European Journal of Pharmacology (v.741, #C)

Sickness behavior is a series of behavioral and psychological changes that develop in inflammatory disease, including infections and cancers. Administration of the bacterial endotoxin lipopolysaccharide (LPS) induces sickness behavior in rodents. Genipin, an aglycon derived from an iridoid glycoside geniposide extracted from the fruit of Gardenia jasminoides, has anti-inflammatory and antidepressant activities. However, the effects of genipin on inflammation-induced changes in emotional behaviors are unknown. In this study, we examined the effects of genipin on LPS-induced inflammation in BV-2 cells and sickness behavior in mice. Pretreatment with genipin inhibited LPS-induced increases in NO production and reduced the mRNA levels of inflammation-related genes (iNOS, COX-2, IL-1β and IL-6) in BV-2 cells. Oral administration of genipin ameliorated LPS-induced depressive-like behavior in the forced swim test and social behavior deficits 24 h after LPS administration in mice. LPS-induced expression of mRNAs for inflammation-related genes and the number of c-fos immunopositive cells decreased in the paraventricular nucleus (PVN) of the hypothalamus and the central nucleus of the amygdala (CeA), suggesting that genipin attenuates LPS-induced changes of emotional behaviors through inhibition of neural activation and inflammatory responses in the PVN and CeA. These novel pharmacological effects of genipin may be useful for treatment of patients with sickness behavior.
Keywords: Genipin; Lipopolysaccharide; Sickness behavior; Inflammation;

Gemcitabine is the first-line treatment for pancreatic adenocarcinoma, but is increasingly used to treat breast, bladder, and non-small cell lung cancers. Despite such broad use, intrinsic and acquired chemoresistance is common. In general, the underlying mechanisms of chemoresistance are poorly understood. Here, current knowledge of gemcitabine metabolism, mechanisms of action, sensitivity and chemoresistance reported over the past two decades are reviewed; and we also offer new perspectives to improve gemcitabine efficacy with particular reference to the treatment of pancreatic cancer.
Keywords: Chemoresistance; Pancreatic tumor; Gemcitabine resistance; Molecular targets; NFκB;

Cyclic phosphatidic acid treatment suppress cuprizone-induced demyelination and motor dysfunction in mice by Shinji Yamamoto; Mari Gotoh; Yuuki Kawamura; Kota Yamashina; Sosuke Yagishita; Takeo Awaji; Motomu Tanaka; Kei Maruyama; Kimiko Murakami-Murofushi; Keisuke Yoshikawa (17-24).
Multiple sclerosis is a chronic demyelinating disease of the central nervous system leading to progressive cognitive and motor dysfunction, which is characterized by neuroinflammation, demyelination, astrogliosis, loss of oligodendrocytes, and axonal pathologies. Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring structure at the sn-2 and sn-3 positions of the glycerol backbone. cPA elicits a neurotrophin-like action and protects hippocampal neurons from ischemia-induced delayed neuronal death. In this study, we investigated the effects of cPA on cuprizone-induced demyelination, which is a model of multiple sclerosis. Mice were fed a diet containing 0.2% cuprizone for 5 weeks, which induces severe demyelination, astrocyte and microglial activation, and motor dysfunction. Simultaneous administration of cPA effectively attenuated cuprizone-induced demyelination, glial activation, and motor dysfunction. These data indicate that cPA may be a useful treatment to reduce the extent of demyelination and the severity of motor dysfunction in multiple sclerosis. cPA is a potential lead compound in the development of drugs for the treatment of this devastating disease.
Keywords: Cyclic phosphatidic acid (cPA); Multiple sclerosis (MS); Cuprizone (CPZ) (PubChem CID: 9723); Demyelination;

Endothelial progenitor cells in diabetic patients with myocardial infarction – Can statins improve their function? by Natália António; Ana Soares; Rosa Fernandes; Francisco Soares; Ana Lopes; Tiago Carvalheiro; Artur Paiva; Luís A. Providência; Lino Gonçalves; Carlos Fontes Ribeiro (25-36).
The effect of statins on endothelial progenitor cells (EPCs) function derived from diabetic patients (DMpts) with acute myocardial infarction (AMI) is unknown. In this study we assess the response of early and late EPCs from diabetic versus non-diabetic patients (NDMpts) with AMI to statins. EPCs were obtained from 10 diabetic and 10 age-matched non-diabetic male patients with AMI. For each patient, cultures of early and late EPCs were performed under 4 different conditions: normal glucose concentration (control); high glucose concentration; normal glucose concentration with atorvastatin supplementation and normal glucose concentration with pravastatin supplementation. To compare the effect of these treatments on EPC function in DMpts versus NDMpts, we performed in vitro: EPC colony-forming units (CFU) assay; cell cycle analysis; viability assessment and expression of the surface markers CXCR4, CD133, CD34 and KDR. Under control conditions, CFU numbers were reduced in DMpts-derived EPCs when compared to those of NDMpts (1.4±0.8 vs 2.6±1.2 CFU/well, P=0.021). When early EPCs from DMpts were cultured in the presence of statins, CFU capacity was restored, surmounting that of NDMpts under control conditions. Statins significantly improved viability of early EPCs and delayed the onset of late EPCs senescence, even in cells from DMpts. In addition, statins induced approximately a 2-fold increase in the proportion of late EPCs in S-phase of the cell cycle (P<0.05). Statins have a beneficial effect on both early and late EPCs from DMpts with AMI. Despite the functional impairment of EPCs from DMpts, they exhibit similar responsiveness to statins as equivalent cells from NDMpts.
Keywords: Endothelial progenitor cells; Diabetes mellitus; Myocardial infarction; Statins; Function;

The effect of dinitrosyl iron complexes with glutathione and S-nitrosoglutathione on the development of experimental endometriosis in rats: A comparative studies by Evgeniya N. Burgova; Nikolai А. Tkachev; Oksana V. Paklina; Vasak D. Mikoyan; Leila V. Adamyan; Anatoly F. Vanin (37-44).
It has been established that intraperitoneal bolus administration of S-nitrosoglutathione (GS-NO) (12.5 μmoles/kg; 10 injections in 10 days), beginning with day 4 after transplantation of two 2-mm autologous fragments of endometrial tissue onto the inner surface of the abdominal wall of rats with surgically induced (experimenta) endometriosis failed to prevent further growth of endometrioid (EMT) and additive tumors, while treatment of animals with dinitrosyl iron complexes (DNIC) with glutathione (12.5 μmoles/kg, 10 injections in 10 days) suppressed tumor growth virtually completely. The histological analysis of EMT samples of GS-NO-treated rats revealed pathological changes characteristic of control (non-treated with GS-NO or DNIC) rats with experimental endometriosis. EPR studies established the presence of the active form of ribonucleotide reductase, a specific marker for rapidly proliferating tumors, in EMT samples of both control and GS-NO-treated animals. Noteworthy, in small-size EMT and adjacent tissues of DNIC-treated rats the active form of ribonucleotide reductase and pathological changes were not found.
Keywords: Endometriosis; Dinitrosyl iron complex; S-nitrosoglutathione;

Neuroprotective effects of rosuvastatin against traumatic spinal cord injury in rats by Ramazan Kahveci; Emre Cemal Gökçe; Bora Gürer; Aysun Gökçe; Uçler Kisa; Duran Berker Cemil; Mustafa Fevzi Sargon; Fatih Ozan Kahveci; Nurkan Aksoy; Bülent Erdoğan (45-54).
Rosuvastatin, which is a potent statin, has never been studied in traumatic spinal cord injury. The aim of this study was to investigate whether rosuvastatin treatment could protect the spinal cord after experimental spinal cord injury. Rats were randomized into the following five groups of eight animals each: control, sham, trauma, rosuvastatin, and methylprednisolone. In the control group, no surgical intervention was performed. In the sham group, only laminectomy was performed. In all the other groups, the spinal cord trauma model was created by the occlusion of the spinal cord with an aneurysm clip. In the spinal cord tissue, caspase-3 activity, tumor necrosis factor-alpha levels, myeloperoxidase activity, malondialdehyde levels, nitric oxide levels, and superoxide dismutase levels were analyzed. Histopathological and ultrastructural evaluations were also performed. Neurological evaluation was performed using the Basso, Beattie, and Bresnahan locomotor scale and the inclined-plane test.After traumatic spinal cord injury, increases in caspase-3 activity, tumor necrosis factor-alpha levels, myeloperoxidase activity, malondialdehyde levels, and nitric oxide levels were detected. In contrast, the superoxide dismutase levels were decreased. After the administration of rosuvastatin, decreases were observed in the tissue caspase-3 activity, tumor necrosis factor-alpha levels, myeloperoxidase activity, malondialdehyde levels, and nitric oxide levels. In contrast, tissue superoxide dismutase levels were increased. Furthermore, rosuvastatin treatment showed improved results concerning the histopathological scores, the ultrastructural score and the functional tests. Biochemical, histopathological, ultrastructural analysis and functional tests revealed that rosuvastatin exhibits meaningful neuroprotective effects against spinal cord injury.
Keywords: Methylprednisolone; Neuroprotection; Rat; Rosuvastatin; Spinal cord injury; Trauma;

Cellular mechanism underlying hydrogen sulfide induced mouse tracheal smooth muscle relaxation: Role of BKCa by Jiehong Huang; Yu-li Luo; Yuan Hao; Yi-lin Zhang; Peng-xiao Chen; Jia-wen Xu; Min-hui Chen; Yong-feng Luo; Nan-shan Zhong; Jun Xu; Wen-liang Zhou (55-63).
Recent studies have suggested that hydrogen sulfide (H2S), an important endogenous signaling gaseous molecule, participates in relaxation of smooth muscle. Nevertheless, the mechanism of this relaxation effect on respiratory system is still unclear. The present study aims to investigate the physiological function as well as cellular mechanism of H2S in tracheal smooth muscle. Application of the H2S donor, sodium hydrosulphide (NaHS) and the precursor of H2S, l-cysteine (l-Cys) induced mouse tracheal smooth muscle (TSM) relaxation in an epithelium-independent manner. The relaxation of TSM induced by NaHS was abrogated by iberiotoxin (IbTX), the large conductance calcium activated potassium channel (BKCa) blocker. In primary cultured mouse TSM cells, NaHS remarkably increased potassium outward currents in whole-cell patch clamp, hyperpolarized TSM cells and inhibited the calcium influx. All of these effects were significantly blocked by IbTX. Consistent with the results in vitro, administration of NaHS in vivo also reduced airway hyperresponsiveness in Ovalbumin (OVA)-challenged asthmatic mice. Our present study indicates that NaHS can induce mouse TSM relaxation by activating BKCa. These observations reveal the physiological function of H2S in airway, which provides a promising pharmacological target for the treatment of asthma and other respiratory diseases associated with over-contraction of TSM.
Keywords: H2S; BKCa channels; Asthma; Tracheal smooth muscle;

Molecular mechanisms contributing to the protective effect of levosimendan in liver ischemia-reperfusion injury by Mohamed A. Ibrahim; Seham A. Abdel-Gaber; Entesar F. Amin; Salwa A. Ibrahim; Rehab K. Mohammed; Aly M. Abdelrahman (64-73).
Ischemia-reperfusion injury (IRI) is an important cause of liver damage in many clinical situations. Levosimendan is a promising therapy for prevention of IRI. The present work investigated the possible contribution of nitric oxide (NO), cyclooxygenase (COX) enzymes, and adenosine triphosphate sensitive potassium channel (K-ATP) in the protective effect of levosimendan in liver IRI in rats. Rats were divided into 7 groups. Sham-operated group (negative control group); IR-nontreated group (positive control group), levosimendan-treated group (treated with levosimendan); indomethacin, nonselective COX inhibitor,+levosimendan group (cotreated with indomethacin+levosimendan); celecoxib (selective COX-2 inhibitor)+levosimendan group; L-NNA (Nitro- ω-L-arginine, nonselective NO synthase inhibitor)+levosimendan group; and glibenclamide (K-ATP blocker)+levosimendan group. Liver injury was evaluated biochemically (by serum level of alanine aminotransferase (ALT)) as well as by histopathology. Hepatic tissue content of oxidative stress markers, tumor necrosis factor-alpha (TNF-α), along with immunohistochemical expression of induced NO synthase (iNOS), endothelial NO synthase (eNOS), and caspase-3 in hepatic tissue were assayed. The study showed that levosimendan attenuated liver IRI as evidenced by a decrease in serum ALT level and confirmed by histopathology. The protective effect of levosimendan was associated with modulation of oxidative stress, TNF-α, iNOS, eNOS, and caspase-3. The hepatoprotective effect of levosimendan was partially attenuated by pretreatment by either nonselective COX inhibitor, NOS inhibitor, or K-ATP channel blocker; indicating that the hepatoprotective effect of levosimendan was attributed, at least in part to activation of COX-1, modulation of NO, and opening of K-ATP channel.
Keywords: Liver ischemia-reperfusion injury; levosimendan;

Protective and biogenesis effects of sodium hydrosulfide on brain mitochondria after cardiac arrest and resuscitation by Hao Pan; Xuemeng Xie; Di Chen; Jincheng Zhang; Yaguang Zhou; Guangtian Yang (74-82).
Mitochondrial dysfunction plays a critical role in brain injury after cardiac arrest and cardiopulmonary resuscitation (CPR). Recent studies demonstrated that hydrogen sulfide (H2S) donor compounds preserve mitochondrial morphology and function during ischemia-reperfusion injury. In this study, we sought to explore the effects of sodium hydrosulfide (NaHS) on brain mitochondria 24 h after cardiac arrest and resuscitation. Male Sprague-Dawley rats were subjected to 6 min cardiac arrest and then resuscitated successfully. Rats received NaHS (0.5 mg/kg) or vehicle (0.9% NaCl, 1.67 ml/kg) 1 min before the start of CPR intravenously, followed by a continuous infusion of NaHS (1.5 mg/kg/h) or vehicle (5 ml/kg/h) for 3 h. Neurological deficit was evaluated 24 h after resuscitation and then cortex was collected for assessments. As a result, we found that rats treated with NaHS revealed an improved neurological outcome and cortex mitochondrial morphology 24 h after resuscitation. We also observed that NaHS therapy reduced intracellular reactive oxygen species generation and calcium overload, inhibited mitochondrial permeability transition pores, preserved mitochondrial membrane potential, elevated ATP level and ameliorated the cytochrome c abnormal distribution. Further studies indicated that NaHS administration increased mitochondrial biogenesis in cortex at the same time. Our findings suggested that administration of NaHS 1 min prior CPR and followed by a continuous infusion ameliorated neurological dysfunction 24 h after resuscitation, possibly through mitochondria preservation as well as by promoting mitochondrial biogenesis.
Keywords: Cardiac arrest and cardiopulmonary resuscitation; Brain mitochondria; Mitochondrial biogenesis;

Indirubin derivatives alter DNA binding activity of the transcription factor NF-Y and inhibit MDR1 gene promoter by Toru Tanaka; Sachiyo Ohashi; Hiroaki Saito; Takashi Higuchi; Keiichi Tabata; Yasuhiro Kosuge; Takashi Suzuki; Shinichi Miyairi; Shunsuke Kobayashi (83-89).
Indirubin derivatives exert antitumor activity. However, their effects on the expression of multidrug resistance gene 1 (MDR1) have not been investigated. Here we found three derivatives that inhibit the MDR1 gene promoter. To investigate the effects of indirubins on the DNA binding of NF-Y, a major MDR1 gene transcription factor that recognizes an inverted CCAAT element in the promoter, gel mobility shift assay was performed using the element as a probe with nuclear extracts from NG108-15, MCF7, HepG2, C2C12, and SK-N-SH cells. Among 17 compounds, 5-methoxyindirubin inhibited the DNA binding of NF-Y significantly, whereas indirubin-3′-oxime and 7-methoxyindirubin 3′-oxime increased the binding considerably. After evaluating a suitable concentration of each compound for transcription analysis using living tumor cells, we performed a reporter gene assay using a reporter DNA plasmid containing EGFP cDNA fused to the MDR1 gene promoter region. Indirubin-3′-oxime exerted a significant inhibitory effect on the MDR1 promoter activity in MCF7 and HepG2 cells, and 5-methoxyindirubin inhibited the activity only in MCF7 cells; 7-methoxyindirubin 3′-oxime suppressed the activity in all of the cell lines. We further confirmed that the compounds reduced endogenous MDR1 transcription without any inhibitory effect on NF-Y expression. Moreover, each compound increased the doxorubicin sensitivity of MCF7 cells. These results indicate that each indirubin derivative acts on the DNA binding of NF-Y and represses the MDR1 gene promoter with tumor cell-type specificity.
Keywords: NF-Y; MDR1 gene promoter; Indirubin derivatives; Tumor cell-type specificity;

Rac1 signaling regulates neutrophil-dependent tissue damage in experimental colitis by Changhui Yu; Su Zhang; Lei Song; Yusheng Wang; Rundk Hwaiz; Lingtao Luo; Henrik Thorlacius (90-96).
Excessive neutrophil recruitment in the colon is a major feature in acute colitis although the signaling mechanisms behind colonic recruitment of neutrophils remain elusive. Herein, we hypothesized that Rac1 activity might play an important role in neutrophil infiltration in the inflamed colon. Female Balb/c mice were treated with the Rac1 inhibitor NSC23766 (0.5 and 5 mg/kg) before and daily after administration of 5% dextran sodium sulfate (DSS). Colonic tissue was collected for quantification of neutrophil recruitment, interleukin-6 (IL-6) and CXC chemokine formation as well as histological damage score five days after challenge with DSS. Rac1 activity was determined by western blot and Mac-1 expression by flow cytometry in neutrophils. Administration of NSC23766 decreased DSS-induced neutrophil recruitment and tissue damage in the colon. Rac1 inhibition decreased colonic formation of IL-6 and CXC chemokines in experimental colitis. Chemokine challenge increased Rac1 activity in neutrophils and NSC23766 markedly reduced this neutrophil activity of Rac1. Inhibition of Rac1 abolished CXC chemokine-induced neutrophil chemotaxis and up-regulation of Mac-1 in vitro. Taken together, Rac1 signaling plays a significant role in controlling accumulation of neutrophils and tissue injury in experimental colitis. Thus, our novel results suggest that targeting Rac1 signaling might be a useful way to protect against neutrophil-mediated tissue injury in acute colitis.
Keywords: Adhesion; Inflammation; Neutrophils; Leukocytes; Migration;

Expression of miR-106b-25 induced by salvianolic acid B inhibits epithelial-to-mesenchymal transition in HK-2 cells by Qiong Tang; Haizhen Zhong; Fengyan Xie; Jiayong Xie; Huimei Chen; Gang Yao (97-103).
Epithelial-to-mesenchymal transition (EMT) is a highly conserved physiological program involved in renal fibrosis. Previous studies have shown that transforming growth factor (TGF)-β1 induces EMT in human kidney proximal tubular epithelial cells (HK-2 cells), whereas salvianolic acid B (Sal B) has a protective effect against EMT. The molecular pathogenesis of such processes is currently not well understood. In this study, a miRCURYTM LNA Array was used to screen HK-2 cells for expression changes of microRNAs (miRNAs) implicated in EMT. After validation by real-time PCR, all three members of the miR-106b-25 cluster (miR-106b, miR-93, and miR-25) were found to be markedly down-regulated during EMT in response to TGF-β1, whereas these miRNAs were up-regulated by Sal B treatment in a dose-dependent manner. Moreover, enhanced expression of miR-106b attenuated EMT by retaining the epithelial morphology of HK-2 cells, reducing the levels of α-smooth muscle actin (α-SMA), and increasing the levels of E-cadherin. To explore the molecular basis underlying the inhibitive effect of the miR-106b-25 cluster against EMT, bioinformatics analysis revealed that TGF-β type II receptor, a regulator of TGF-β signaling, might be a direct target of the miR-106b-25 cluster. In turn, low levels of TGF-β type II receptor in EMT of HK-2 cells were shown under the increase of miR-106b. In conclusion, our data suggest that the miR-106b-25 cluster may contribute to EMT in the kidney, and is involved in the protective effect of Sal B. Targeting of specific miRNAs may be a novel therapeutic approach to treat renal fibrosis.
Keywords: Epithelial-to-mesenchymal transition; miR-106b-25; Salvianolic acid B; Transforming growth factor-β1;

Lycopene has attracted significant research interest due to its beneficial therapeutic effects, which include anti-oxidant, neuro-protective and anti-cancer effects, but the mechanisms of its beneficial action are not clear so far. The present study was carried out to elucidate the neuroprotective effect of lycopene against the β-amyloid induced cognitive impairment and mitochondria oxidative damage in rats. β-amyloid (β-A1-42) was administered through intracerebroventricular (ICV) by using stereotaxic instrument in male Wistar rats. Lycopene (2.5 and 5 mg/kg) was administrated for three weeks. Behavioral performances were conducted during the study. The rats were sacrificed on the 21st day following the last behavioral test and cytoplasmic fractions of hippocampus were prepared for the quantification of acetylcholinesterase, oxidative stress parameter, mitochondrial enzymes, and inflammatory mediator like TNF-α, Il-6 activities, caspase-3 and BDNF. ICV β-A1-42 resulted in poor memory retention in Morris water maze and caused marked oxidative stress as indicated by significant increase in oxidative, mitochondria damage, TNF-α, IL-6 and Caspase-3 activity. We also found that β-A1-42 induced animal altered BDNF level than control animals. Chronic administration of lycopene resulted in an improvement in memory retention, attenuation of mitochondrial-oxidative damage, reduced neuro-inflammation and restoration of BDNF level in β-A1-42 treated rats. These studies indicated that lycopene helps to protect β-A1-42 induced cognitive dysfunction and modulates amyloidogenesis.Display Omitted
Keywords: Alzheimer׳s disease; BDNF; β-amyloid; Lycopene; Anti-oxidant;

Oxytocin has therapeutic effects on cancer, a hypothesis by Mohammad Hossein Imanieh; Fereshte Bagheri; Ali Mohammad Alizadeh; Soheil Ashkani-Esfahani (112-123).
Oxytocin (OT) is the first peptide hormone structurally assessed and chemically synthesized in biologically active form. This hormone acts as an important factor in a human reproductive system particularly during pregnancy and lactation in women. So far, different therapeutic roles for OT have been identified as a spectrum from central and peripheral actions on male and female reproductive systems, circulatory system, musculoskeletal system, etc. Some in vitro and in vivo studies also revealed that OT is responsible for bivariate biological functions involved in cancer as following. By activating OT receptor in tumoral cells, OT enacts as a growth regulator, whether activator or inhibitor. Regarding the increase of OT in some conditions such as breastfeeding, exercise, and multiparity, we can relate the effect of these conditions on cancer with OT effects. Based on this hypothesis, we present a review on the effects of this neuropeptide on various types of cancer and also the influence of these conditions on the same cancer.
Keywords: Oxytocin; Cancer; Review; Neuropeptide;

The analgesic effect of dipyrone in peripheral tissue involves two different mechanisms: Neuronal KATP channel opening and CB1 receptor activation by Gilson Gonçalves dos Santos; Elayne Vieira Dias; Juliana Maia Teixeira; Maria Carolina Pedro Athie; Ivan José Magayewski Bonet; Cláudia Herrera Tambeli; Carlos Amilcar Parada (124-131).
Dipyrone (metamizole) is an analgesic pro-drug used to control moderate pain. It is metabolized in two major bioactive metabolites: 4-methylaminoantipyrine (4-MAA) and 4-aminoantipyrine (4-AA). The aim of this study was to investigate the participation of peripheral CB1 and CB2 cannabinoid receptors activation in the anti-hyperalgesic effect of dipyrone, 4-MAA or 4-AA. PGE2 (100 ng/50 µL/paw) was locally administered in the hindpaw of male Wistar rats, and the mechanical nociceptive threshold was quantified by electronic von Frey test, before and 3 h after its injection. Dipyrone, 4-MAA or 4-AA was administered 30 min before the von Frey test. The selective CB1 receptor antagonist AM251, CB2 receptor antagonist AM630, cGMP inhibitor ODQ or KATP channel blocker glibenclamide were administered 30 min before dipyrone, 4-MAA or 4-AA. The antisense-ODN against CB1 receptor expression was intrathecally administered once a day during four consecutive days. PGE2-induced mechanical hyperalgesia was inhibited by dipyrone, 4-MAA, and 4-AA in a dose-response manner. AM251 or ODN anti-sense against neuronal CB1 receptor, but not AM630, reversed the anti-hyperalgesic effect mediated by 4-AA, but not by dipyrone or 4-MAA. On the other hand, the anti-hyperalgesic effect of dipyrone or 4-MAA was reversed by glibenclamide or ODQ. These results suggest that the activation of neuronal CB1, but not CB2 receptor, in peripheral tissue is involved in the anti-hyperalgesic effect of 4-aminoantipyrine. In addition, 4-methylaminoantipyrine mediates the anti-hyperalgesic effect by cGMP activation and KATP opening.
Keywords: Dipyrone; 4-methylaminoantipyrine; 4-aminoantipyrine; Cannabinoid receptors; cGMP; KATP channel; Dipyrone (Metamizol) (PubChem CID: 23674183);

Nicotinic systems have been shown by a variety of studies to be involved in cognitive function. Nicotinic receptors have an inherent property to become desensitized after activation. The relative role of nicotinic receptor activation vs. net receptor inactivation by desensitization in the cognitive effects of nicotinic drugs remains to be fully understood. In these studies, we tested the effects of the α7 nicotinic receptor antagonist methyllycaconitine (MLA), the α4β2 nicotinic receptor antagonist dihydro-β-erythroidine (DHβE), the nonspecific nicotinic channel blocker mecamylamine and the α4β2 nicotinic receptor desensitizing agent sazetidine-A on learning in a repeated acquisition test. Adult female Sprague–Dawley rats were trained on a repeated acquisition learning procedure in an 8-arm radial maze. MLA (1–4 mg/kg), DHβE (1–4 mg/kg), mecamylamine (0.125–0.5 mg/kg) or sazetidine-A (1 and 3 mg/kg) were administered in four different studies either alone or together with the NMDA glutamate antagonist dizocilpine (0.05 and 0.10 mg/kg). MLA significantly counteracted the learning impairment caused by dizocilpine. The overall choice accuracy impairment caused by dizocilpine was significantly attenuated by co-administration of DHβE. Low doses of the non-specific nicotinic antagonist mecamylamine also reduced dizocilpine-induced repeated acquisition impairment. Sazetidine-A reversed the accuracy impairment caused by dizocilpine. These studies provide evidence that a net decrease in nicotinic receptor activity can improve learning by attenuating learning impairment induced by NMDA glutamate blockade. This adds to evidence in cognitive tests that nicotinic antagonists can improve cognitive function. Further research characterizing the efficacy and mechanisms underlying nicotinic antagonist and desensitization induced cognitive improvement is warranted.
Keywords: Nicotinic; Antagonist; Learning; α4β2; α7; Dizocilpine;

3β-Acetyl tormentic acid reverts MRP1/ABCC1 mediated cancer resistance through modulation of intracellular levels of GSH and inhibition of GST activity by Gleice da Graça Rocha; Rodrigo Rodrigues Oliveira; Maria Auxiliadora Coelho Kaplan; Cerli Rocha Gattass (140-149).
ABC transporter overexpression is an important mechanism of multidrug resistance (MDR) and one of the main obstacles to successful cancer treatment. As these proteins actively remove chemotherapeutics from the tumor cells, the pharmacological inhibition of their activity is a possible strategy to revert drug resistance. Moreover, the ability of MDR inhibitors to sensitize resistant cells to conventional drugs is important for their clinical use. Evidence has shown that the multidrug resistance protein 1 (MRP1/ABCC1) is a negative prognostic marker in patients with lung, gastric, or breast cancers or neuroblastoma. Previous data have shown that 3β-acetyl tormentic acid (3ATA) inhibits the transport activity of the protein MRP1/ABCC1. In this study, we evaluated the ability of 3ATA to sensitize an MDR cell line (GLC4/ADR), which overexpresses MRP1, and investigated the anti-MRP1 mechanisms activated by 3ATA. The results showed that 3ATA is able to reverse the resistance of the MDR cell line to doxorubicin and vincristine, two drugs that are commonly used in cancer chemotherapy. Regarding the sensitizing mechanism induced by 3ATA, this work shows that the triterpene does not modulate the expression of MRP1/ABCC1 but is able to reduce total intracellular glutathione (GSH) levels and decrease the activity of glutathione-s-transferase (GST), the enzyme responsible for the glutathione conjugation of xenobiotics. Together, these results show that 3ATA sensitizes the MDR cell line overexpressing MRP1/ABCC1 to antineoplastic drugs and that this effect is mediated by the modulation of intracellular levels of GSH and GST activity.Mechanisms involved in sensitization of cancer cells by 3ATA. (1) By inhibiting the MRP1 activity, 3ATA increases the intracellular pool of drug allowing it to reach effective cytotoxic/lethal concentration. (2) This effect is also due to the ability of 3ATA in inhibiting GST activity and therefore, decreasing the amount of conjugated drug to be transported. (3) 3ATA decreases the intracellular pool of GSH contributing to limit the availability of cellular GSH needed for metabolizing and transporting some alkylating agents out of the cell. (4) By affecting/altering the GSH pool, 3ATA also decreases the antioxidant capacity of the cell, favors ROS accumulation. The overall result of these mechanisms is increased accumulation of drugs and cell death. Display Omitted
Keywords: Cancer; Triterpenes; MRP1/ABCC1 inhibitor; Multidrug resistance reversal; 3β-acetyl tormentic acid;

To investigate the effects of dobutamine hydrochloride on early postoperative cognitive dysfunction (POCD) and plasma tumor necrosis factor (TNF)-α concentration in patients undergoing hip arthroplasty, 124 patients undergoing unilateral total hip arthroplasty, aged 70–92 years old, were randomly assigned to four groups (n=31) as follows: a control group of patients receiving only saline (intravenous infusion, i.v.); and groups receiving 2, 4, or 6 μg kg−1  min−1 (i.v.) of dobutamine hydrochloride. Cognitive functions were assessed on the day before surgery (T1), and the 1st day (T2), 3rd day (T3), and 7th day (T4) postsurgery using the Mini Mental State Examination (MMSE). The plasma TNF-α protein level was determined 10 min before anesthesia (Ta), and 10 min (Tb), 30 min (Tc), and 60 min (Td) after anesthesia by an enzyme-linked immunosorbent assay. Cognitive disorder was observed within the first 3 days after hip arthroplastic surgery, and it had recovered 7 days after the operation in the control group of patients. Administration of 2 or 4 μg kg−1  min−1 dobutamine hydrochloride was able to reverse the early POCD. Simultaneously, an increase of plasma TNF-α levels 30 min after anesthesia was observed (41.34±9.61 vs. 27.75±5.45), which was significantly suppressed by the administration of low-dose dobutamine hydrochloride (29.23±7.32 vs. 41.34±9.61) but not by high-dose dobutamine hydrochloride (45.9±12.11 vs. 41.34±9.61). Together, our data indicated that the plasma concentration of TNFα was engaged in the effect of dobutamine hydrochloride on POCD.
Keywords: Dobutamine hydrochloride; Elderly patient; Hip arthroplasty; Early postoperative cognitive dysfunction; TNF-α;

Coronary artery disease is one of the leading causes of death worldwide. One of the significant causes of this disease is hypercholesterolemia which is the result of various genetic alterations that are associated with the accumulation of specific classes of lipoprotein particles in plasma. A number of drugs are used to treat hypercholesterolemia like statin, fibrate, bile acid sequestrants, niacin, ezetimibe, omega-3 fatty acids and natural extracts. It has been observed that these drugs show diverse response in different individuals. The present review explains the mechanism of action of these drugs as well as mechanism of its lesser effectiveness or resistance in some individuals. There are various identified genetic variations that are associated with diversity in the drugs response. Therefore, present study helps to understand the ethiology of drug mechanism and resistance developed against drugs used to treat hypercholesterolemia.
Keywords: Hypercholesterolemia; Statin resistance; Fibrate resistance; Anti-hypercholesterolemia drugs;

Previous work on rat aorta has shown that L-NMMA and ADMA each enhance vasoconstrictor-induced tone, consistent with blockade of basal nitric oxide activity, whereas they exert little inhibitory effect on acetylcholine-induced relaxation when tone is matched carefully to that of control tissues. The aim of this study was to determine if the ability of L-NMMA or ADMA to inhibit nitric oxide-mediated relaxation was critically determined by the efficacy of the relaxant stimulus. The effects of L-NMMA or ADMA were examined on relaxation to a range of agonists producing different maximal responses, namely, acetylcholine, the muscarinic partial agonist, butyrylcholine, and calcitonin gene-related peptide-1 (CGRP-1). The effects of L-NMMA or ADMA were also examined on relaxation to acetylcholine when its apparent efficacy at the M3 muscarinic receptor was reduced using the irreversible receptor blocking agent, phenoxybenzamine. Maximal relaxation induced by butyrylcholine or CGRP-1 was lower than to acetylcholine. While acetylcholine-induced relaxation was largely resistant to blockade by L-NMMA or ADMA (0.1 or 1 mM), relaxation to butyrylcholine or CGRP-1 was powerfully suppressed. Phenoxybenzamine (0.1–10 μM for 30 min) concentration-dependently reduced maximal acetylcholine-induced relaxation. When the efficacy of acetylcholine was reduced by phenoxybenzamine, its residual relaxant effect was powerfully inhibited by L-NMMA or ADMA (0.1 or 1 mM). Thus, in rat aorta, the ability of L-NMMA or ADMA to block agonist-induced nitric oxide activity is critically determined by the efficacy of the relaxant stimulus.

Dissection of mechanisms that account for imidazoline-induced lowering of blood glucose in mice by Karin Stadlbauer; Zsuzsanna Lehner; Natasa Stamenkovic; Ingo Rustenbeck; Lidia Surman; Anton Luger; Clemens Fürnsinn (178-185).
Multiple mechanisms have been suggested to be responsible for the insulinotropic and blood glucose lowering effects of imidazoline compounds. This study was to unravel which mechanism predominantly accounts for glucose lowering by the prototypical imidazolines idazoxan and phentolamine. To this end, an α2-adrenoceptor agonist (UK14,304) and a KATP channel opener (diazoxide) were used to inhibit insulin release from isolated perifused mouse islets and to induce hyperglycaemia in conscious mice. Potentials of idazoxan and phentolamine to counteract these effects were examined in a comparative manner. In perifused islets, idazoxan increased insulin release only in the presence of the α2-agonist, whereas phentolamine strongly counteracted both inhibitors of insulin release. In vivo, a lower dose of idazoxan was necessary to ameliorate hyperglycaemia induced by the α2-agonist than by the KATP channel opener, indicating α2A-antagonism as the predominant mechanism of action (decrease in incremental area under the glucose curve induced by 0.1 mg/kg idazoxan: under diazoxide, −3±7%, vs. under UK14,304, −34±9%, P<0.02). In contrast, identical doses of phentolamine were required to counteract hyperglycaemia induced by the two inhibitors of insulin release, implicating involvement of another mechanism beside α2A-antagonism (2 mg/kg phentolamine: diazoxide, −11±8%, vs. UK14,304, −15±9%, ns; 4 mg/kg phentolamine: diazoxide, −48±6%, vs. UK14,304, −48±8%, ns). The results show that imidazolines can lower blood glucose via more than one mechanism of action, with the relative contributions of the mechanisms varying considerably between individual compounds. Dissection of the involved mechanisms could help to develop imidazoline drugs for the treatment of type 2 diabetes.
Keywords: Imidazoline; Phentolamine; Idazoxan; α2-adrenoceptor; K ATP channel;

Contribution of receptor for advanced glycation end products to vasculature-protecting effects of exercise training in aged rats by Qi Gu; Bing Wang; Xiao-Feng Zhang; Yan-Ping Ma; Jian-Dong Liu; Xiao-Ze Wang (186-194).
The aim of present work was to investigate the underlying mechanism of vasculature-protecting effects of exercise training in aged rats. Experiment 1: aged rats were given moderate-intensity exercise for 12 weeks. Exercise training suppressed advanced glycation evidenced by reduced activity of aldose reductase, increased activity of glyoxalase 1, reduced levels of methylglyoxal and Nε-(carboxymethyl) lysine, and decreased expression of receptor for advanced glycation end products (RAGE) in aged aortas. Experiment 2: aged rats were given moderate-intensity exercise for 12 weeks or treated with FPS-ZM1, an inhibitor of RAGE. Exercise training attenuated aortic stiffening with age marked by reduced collagen levels, increased elastin levels and reduced pulse wave velocity (PWV), and prevented aging-related endothelial dysfunction marked by restored endothelium-mediated vascular relaxation of aortas in response to acetylcholine. Exercise training in aging aortas reduced formation of malondialdehyde, 3-nitrotyrosin and reactive oxygen species, increased GSH/GSSG ratio, suppressed activation of NFκB, and reduced levels of IL-6 and chemokine (C-C motif) ligand 2. Similar effects were demonstrated in aged rats treated with FPS-ZM1. Collectively, exercise suppressed advanced glycation in the aortas of aged rats, which, at least in part, explained the vasculature-protecting effects of exercise training in aged population.
Keywords: Exercise training; Aging; Vascular dysfunction; Advanced glycation end products;

Beta-asarone attenuates amyloid beta-induced autophagy via Akt/mTOR pathway in PC12 cells by Zhongfeng Xue; Yalei Guo; Sheng Zhang; Liping Huang; Yuping He; Ruoming Fang; Yongqi Fang (195-204).
Alzheimer′s disease (AD) is an age related and progressive neurodegenerative disease. Autophagy is a self-degradative process and plays a critical role in removing long-lived proteins and damaged organelles. Recent evidence suggests that autophagy might be involved in the pathogenesis of AD. β-asarone have various neuroprotective effects. However, the effect of β-asarone on autophagy in amyloid β-peptide (Aβ) induced cell injury is unclear, and little is known about the signaling pathway of β-asarone in autophagy regulation. The aim of the present study was to determine whether β-asarone protects cells from Aβ1–42 induced cytotoxicity via regulation of Beclin-1 dependent autophagy and its regulating signaling pathway. We examined effects of β-asarone on cell morphology, cell viability, neuron specific enolase (NSE) levels, autophagosomes and regulating Beclin-1, p-Akt and p-mTOR expressions in Aβ1–42 treated PC12 cells. We found that β-asarone could maintain the original morphology of cells and increase cell viability and decrease NSE levels significantly. Meanwhile, β-asarone decreased Beclin-1 expression significantly. In addition, β-asarone can increase levels of p-Akt and p-mTOR. These results showed that β-asarone protected cells from Aβ1–42 induced cytotoxicity and attenuated autophagy via activation of Akt-mTOR signaling pathway, which could be involved in neuroprotection of β-asarone against Aβ toxicity. Our findings suggest that β-asarone might be a potential preventive drug for AD.Display Omitted
Keywords: Alzheimer′s disease; Akt; Autophagy; Beclin-1; Beta-asarone; Mammalian target of rapamycin;

Altered airway cell proliferation plays an important role in the pathogenesis of human bronchial asthma and chronic obstructive pulmonary disease (COPD) as well as the equine recurrent airway obstruction (RAO) with consistent changes, i.e. narrowing the airway wall, explained by proliferation and differentiation of fibroblasts. In permanent cell lines, it has been suggested that β2-adrenoceptor agonists and glucocorticoids regulate cell proliferation via the β2-adrenoceptor pathway; indeed, no study was carried out in fresh isolated primary equine bronchial fibroblasts (EBF). We characterized the β-adrenoceptors in EBF, and compared effects of long-acting (clenbuterol) and short-acting (salbutamol and isoproterenol) β2-agonists and dexamethasone on proliferation, differentiation and collagen synthesis. High density (B max; 5037±494 sites/cell) of β2-adrenoceptor subtype was expressed in EBF. β2-agonists inhibited concentration-dependently EBF proliferation with potency of clenbuterol>salbutamol »isoproterenol which was inhibited by ICI 118.551 and propranolol but not by CGP 20712A. In contrast, dexamethasone alone inhibited less EBF proliferation, but the effect was high when dexamethasone was combined with β2-agonists. Transforming growth factor-β1 (TGF-β1) increased transformation of fibroblasts into myofibroblasts, which was inhibited by clenbuterol and dexamethasone alone and drug combination resulted in high inhibition rate. Collagen synthesis in EBF was rather hampered by dexamethasone than by β-agonists. Collectively, the expression of β2-adrenoceptor subtype in EBF and the anti-proliferative effect of clenbuterol suggest that β2-adrenoceptors are growth inhibitory and anti-fibrotic in EBF. These β2-agonist effects in EBF were synergistically enhanced by dexamethasone, providing the additive effects of glucocorticoids to counteract airway remodelling and morbidity of asthma and RAO.
Keywords: Primary bronchial fibroblasts; Equine; Proliferation; Differentiation; β2-agonists;

Sesamin increases heme oxygenase-1 protein in RAW 264.7 macrophages through inhibiting its ubiquitination process by Mizuki Fukunaga; Masatoshi Ohnishi; Ayano Shiratsuchi; Takuya Kawakami; Madoka Takahashi; Misato Motomura; Kyohei Egusa; Tomoka Urasaki; Atsuko Inoue (214-221).
Sesamin is a major component in lignans of sesame seed oil, known to possess potent anti-oxidative capacity. In this study, the variation of heme oxygenase (HO)-1, a kind of anti-oxidative enzyme, by sesamin in murine macrophage cell line RAW 264.7 cells was investigated. Lipopolysaccharide (LPS; 10 μg/ml) exposure tended to increase HO-1 protein expression. Co-treatment with 100 μM sesamin for 12 h up-regulated the HO-1 protein level increased by LPS; however, HO-1 mRNA was unaffected. Sesamin delayed the reversal, by the protein synthesis inhibitor cycloheximide (1 μM), of the LPS-induced increase of HO-1 protein level. Meanwhile, sesamin suppressed LPS-induced expression of inducible nitric oxide (NO) synthase (iNOS) protein and associated NO release. LPS-induced increase of iNOS protein expression was also reversed by cycloheximide, which was not affected by sesamin, unlike HO-1. To clarify the mechanisms that underlie the up-regulation of HO-1 protein level by sesamin, the human embryonic kidney (HEK) 293 T cell line transfected with Flag-tagged HO-1 was used. A proteasome inhibitor, MG-132 (10 μM), stabilized HO-1 protein in HEK 293T cells. Co-treatment with sesamin decreased ubiquitinated HO-1 protein accumulation by MG-132. However, sesamin did not affect the proteasome activity. These findings suggest that sesamin disturbs the degradation of HO-1 protein through inhibiting its ubiquitination, resulting in HO-1 protein up-regulation.
Keywords: Sesamin; Heme oxygenase-1; Ubiquitination; Macrophage;

Mechanisms underlying the hypotensive and vasodilator effects of Ru(terpy)(bdq)NO]3+, a nitric oxide donor, differ between normotensive and spontaneously hypertensive rats by Simone R. Potje; Felipe C. Munhoz; Ligia A. Perassa; Murilo E. Graton; Ariana A.F. Pereira; Ana Claúdia M.S. Nakamune; Roberto S. da Silva; Lusiane M. Bendhack; Doris H. Sumida; Cristina Antoniali (222-229).
The endothelium impairs the vasodilator effect of Ru(terpy)(bdq)NO]3+ (TERPY) in Wistar rat aortas. We hypothesized that endothelial dysfunction could modulate TERPY׳s effect in spontaneously hypertensive rats. The present study investigated the role of the endothelium in the hypotensive and vasodilator effects of TERPY in spontaneously hypertensive rats. We observed a higher hypotensive effect of TERPY in spontaneously hypertensive than in Wistar rats. l-NG-Nitroarginine methyl ester, a nitric oxide synthase inhibitor, increased TERPY׳s hypotensive effect in Wistar but not in spontaneously hypertensive rats. TERPY induced a concentration-dependent vasodilator effect in aortas of both rat models. Endothelium removal or l-NAME increased TERPY׳s potency in Wistar rat aortas; this effect was decreased in spontaneously hypertensive rats. TERPY increased nitric oxide level in spontaneously hypertensive rat endothelial cells; this increase was abolished in the presence of l-NAME. In contrast, this effect was increased in Wistar rats. TERPY, with or without l-NAME, decreased levels of reactive oxygen species in spontaneously hypertensive rat endothelial cells. However, it increased these levels in Wistar rats. TERPY reduced aortic endothelial nitric oxide synthase expression in Wistar rats, but did not alter its expression in spontaneously hypertensive rats. In conclusion, different mechanisms underlie the hypotensive and vasodilator effects of TERPY in these two rat models. TERPY reduced endothelial nitric oxide synthase expression and increased reactive oxygen species production in Wistar rat aortas, but did not alter these in spontaneously hypertensive rats. Furthermore, the nitric oxide released by TERPY reacts with reactive oxygen species, decreasing their bioavailability in spontaneously hypertensive rats.
Keywords: Spontaneously hypertensive rats; Endothelium; Nitric oxide donor; Hypotensive effect; Vasodilator effect;

Phytoestrogens and their effects by Alexander V. Sirotkin; Abdel Halim Harrath (230-236).
The chemical structure, classification, source, metabolism, physiological and health effects of plant phytoestrogens and mechanisms of their action are reviewed. The available knowledge suggests that phytoestrogens can affect a number of physiological and pathological processes related to reproduction, bone remodeling, skin, cardiovascular, nervous, immune systems and metabolism. Due to these effects, phytoestrogens and phytoestrogen-containing diet can be useful for the prevention and treatment of menopausal symptoms, skin aging, osteoporosis, cancer, cardiovascular, neurodegenerative, immune and metabolic diseases. Possible problems in understanding and application of phytoestrogens (multiple targets and multiple estrogen receptor –dependent and –independent mechanisms of action, the discrepancy between the results of experimental and clinical studies, adequate source of phytoestrogen) have been discussed.
Keywords: Phytoestrogen; Reproduction; Bone; Cardiovascular; Nervous system; Immune system;

The nature of the pharmacodynamic interactions of drugs is influenced by the drugs׳ mechanisms of action. It has been hypothesized that drugs with different mechanisms are likely to interact synergistically, whereas those with similar mechanisms seem to produce additive interactions. In this review, we describe an extensive investigation of the published literature on drug combinations of anticonvulsants, the nature of the interaction of which has been evaluated by type I and II isobolographic analyses and the subthreshold method. The molecular targets of antiepileptic drugs (AEDs) include Na+ and Ca2+ channels, GABA type-A receptor, and glutamate receptors such as NMDA and AMPA/kainate receptors. The results of this review indicate that the nature of interactions evaluated by type I isobolographic analyses but not by the two other methods seems to be consistent with the above hypothesis. Type I isobolographic analyses may be used not only for evaluating drug combinations but also for predicting the targets of new drugs.
Keywords: Drug combinations; Anticonvulsant; Type I isobolographic analysis; Type II isobolographic analysis; Subthreshold method; Animal model;

Programmed cell death 4 (PDCD4) is a bona fide tumor suppressor protein and plays a critical role in controlling the rate of protein synthesis. Here, we show that TPA selectively activated the S6K1 and ERK1/2 kinases, contributing to PDCD4 proteolysis and Pdcd4 mRNA degradation in HepG2 cells, respectively. In addition, we observed that sulforaphane suppression of TPA-induced S6K1 and ERK1/2 activation played a critical role in attenuating PDCD4 poly-ubiquitination and Pdcd4 mRNA downregulation. Moreover, we observed that silencing Pdcd4 led to not only an increased expression of c-Jun, but also a decreased expression of p21, the latter of which contributed to suppression of Keap1-dependent Nrf2 poly-ubiquitination. Finally, we demonstrate that the expression of PDCD4, p21 and Nrf2 is higher, but that of c-Jun is lower in normal human liver tissues, compared with hepatoma tissues. Collectively, our study illustrates that attenuating the rate of PDCD4 proteolysis and Pdcd4 mRNA degradation serves as a novel anti-inflammatory and cytoprotective mechanism of sulforaphane.
Keywords: Sulforaphane; PDCD4; S6K1; ERK1/2; Nrf2;

The DPP-IV inhibitor linagliptin and GLP-1 induce synergistic effects on body weight loss and appetite suppression in the diet-induced obese rat by Henrik H. Hansen; Gitte Hansen; Sarah Paulsen; Niels Vrang; Michael Mark; Jacob Jelsing; Thomas Klein (254-263).
Linagliptin is a dipeptidyl peptidase (DPP)-IV inhibitor approved for the treatment of type 2 diabetes. DPP-IV inhibitors are considered weight neutral, suggesting that elevation of endogenous incretin levels is not sufficient to promote weight loss per se. Here we evaluated the effect of linagliptin in combination with subcutaneous treatment of GLP-1(7-36) on body weight regulation in diet-induced obese (DIO) rats. Linagliptin administered perorally (1.5 mg/kg, b.i.d.), but not subcutaneously (0.5 mg/kg, b.i.d.), evoked a very modest body weight loss (2.2%) after 28 days of treatment. GLP-1 (0.5 mg/kg, s.c.) treatment alone induced a body weight loss of 4.1%. In contrast, combined linagliptin (1.5 mg/kg, p.o., or 0.5 mg/kg, s.c.) and GLP-1 (0.5 mg/kg) treatment evoked a marked anorectic response with both routes of linagliptin administration being equally effective on final body weight loss (7.5–8.0%). In comparison, liraglutide monotherapy (0.2 mg/kg, s.c., b.i.d.) reduced body weight by 10.1%. Interestingly, the weight lowering effect of combined linagliptin and GLP-1 treatment was associated with a marked increase in chow preference, being more pronounced as compared to liraglutide treatment. In addition, linagliptin and GLP-1 co-treatment, but not liraglutide, specifically increased prepro-dynorphin mRNA levels in the caudate–putamen, an effect not obtained with administration of the compounds individually. In conclusion, co-treatment with linagliptin and GLP-1 synergistically reduces body weight in obese rats. The anti-obesity effect was caused by appetite suppression with a concomitant change in diet preference, which may potentially be associated with increased dynorphin activity in forebrain regions involved in reward anticipation and habit learning.
Keywords: Obesity; Linagliptin; DPP-IV inhibitor; GLP-1; Weight loss; Dynorphin;

Pharmacogenomic study of the role of the nociceptin/orphanin FQ receptor and opioid receptors in diabetic hyperalgesia by Kris Rutten; Thomas M. Tzschentke; Thomas Koch; Klaus Schiene; Thomas Christoph (264-271).
Targeting functionally independent receptors may provide synergistic analgesic effects in neuropathic pain. To examine the interdependency between different opioid receptors (µ-opioid peptide [MOP], δ-opioid peptide [DOP] and κ-opioid peptide [KOP]) and the nociceptin/orphanin FQ peptide (NOP) receptor in streptozotocin (STZ)-induced diabetic polyneuropathy, nocifensive activity was measured using a hot plate test in wild-type and NOP, MOP, DOP and KOP receptor knockout mice in response to the selective receptor agonists Ro65-6570, morphine, SNC-80 and U50488H, or vehicle. Nocifensive activity was similar in non-diabetic wild-type and knockout mice at baseline, before agonist or vehicle administration. STZ-induced diabetes significantly increased heat sensitivity in all mouse strains, but MOP, DOP and KOP receptor knockouts showed a smaller degree of hyperalgesia than wild-type mice and NOP receptor knockouts. For each agonist, a significant antihyperalgesic effect was observed in wild-type diabetic mice (all P<0.05 versus vehicle); the effect was markedly attenuated in diabetic mice lacking the cognate receptor compared with wild-type diabetic mice. Morphine was the only agonist that demonstrated near-full antihyperalgesic efficacy across all non-cognate receptor knockouts. Partial or near-complete reductions in efficacy were observed with Ro65-6570 in DOP and KOP receptor knockouts, with SNC-80 in NOP, MOP and KOP receptor knockouts, and with U50488H in NOP and DOP receptor knockouts. There was no evidence of NOP and MOP receptor interdependency in response to selective agonists for these receptors. These findings suggest that concurrent activation of NOP and MOP receptors, which showed functional independence, may yield an effective and favorable therapeutic analgesic profile.
Keywords: Antihyperalgesia; Knockout mice; Nociceptin/orphanin FQ receptor; Opioid receptors; ORL1; Neuropathic pain;

Ellagic acid enhances morphine analgesia and attenuates the development of morphine tolerance and dependence in mice by Mohammad Taghi Mansouri; Bahareh Naghizadeh; Behnam Ghorbanzadeh (272-280).
According to our previous study, ellagic acid has both dose-related central and peripheral antinociceptive effect through the opioidergic and l-arginine-NO-cGMP-ATP sensitive K+ channel pathways. In the present study, the systemic antinociceptive effects of ellagic acid in animal models of pain, and functional interactions between ellagic acid and morphine in terms of analgesia, tolerance and dependence were investigated. Ellagic acid (1–30 mg/kg; i.p.) showed significant and dose-dependent antinociceptive effects in the acetic acid-induced writhing test. Intraperitoneal ellagic acid acutely interacted with morphine analgesia in a synergistic manner in this assay. Ellagic acid (1–10 mg/kg; i.p.) also exerted analgesic activity in the hot-plate test. Pre-treatment with naloxone (1 mg/kg; i.p.) significantly reversed ellagic acid, morphine as well as ellagic acid-morphine combination-induced antinociceptin in these two tests. More importantly, when co-administered with morphine, ellagic acid (1–10 mg/kg) effectively blocked the development of tolerance to morphine analgesia in the hot-plate test. Likewise, ellagic acid dose-dependently prevented naloxone-precipitated withdrawal signs including jumping and weight loss. Ellagic acid treatment (1–30 mg/kg; i.p.) had no significant effect on the locomotion activity of animals using open-field task. Therefore, these results showed that ellagic acid has notable systemic antinociceptive activity for both tonic and phasic pain models. Altogether, ellagic acid might be used in pain relief alone or in combination with opioid drugs because of enhancing morphine analgesia and preventing morphine-induced tolerance to analgesia and dependence.
Keywords: Ellagic acid; Morphine; Antinociceptive; Tolerance; Dependence; Isobolography;

The role of Bcl-2 family proteins in pulmonary fibrosis by Leila Safaeian; Alireza Abed; Golnaz Vaseghi (281-289).
Pulmonary fibrosis is characterized by epithelial injury, abnormal tissue repair, fibroproliferation and loss of pulmonary function as a result of a complex interaction of multiple cellular and molecular processes. There is accumulating evidence in support of a role for apoptosis in the pathogenesis of interstitial lung diseases. The Bcl-2 (B-cell lymphoma-2) family of proteins, which consists of antiapoptotic and pro-apoptotic members, is a critical regulator for apoptosis and development of pulmonary fibrosis. The association between Bcl-2 family members and various pathways and mediators has been also described in the pulmonary fibrosis. This article reviews the recent advances regarding the roles of Bcl-2 family as the apoptosis-regulatory factors in pulmonary fibrosis from human tissue studies, animal models, ex vivo and in vitro studies. Further understanding of apoptosis signaling regulation through Bcl-2 family proteins in the lung tissue may lead to better design of new therapeutic interventions for pulmonary fibrosis.
Keywords: Pulmonary fibrosis; Apoptosis; Bcl-2 family proteins;

Crocetin is one of the major active constituents of saffron extract, and it is a carotenoid compound that prevents reactive oxygen species and has anti-inflammation and anti-apoptosis characteristics. This study aims to investigate whether crocetin repairs myocardial damage in vivo after ischemia reperfusion (I/R) and the mechanisms underlying its cardioprotective effects. Male Wistar rats were randomly allocated into three groups: Sham, I/R, CRO. Crocetin (50 mg/kg/day, i.g.) or sodium carboxymethylcellulose (CMC-Na) was intragastrically administered to Wistar rats for 7 days before operation. Myocardial ischemia reperfusion injury (MIRI) was induced by occluding the left anterior descending (LAD) coronary artery for 45 min and subsequent reperfusion for 3 h. The cardioprotective effects of crocetin were evaluated by biochemical values, histopathological observations and the antiapoptotic relative proteins and gene expressions. In the rat model, pretreatment with 50 mg/kg crocetin reduced the cardiac injury, oxidative stress and inflammation compared with that of the non-treated rats, as shown by the decreased levels of infarct size, creatine kinase-MB (CK-MB), malondialdehyde (MDA), and tumor necrosis factor-alpha (TNF-α) activity and the increased levels of total superoxide dismutase (T-SOD) and inflammation cytokines interleukin-10 (IL-10) activity. Crocetin activation also decreases the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining positive percentage and Bax expression, elevated Bcl-2 and endothelial nitric oxide synthase (eNOS) expression and nitrite (NO) production, these indicating that crocetin can suppress the apoptosis damage. These results indicate that crocetin can provide protection against MIRI in rats by inhibiting ROS production, blocking inflammation, and reducing myocardium apoptosis.Display Omitted
Keywords: Crocetin; Ischemia reperfusion injury; Oxidative stress; Inflammation; Apoptosis;

Effect of ursolic acid and Rosiglitazone combination on hepatic lipid accumulation in high fat diet-fed C57BL/6J mice by Arjunan Sundaresan; Thangaiyan Radhiga; Kodukkur Viswanathan Pugalendi (297-303).
This study investigated the combined effect of ursolic acid (UA) and Rosiglitazone (RSG) on lipid regulatory genes in high fat diet (HFD)-fed mice. Male C57BL/6J mice were fed either normal diet or HFD for 10 weeks, after which animals in each dietary group were divided into following six groups, (normal diet, normal diet plus UA and RSG, HFD alone, HFD plus UA, HFD plus RSG, and HFD plus UA and RSG), for the next 5 weeks. UA (5 mg/kg BW) and RSG (4 mg/kg BW) were administered as suspensions directly into the stomach using a gastric tube. At the end of the study (106th day), their liver was analyzed for lipid content. RT-PCR and western blotting methods were used to analyze lipid regulatory genes. HFD-fed mice showed increased activities of hepatic marker enzymes (aspartate aminotransferase and alanine aminotransferase) in plasma and an increased concentration of total cholesterol, triglyceride and free fatty acid in liver. These results were confirmed by upregulated mRNA expression of lipogenic genes such as sterol-regulatory-element-binding protein-1c, fatty acid synthase and acetyl-CoA carboxylase and downregulated mRNA expression of fatty acid oxidative genes such as carnitine palmitoyltransferase-1, acetyl-CoA carboxylase and peroxisome proliferator activated receptor-α in HFD-fed mice. Combined treatment (UA/RSG) significantly reduced the hepatic marker enzyme activities and decreased the lipid accumulation in liver. Furthermore, combination treatment (UA/RSG) down-regulated lipogenic genes and upregulated fatty acid oxidative genes in HFD-fed mice. This study suggests that UA in combination with RSG reduced lipid accumulation in liver.
Keywords: High fat diet; Ursolic acid; Rosiglitazone; C57BL/6J mice; Dyslipidemia;

The H3 receptor agonist immepip does not affect l-dopa-induced abnormal involuntary movements in 6-OHDA-lesioned rats by Maria Papathanou; Peter Jenner; Mahmoud Iravani; Michael Jackson; Kim Stockwell; Isabel Strang; Bai-Yun Zeng; Andrew C. McCreary; Sarah Rose (304-310).
The treatment of dyskinesia in Parkinson׳s disease remains poor but H3 receptor agonists have been suggested as a novel pharmacological approach. We examined the effects of the H3 agonist, immepip, in 6-OHDA-lesioned rats exhibiting AIMs (abnormal involuntary movements), a rat analogue of dyskinesia, in response to l-dopa compared to the known anti-dyskinetic agents amantadine, MK-801 and 8-OHDPAT. We then attempted to extend these studies in to dyskinetic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated common marmosets.Amantadine, MK-801 and 8-OHDPAT all dose-dependently reduced l-dopa-induced axial, lingual and oral (ALO) AIMs in 6-OHDA-lesioned animals accompanied by a reduction in contralateral rotation with higher doses of amantadine and MK-801. By contrast, immepip had no effect on AIMs expression or contralateral rotation. In the MPTP-treated common marmoset exhibiting dyskinesia to l-dopa, immepip alone induced retching and in combination with l-dopa administered subcutaneously or orally induced the rapid onset of retching and vomiting which was not controlled by pretreatment with domperidone. Administration of the unrelated H3 agonist, imetit had the same effect. Despite causing negative side-effects, it appears that both agonists reduced the antiparkinsonian response to l-dopa resulting in reduced dyskinesia.H3 agonists appear unlikely candidates for the treatment of dyskinesia in PD based on lack of evidence of efficacy and potential adverse effects.
Keywords: Parkinson׳s disease; l-Dopa; Dyskinesia; NMDA; 5HT1A and H3 receptors;

The increase in insulin response to oral glucose compared with glucose given by intravenous injection is termed the incretin effect and is mediated by two peptide hormones secreted from the gut in response to nutrient intake: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic β-cells. Both the GLP-1 receptor and the GIP receptor are members of the secretin family of G protein-coupled receptors and couple positively with adenylate cyclase, resulting in an increase in intracellular cAMP. In the present study, we investigated the activity of six previously reported peptide ligands at both the GLP-1 and GIP receptors expressed on HEK-293 cells using a highly sensitive reporter gene assay. GLP-1 and GIP demonstrated almost 100,000-fold selectivity for their respective receptors. Exendin 4 (Ex-4), a long-acting GLP-1 receptor agonist, displayed considerable activity at the GIP receptor. Exendin 9–39 (Ex 9–39) was able to block activity at both the GLP-1 and GIP receptors, and Pro3GIP, a previously-reported GIP receptor antagonist, was shown to act as a partial agonist at the GIP receptor. These data highlight the need for more selective antagonists to study these therapeutically important receptors.
Keywords: G protein-coupled receptor; Pro3GIP; GLP-1; GIP;

Activation of spinal α2 adrenergic receptors induces hyperglycemia in mouse though activating sympathetic outflow by Yun-Beom Sim; Soo-Hyun Park; Sung-Su Kim; Su-Min Lim; Jun-Sub Jung; Hong-Won Suh (316-322).
The roles of α2-adrenergic receptors located in the spinal cord in the regulation of blood glucose levels were studied in imprinting control region (ICR) mice. Mice were treated intrathecally (i.t.) with clonidine or yohimbine, and the blood glucose levels were measured at 0, 30, 60 and 120 min after i.t. administration. The i.t. injection with clonidine caused a pronounced elevation of the blood glucose levels in a dose-dependent manner. Clonidine-induced hyperglycemic effect was dose-dependently attenuated by i.t. pretreatment with yohimbine. Furthermore, plasma insulin level was attenuated by clonidine, and yohimbine pretreatment reversed partially, but significantly, clonidine-induced down-regulation of the plasma insulin level. I.t. pretreatment with pertussis toxin (PTX) almost abolished the hyperglycemic effect induced by clonidine. PTX pretreatment reversed the induced down-regulation of the insulin level. In addition, i.t. pretreatment with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) or intraperitoneal (i.p.) pretreatment with mifepristone, hexamethonium and 6-hydroxydopamine (6-OHDA) attenuated the hyperglycemic effect induced by clonidine. I.t. injected clonidine significantly increased plasma corticosterone level. The elevated blood glucose level induced by clonidine was significantly decreased in adrenalectomized (ADX) mice. Our results suggest that the α2-adrenergic receptors located in the spinal cord play important roles for the elevation of the blood glucose level. The hyperglycemic effect induced by clonidine appears to be mediated by a reduction of the plasma insulin level. In addition, glucocortioid system appears to be involved in clonidine-induced hyperglycemic effect. Furthermore, the clonidine-induced hyperglycemia appears to be mediated via activating the spinal nerves or peripheral sympathetic nervous system.
Keywords: Spinal cord; Adrenergic receptors; Blood glucose; Pertussis toxin; Glucocorticoid; Adrenalectomy;

Differential regulation of astrocyte prostaglandin response by kinins: Possible role for mitogen activated protein kinases by Nofar Torika; Talia Filipovich-Rimon; Keren Asraf; Ella Roasso; Abraham Danon; Sigal Fleisher-Berkovich (323-329).
The role of kinins, well known as peripheral inflammatory mediators, in the modulation of brain inflammation is not completely understood. The present data show that bradykinin, a B2 receptor agonist, enhanced both basal and lipopolysaccharide (LPS)-induced cyclooxygenase-2 mRNA and protein levels and prostaglandin E 2 synthesis in primary rat astrocytes. By contrast, Lys-des-Arg9-bradykinin, which is a bradykinin breakdown product and a selective kinin B1 receptor agonist, attenuated both basal and LPS-induced astrocyte cyclooxygenase-2 mRNA levels and prostaglandin E 2 production. Pre-treating the cells with p42/p44 MAPK but not with JNK or p38 inhibitors completely abrogated PGE2 synthesis in cells stimulated with LPS in the presence of bradykinin or bradykinin B1 receptor agonist. Bradykinin, but not the bradykinin B1 receptor agonist, augmented p42/p44 MAPK phosphorylation. The phosphorylation of JNK and p38 was not altered upon exposure to Bradykinin or the bradykinin B1 receptor agonist. These results suggest that the dual delayed effect of kinins on PGE2 synthesis may be due to differential regulation of COX-2 and signaling molecules such as p42/p44 MAPKs. Thus, kinins may exert opposing actions on brain inflammation and neurodegenerative diseases.
Keywords: Bradykinin; Bradykinin B1 receptor agonist; Lipopolysaccaride; Primary astrocytes;

Phosphodiesterase isoenzymes in the human urethra: A molecular biology and functional study by George T. Kedia; Matthias Oelke; Joachim E. Sonnenberg; Michael Sohn; Andreas Bannowsky; Markus A. Kuczyk; Stefan Ückert (330-335).
Experimental and clinical studies have suggested a role for phosphodiesterase (PDE) isoenzymes in the control of the human lower urinary tract. This study aimed to investigate the expression of PDE isoenzymes and the effects of PDE inhibitors (PDE-Is) in isolated human urethral smooth muscle (USM). The expression of messenger ribonucleic acid (mRNA) specifically encoding for PDE isoenzymes and isoforms (1A, 1B, 1C, 2A, 4A, 4B, 4C, 4D, 5A and 11A) was analyzed by means of reverse transcriptase polymerase chain reaction (RT-PCR). Using a tissue bath technique, the effects of vinpocetine (PDE1-I), erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (EHNA-HCl=MEP1) (PDE2-I), rolipram (PDE4-I), sildenafil, vardenafil and tadalafil (PDE5-Is) (0.01–10 µM) on the tension of USM induced by norepinephrine were investigated. The production of cyclic guanosine monophosphate (cyclic GMP) and cyclic adenosine monophosphate (cyclic AMP) was measured by means of radioimmunoassays. RT-PCR analysis revealed the expression of PDE1B, PDE1C, PDE4A, PDE4C, PDE4D, PDE5A and PDE11A. The tension induced by norepinephrine (NE) was reversed by the PDE inhibitors with the following rank order of efficacy: rolipram (mean: −39%)≥sildenafil (−35%)>vardenafil (−26%)>tadalafil (−20%)>vinpocetine (−16%)>MEP1 (−2%). The relaxing effects of the drugs were paralleled by an elevation in tissue levels of cyclic AMP and cyclic GMP. Selective inhibitors of PDE4 and PDE5 can antagonize the tension induced by alpha-adrenergic stimulation of USM. PDE inhibition might represent an interesting option to facilitate the relaxation of the human outflow region.
Keywords: Human male urethra; Phosphodiesterase enzymes; Cyclic AMP; Cyclic GMP;

hERG trafficking inhibition in drug-induced lethal cardiac arrhythmia by Hisashi Nogawa; Tomoyuki Kawai (336-339).
Acquired long QT syndrome induced by non-cardiovascular drugs can cause lethal cardiac arrhythmia called torsades de points and is a significant problem in drug development. The prolongation of QT interval and cardiac action potential duration are mainly due to reduced physiological function of the rapidly activating voltage-dependent potassium channels encoded by human ether-a-go-go-related gene (hERG). Structurally diverse groups of drugs are known to directly inhibit hERG channel conductance. Therefore, the ability of acute hERG inhibition is routinely assessed at the preclinical stages in pharmaceutical testing. Recent findings indicated that chronic treatment with various drugs not only inhibits hERG channels but also decreases hERG channel expression in the plasma membrane of cardiomyocytes, which has become another concern in safety pharmacology. The mechanisms involve the disruption of hERG trafficking to the surface membrane or the acceleration of hERG protein degradation. From this perspective, we present a brief overview of mechanisms of drug-induced trafficking inhibition and pathological regulation. Understanding of drug-induced hERG trafficking inhibition may provide new strategies for predicting drug-induced QT prolongation and lethal cardiac arrhythmia in pharmaceutical drug development.
Keywords: Acquired long QT syndrome; hERG channel; hERG trafficking inhibition; Safety pharmacology;