus, the study shows the antihyperglycaemic, antilipid peroxidative and antioxidant effects of gallic acid on streptozotocin induced diabetic rats. The effect exerted by 20mg/kg body weight of gallic acid was more effective than 10 mg/kg body weight of gallic acid.
Keywords: Antioxidant; Blood glucose; Diabetes mellitus; Gallic acid; Pancreas; Streptozotocin;

Effect of a novel biphenyl compound, VMNS2e on ob/ob mice by Sucheta B. Kurundkar; Narsingh Sachan; Kisan M. Kodam; Vithal M. Kulkarni; Subhash L. Bodhankar; Vikram S. Ghole (472-478).
VMNS2e is a novel biphenyl compound, which in previous studies had showed most favourable interactions with the active site of protein tyrosine phosphatase 1B (PTP1B). The effect of acute and chronic treatment of VMNS2e (30 mg/kg) was investigated in ob/ob mice. Plasma glucose was measured after acute administration of VMNS2e (30 mg/kg) in both lean and ob/ob mice. In the chronic study, VMNS2e (30 mg/kg) was given orally, once daily for 60 days. Metformin (300 mg/kg) was taken as standard therapy. Body weight, food intake and blood glucose was measured weekly while glycosylated hemoglobin A1c (HbA1c), insulin, triglyceride, total cholesterol, low density lipoprotein (LDL), fructosamine, non esterified fatty acid and organ weight were estimated after the completion of treatment period. Oral glucose tolerance test was performed on the last day of treatment. Liver and epididymal fat weights were taken. Acute dose of VMNS2e elicited an anti hyperglycemic effect. It reduced blood glucose by 14% (0.5 h) and 35.6% (6 h). Chronic VMNS2e treatment improved glucose tolerance by 25.3%. It decreased blood glucose levels. Hyperinsulinemia was reduced (19.6%). VMNS2e treatment had no significant effect on body weight and food consumption. VMNS2e treatment exhibited significant reduction (28.2%) in HbA1c, plasma triglyceride (49%), LDL (24%) and fructosamine (13%) levels. VMNS2e treatment did not alter total cholesterol and non esterified fatty acid levels. Epididymal fat/body weight ratio was reduced (26.3%). VMNS2e exhibited both acute and chronic anti hyperglycemic effect, insulin sensitivity along with improvement in various lipid parameters and glycemic control.
Keywords: PTP1B inhibitor; VMNS2e; Anti hyperglycaemic; Insulin sensitizer; Lipid metabolism; (ob/ob mouse);

Recently, it has been demonstrated that fibroin and fibroin-derived peptides enhances insulin sensitivity and glucose metabolism in adipocytes. Here, we show that a synthetic hexapeptide Gly-Ala-Gly-Val-Gly-Tyr (GAGVGY) derived from repetitive amino acid sequence of fibroin improves glucose transport and exerts beneficial lipid metabolic effects in 3T3-L1 adipocytes. GAGVGY increases both basal and insulin-stimulated glucose uptake through enhancement of GLUT1 expression and PI 3-K-dependent GLUT4 translocation, respectively. GAGVGY treatment also led to a significant reduction in the expression of lipogenic genes including sterol regulatory element binding protein-1c (SREBP1c), peroxisome proliferator-activated receptor-γ (PPARγ), and fatty acid synthase (FAS) in mature 3T3-L1 adipocytes, which was corroborated with decreased lipid accumulation by GAGVGY treatment. Additionally, in cells undergoing differentiation, mRNA levels of adipogenic genes including PPARγ and CCAAT/enhancer binding protein α (C/EBPα), stearoyl-CoA desaturase 1 (SCD1), and FAS were suppressed by GAGVGY. Furthermore, GAGVGY increased AMP-activated protein kinase (AMPK) phosphorylation and adiponectin secretion in 3T3-L1 adipocytes. The latter effect was supported with evidence showing increased AMPK activation in C2C12 myocytes treated with 3T3-L1-adipocyte-conditioned medium. Together, our data suggest that GAGVGY has multiple beneficial effects on glucose and lipid metabolism, and would control hyperglycemia without the adverse effect of weight gain.
Keywords: Peptide; Glucose transport; Lipid metabolism; AMPK; Adiponectin; 3T3-L1 adipocyte;

Erratum to “The natural antioxidant alpha-lipoic acid induces p27Kip1-dependent cell cycle arrest and apoptosis in MCF-7 human breast cancer cells” [Eur. J. Pharmacol. 641 (2010) 29-34] by Elena Dozio; Massimiliano Ruscica; Luca Passafaro; Giada Dogliotti; Liliana Steffani; Paola Marthyn; Alessandra Pagani; Germana Demartini; Daniele Esposti; Franco Fraschini; Paolo Magni (486).