European Journal of Pharmacology (v.776, #C)

Our previous study suggested that co-administration of celecoxib increased chemo-sensitivity of multidrug-resistant human gastric cancer SGC-7901/DDP cells to cisplatin (DDP) in vitro. The present study was designed to investigate whether celecoxib had the similar activities in vivo. SGC-7901/DDP and SGC-7901 xenograft mouse models were established. At the end of the experiment, cisplatin treatment alone significantly inhibited tumor growth in SGC-7901 xenograft, as compared with that in SGC-7901/DDP xenograft, suggesting that it maintained cisplatin sensitivity. When cisplatin and celecoxib were co-administrated, their antitumor activities were augmented in SGC-7901/DDP xenograft. The levels of Ki67 and PCNA after combination therapy were significantly decreased in SGC-7901/DDP xenograft, as compared with those of cisplatin treatment alone. Moreover, examining the apoptotic index by TUNEL assay showed similar results. Further studies demonstrated the inhibitory effect of celecoxib on cyclooxygenase-2 and P-glycoprotein expression was the possible reason to increase sensitivity of SGC-7901/DDP cells to cisplatin in vivo. However, the ratio of thromboxane B2 and prostaglandin F was elevated after celecoxib treatment in mice. This has been proposed to increase the risk of thrombogenesis. Further studies are required to evaluate the efficacy and safety of celecoxib for reducing chemo-resistance in gastric cancer.
Keywords: Celecoxib; Xenograft; Cisplatin; Drug-resistance;

Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24 h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ9 desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ9-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer
Keywords: Cis-vaccenic acid; Fetal hemoglobin; Sickle cell anemia; Erythroid progenitor stem cells; Mono-unsaturated fatty acid; Gamma globin;

Endothelial dysfunction is believed the early stage of development of diabetic cardiovascular complications. Sphingosine-1-phosphate (S1P) regulates various biological activities by binding to sphingosine-1-phosphate receptors (S1PRs) including S1PR1-S1PR5. In the present study, the role of S1P receptors in S1P-induced human coronary artery endothelial cells (HCAECs) dysfunction under high glucose condition was investigated and the underlying mechanism was explored. S1PR1-S1PR5 mRNA levels were detected by quantitative Real-time PCR. NO level and polymorphonuclear neutrophils (PMN)-endothelial cells adhesion were measured by nitrate reductase and myeloperoxidase colorimetric method, respectively. Protein levels of endothelial nitric oxide synthase (eNOS), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1(ICAM-1), phosphatidylinositol 3-kinase (PI3K) and Akt were measured by Western blot analysis. S1PR2 were found the predominant S1P receptor expressed in HCAECs exposed to high glucose. NO level and eNOS activity were remarkably decreased, while PMN adhesion, VCAM-1 and ICAM-1 protein levels were increased significantly by S1P treatment in HCAECs exposed to high glucose and normal glucose. Blockage of S1PR2 with specific antagonist JTE-013 and small interfering RNA (siRNA) resulted in enhanced NO level and eNOS activity as well as decreased PMN adhesion, reduced protein levels of VCAM-1 and ICAM-1 induced by S1P. Furthermore, Phosphor-PI3K and phosphor-Akt level were markedly increased by S1PR2 blockade in S1P-treated cells exposed to high glucose, which were suppressed by PI3K inhibitor wortmannin. In conclusion, S1P/S1PR2 mediated endothelial dysfunction partly by inhibiting PI3K/Akt signaling pathway under high glucose condition. S1PR2 blockage could ameliorate endothelial dysfunction which might provide a potential therapeutic strategy for diabetic vascular complications.
Keywords: Sphingosine-1-phosphate; Sphingosine-1-phosphate receptor 2; Endothelial cells; PI3K; Akt; Diabetes;

Restoration of blood supply to ischemic myocardium causes cardiomyocyte damage, a process known as ischemia-reperfusion injury. Excess reactive oxygen species and intracellular calcium contribute to cell damage but the involvement of sirt1, a versatile protein deacetylase in reperfusion-induced cell damage remains unknown. Here, we found that hypoxia-reoxygenation, an in vitro model of ischemia-reperfusion injury, induced H9c2 cardiomyocyte apoptosis as revealed by caspase-3 assay, Hoechst 33258 staining, flow cytometric analysis and JC-1 staining. Molecular docking analysis showed that, pterostilbene, a natural dimethyl ether derivative of resveratrol, binds to the enzymatic active pocket of sirt1. Importantly, application of pterostilbene at low concentrations of 0.1–3.0 μM rescued H9c2 cells from apoptosis, an effect comparable with resveratrol at 20 μM. Mechanistically, pterostilbene exerted its cardioprotective effects via 1) stimulation of sirt1 activity, since pretreatment of H9c2 cells with splitomicin, an antagonist of sirt1, removed the effects of pterostilbene, and 2) enhancement of sirt1 expression. Therefore, the present study demonstrates that activation of sitr1 during ischemia-reperfusion is cardioprotective and that the natural compound-pterostilbene-could be used therapeutically to alleviate ischemia-reperfusion injury.
Keywords: Ischemia-reperfusion injury; Pterostilbene; Cardiomyocyte apoptosis; Sirt1;

Mangiferin protect myocardial insults through modulation of MAPK/TGF-β pathways by Kapil Suchal; Salma Malik; Nanda Gamad; Rajiv Kumar Malhotra; Sameer N. Goyal; Shreesh Ojha; Santosh Kumari; Jagriti Bhatia; Dharamvir Singh Arya (34-43).
Mangiferin, a xanthone glycoside isolated from leaves of Mangifera indica (Anacardiaceae) is known to modulate many biological targets in inflammation and oxidative stress. The present study was designed to investigate whether mangiferin exerts protection against myocardial ischemia-reperfusion (IR) injury and possible role of Mitogen Activated Protein Kinase (MAPKs) and Transforming Growth Factor-β (TGF-β) pathways in its cardioprotection. Male albino Wistar rats were treated with mangiferin (40 mg/kg, i.p.) for 15 days. At the end of the treatment protocol, rats were subjected to IR injury consisting of 45 min ischemia followed by 1 h reperfusion. IR-control rats caused significant cardiac dysfunction, increased serum cardiac injury markers, lipid peroxidation and a significant decrease in tissue antioxidants as compared to sham group. Histopathological examination of IR rats revealed myocardial necrosis, edema and infiltration of inflammatory cells. However, pretreatment with mangiferin significantly restored myocardial oxidant-antioxidant status, maintained membrane integrity, and attenuated the levels of proinflammatory cytokines, pro-apoptotic proteins and TGF-β. Furthermore, mangiferin significantly reduced the phosphorylation of p38, and JNK and enhanced phosphorylation of ERK1/2. These results suggest that mangiferin protects against myocardial IR injury by modulating MAPK mediated inflammation and apoptosis.
Keywords: Apoptosis; Inflammation; Ischemia-reperfusion injury; Mangiferin; MAPK; Oxidative stress;

Inhibition of human α7 nicotinic acetylcholine receptors by cyclic monoterpene carveol by Yosra Lozon; Ahmed Sultan; Stuart J. Lansdell; Tatiana Prytkova; Bassem Sadek; Keun-Hang Susan Yang; Frank Christopher Howarth; Neil S. Millar; Murat Oz (44-51).
Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order carveol>thymoquinone>carvacrol>menthone>thymol>limonene>eugenole>pulegone≥carvone≥vanilin. Among the monoterpenes, carveol showed the highest potency on acetylcholine-induced responses, with IC50 of 8.3 µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies.
Keywords: Nicotinic acetylcholine receptor; Monoterpenes; Carveol; Xenopus oocyte;

Enhanced anti-tumor activity and reduced toxicity by combination andrographolide and bleomycin in ascitic tumor-bearing mice by Huizhen Guo; Zhenbiao Zhang; Zuqing Su; Chaoyue Sun; Xie Zhang; Xiaoning Zhao; Xiaoping Lai; Ziren Su; Yucui Li; Janis Yaxian Zhan (52-63).
Bleomycin (BLM) is an effective anti-carcinogen. With the main detrimental effects of inducing pulmonary fibrosis on patients, its clinical use is limited. Developing agents that enhance the efficacy and attenuate the side effects of cancer chemotherapy are critical. Andrographolide (Andro), an active diterpenoid labdane component extracted from Andrographis panicula, is generally prescribed for treatment of inflammatory associated diseases. The study showed that BLM combined with Andro was significantly more effective than BLM alone on inhibiting the tumor growth, arresting the cell cycle at G0/G1 phase, promoting the capase−3 and capase−8 activity to induce cancer cell apoptosis. The underlying mechanisms may be related to the transcriptional regulation of P53/P21/Cyclin pathways. Moreover, BLM induced pulmonary fibrosis in tumor-bearing mice, but BLM combined with Andro dramatically alleviated the lesion in pulmonary fibrosis by activating the SOD, suppressing MDA and HYP production, in the meanwhile attenuating the IL−1β, TNF- α, IL−6 and TGF-β1 level. These mechanisms were associated with its effect on inhibition of protein expression of TGF-β, α-SMA, p–Smad2/3, enhanced expression of Smad7. Thus, it demonstrated that Andro might be a potential adjuvant therapeutic agent for BLM.Display Omitted
Keywords: Bleomycin; Andrographolide; Anticancer; Pulmonary fibrosis; Combination;

Neuropeptide Y is an angiogenic factor in cardiovascular regeneration by Rabya Saraf; Feroze Mahmood; Rabia Amir; Robina Matyal (64-70).
In diabetic cardiomyopathy, there is altered angiogenic signaling and increased oxidative stress. As a result, anti-angiogenic and pro-inflammatory pathways are activated. These disrupt cellular metabolism and cause fibrosis and apoptosis, leading to pathological remodeling. The autonomic nervous system and neurotransmitters play an important role in angiogenesis. Therapies that promote angiogenesis may be able to relieve the pathology in these disease states. Neuropeptide Y (NPY) is the most abundantly produced and expressed neuropeptide in the central and peripheral nervous systems in mammals and plays an important role in promoting angiogenesis and cardiomyocyte remodeling. It produces effects through G-protein-coupled Y receptors that are widely distributed and also present on the myocardium. Some of these receptors are also involved in diseased states of the heart. NPY has been implicated as a potent growth factor, causing cell proliferation in multiple systems while the NPY3-36 fragment is selective in stimulating angiogenesis and cardiomyocyte remodeling. Current research is focusing on developing a drug delivery mechanism for NPY to prolong therapy without having significant systemic consequences. This could be a promising innovation in the treatment of diabetic cardiomyopathy and ischemic heart disease.
Keywords: Neuropeptide Y; NPY3-36; Angiogenesis; Cardiovascular regeneration; Diabetic cardiomyopathy;

Muscarinic and dopamine brain systems interact intimately, and muscarinic receptor ligands, like dopamine ligands, can modulate the reinforcing and discriminative stimulus (SD) effects of cocaine. To enlighten the dopamine/muscarinic interactions as they pertain to the SD effects of cocaine, we evaluated whether muscarinic M1, M2 or M4 receptors are necessary for dopamine D1 and/or D2 antagonist mediated modulation of the SD effects of cocaine. Knockout mice lacking M1, M2, or M4 receptors, as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10 mg/kg cocaine from saline in a food-reinforced drug discrimination procedure. Effects of pretreatments with the dopamine D1 antagonist SCH 23390 and the dopamine D2 antagonist eticlopride were evaluated. In intact mice, both SCH 23390 and eticlopride attenuated the cocaine discriminative stimulus effect, as expected. SCH 23390 similarly attenuated the cocaine discriminative stimulus effect in M1 knockout mice, but not in mice lacking M2 or M4 receptors. The effects of eticlopride were comparable in each knockout strain. These findings demonstrate differences in the way that D1 and D2 antagonists modulate the SD effects of cocaine, D1 modulation being at least partially dependent upon activity at the inhibitory M2/M4 muscarinic subtypes, while D2 modulation appeared independent of these systems.
Keywords: Muscarinic receptors; Dopamine receptors; Knockout mice; Drug discrimination; M1; M2; M4; D1; D2;

Nerol alleviates pathologic markers in the oxazolone-induced colitis model by Adriana Estrella González-Ramírez; María Eva González-Trujano; Sandra A. Orozco-Suárez; Noé Alvarado-Vásquez; Francisco Javier López-Muñoz (81-89).
Nerol is a natural monoterpene with antinociceptive and anti-inflammatory properties. Its possible beneficial effects in ulcerative colitis and its corresponding mechanism of action have not been determined to date. The aim of this study was to investigate whether nerol prevents the appearance of pathological markers and hyperalgesia in oxazolone-induced colitis, and protects against gastric damage produced by ethanol. The experimental design included groups of oxazolone-treated mice receiving nerol at 10–300 mg/kg, p.o., or a reference drug (sulfasalazine, 100 mg/kg, p.o.) compared to sham and untreated groups. Gastric damage was evaluated in the absolute ethanol-induced ulcer model in rats. Variables measured in animals with oxazolone-induced colitis included weight loss, stool consistency and macroscopic colon damage; mechanical nociception was determined by the use of von Frey filaments, whereas levels of inflammatory cytokines were assessed by enzyme-linked immunosorbent assay. Nerol (30−300 mg/kg, p.o.) prevented or significantly decreased the pathological alterations observed in the oxazolone- induced colitis model. It also showed antinociceptive effects and reduced the increased levels of inflammatory cytokines (IL-13 and TNF-α). Gastric damage was also prevented starting at 10 mg/kg, p.o. In conclusion, our results provide evidence for a beneficial effect of nerol after colitis induction involving tissue protection, antinociception and modulation of the immunological system, suggesting the therapeutic potential of this monoterpene as a novel alternative in controlling ulcerative colitis.Display Omitted
Keywords: Gastroprotection; Inflammatory cytokines; Nerol; Oxazolone; Ulcerative colitis;

Orientin, a flavonoid exists in Chinese traditional herbal Polygonum orientale L., has been previously demonstrated to protect against myocardial ischemia reperfusion injury (MIRI) through inhibition of apoptosis. However, the underlying mechanisms remain to be elucidated and we therefore in this study investigated the effects of orientin on autophagy during MIRI in rats. The results indicate that orientin, at the concentrations of 10 and 30 μM in the cultures of neonatal rat cardiomyocytes, promoted the induction of autophagy, increasing the formation of autophagosomes and enhancing the expression of LC3 puncta, LC3-II/LC3-I ratio and Beclin 1 after hypoxia/reoxygenation. The induction of autophagy by orientin correlated with enhanced cell viability and decreased apoptosis, which was significantly attenuated by autophagy inhibitor wortmannin, a phosphatidylinositol-3-kinase (PI3K) inhibitor. Moreover, application of orientin increased the activation of AMPK and Akt, downregulated the phosphorylation of mammalian target of rapamycin (mTOR) and the expression of Raptor, and enhanced the interaction between Beclin 1 and Bcl-2 in endoplasmic reticulum due to increased phosphorylation of Beclin 1 and decreased phosphorylation of Bcl-2. Our investigation suggests that the cardioprotective effects of orientin during MIRI may be mediated through the balance of autophagy through regulating AMPK, Akt, mTOR, and Bcl-2 associated signaling pathways.Display Omitted
Keywords: Orientin; Hypoxia/reoxygenation; Autophagy; AMPK; mTORC1; Beclin 1;

Antihyperuricemic effects of thiadiazolopyrimidin-5-one analogues in oxonate treated rats by Kadanuru R. Sathisha; Shubha Gopal; Kanchugarakoppal S. Rangappa (99-105).
Hyperuricemia is a risk factor for not only gout, but also to a variety of disorders that affect the vital organ systems of the human body. The xanthine oxidase (XO) is the key enzyme in the production of uric acid and its inhibition can inhibit hyperuricemia. Although, XO inhibitor allopurinol is widely prescribed antigout agent but its use is not without any side effects. Previously, we described the synthesis of four novel thiadiazolopyrimidin-5-one analogues as effective XO inhibitors and molecular docking studies also confirmed this. When these analogues were tested in potassium oxonate treated rats, their serum uric acid and creatinine levels were dropped significantly from 4.85±0.03 mg/dl to 1.21±0.01 mg/dl and 0.92±0.02 mg/dl to 0.40±0.02 mg/dl respectively. Among the pyrimidine analogues tested, 6a was most potent. Histological examinations of both liver and kidney tissues exhibited severe necrosis in oxonate treated rats and pyrimidine analogues could significantly attenuate this with a correlative inhibitory profile of hepatic XO from the same rats. Our results demonstrate antihyperuricemic effect of novel thiadiazolopyrimidin-5-one analogues in oxonate treated rats, which can be further explored not only as antigout therapeutics but also in other systems where hyperuricemia is the driving cause of the disease.
Keywords: Thiadiazolopyrimidin-5-one analogues; Potassium oxonate; Hyperuricemia; Xanthine oxidase; Gout;

Telmisartan, one unique angiotensin II type 1 receptor blocker, has been attracting attention due to its putative peroxisome proliferator-activated receptor (PPAR)-γ or β/δ actions. Recently, telmisartan has been reported to exert neuroprotective effects in animal models of Parkinson's disease (PD). However, the underlying mechanisms have not been fully clarified. Recently, accumulating evidence has shown that endoplasmic reticulum (ER) stress plays a crucial role in rotenone-induced neuronal apoptosis. Additionally, studies have revealed that inositol-requiring enzyme/endonuclease 1α (IRE1α) is necessary and sufficient to trigger ER stress. In the present study, we aimed to determine whether ER stress-activated IRE1α-mediated apoptotic pathway is involved in the neuroprotection of telmisartan in the rotenone rats of PD and explore the possible involvement of PPAR-β/δ activation. The catalepsy tests were performed to test the catalepsy symptom. The dopamine content and α-synuclein expression were ascertained through high-performance liquid chromatography and immunohistochemistry, respectively. The expression of IRE1α, TNF receptor associated factor 2 (TRAF2), caspase-12 and PPAR-β/δ was detected by western blot. Neuronal apoptosis was assessed by TUNEL and immunohistochemistry. Our results show that telmisartan ameliorated the catalepsy symptom and attenuated dopamine depletion as well as α-synuclein accumulation. Moreover, telmisartan decreased ER stress-mediated neuronal apoptosis. Furthermore, telmisartan inhibited IRE1α-TRAF2-caspase-12 apoptotic signaling pathway. Additionally, telmisartan activated PPAR β/δ, implying that PPAR-β/δ activation properties of telmisartan are possibly or partially involved in the neuroprotective effects. In conclusion, our findings suggest that suppressing ER stress-activated IRE1α-TRAF2-caspase-12 apoptotic pathway is involved in the neuroprotective effects of telmisartan in the rotenone rats of PD.
Keywords: Parkinson's disease; Telmisartan; Inositol-requiring enzyme 1α; Apoptosis; Peroxisome proliferator-activated receptor-β/δ;

Cholinergic activation of neurons in the medulla oblongata changes urinary bladder activity by plasma vasopressin release in female rats by Eduardo M. Cafarchio; Luiz A. da Silva; Luciana C. Auresco; Cristiana A. Ogihara; Roberto L. Almeida; Gisele Giannocco; Maria C.B. Luz; Fernando L.A. Fonseca; Monica A. Sato (116-123).
The central control of the micturition is dependent on cortical areas and other ascending and descending pathways in the brain stem. The descendent pathways from the pons to the urinary bladder (UB) can be direct or indirect through medullary neurons (MN). Chemical stimulation with l-glutamate of MN known for their involvement in cardiovascular regulation evokes changes in pelvic nerves activities, which innervate the urinary bladder. Different neurotransmitters have been found in medullary areas; nevertheless, their involvement in UB control is few understood. We focused to investigate if cholinergic activation of neurons in the medulla oblongata changes the urinary bladder activity. Carbachol (cholinergic agonist) or atropine (cholinergic antagonist) was injected into the 4thV in anesthetized female Wistar rats and the intravesical pressure (IP), mean arterial pressure (MAP), heart rate (HR) and renal conductance (RC) were recorded for 30 min. Carbachol injection into the 4thV increased IP with peak response at 30 min after carbachol and yielded no changes in MAP, HR and RC. Atropine injection into the 4thV decreased IP and elicited no changes in MAP, HR and RC. Plasma vasopressin levels evaluated by ELISA kit assay increased after carbachol into the 4th V. Intravenous blockade of V1 receptors prior to carbachol into the 4thV abolished the increase in IP evoked by carbachol. Therefore, our findings suggest that cholinergic activation of neurons in the medulla oblongata by carbachol injections into the 4thV increases IP due to plasma vasopressin release, which acts in V1 receptors in the UB.
Keywords: Cholinergic; Medulla oblongata; Fourth ventricle; Urinary bladder; Vasopressin;

Ghrelin induces colon cancer cell proliferation through the GHS-R, Ras, PI3K, Akt, and mTOR signaling pathways by Gi-Shih Lien; Chien-Huang Lin; You-Lan Yang; Ming-Shun Wu; Bing-Chang Chen (124-131).
Colon cancer is the third most common malignancy worldwide. Recently, some interesting associations between ghrelin and cancer were reported, and it may participate in colon cancer development. In the present report, we explored the role of the growth hormone secretagogue receptor (GHS-R), Ras, phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) pathways in the ghrelin-induced proliferation of human colon cancer cells. Ghrelin-caused HT-29 proliferation was reduced by [D-Lys3]-GHRP-6 (a GHS-R inhibitor). We also found that a dominant negative mutant of Ras (Ras DN), a PI3K inhibitor (LY 294002), an Akt DN, and an mTOR inhibitor (rapamycin) attenuated ghrelin-caused colon cancer cell proliferation. We found that ghrelin induced time-dependent increases in Ras activity. Moreover, ghrelin-mediated Akt Ser473 phosphorylation was attenuated by a Ras DN and LY 294002. Furthermore, a Ras DN, LY 294002, and an Akt DN all inhibited ghrelin-caused mTOR Ser2448 phosphorylation. These results indicate that the Ras/PI3K/Akt/mTOR cascade plays a critical role in ghrelin-induced colon cancer cell proliferation.Display Omitted
Keywords: Colon cancer; Ghrelin; Proliferation; Ras; PI3K/Akt; mTOR;

Cannabinoids are known to inhibit neuronal activity and have significant immunomodulatory effects which suggest a role in inflammatory airway diseases. In the present study, we tested the hypothesis that cannabinoids have both acute and chronic modulatory effects on nerve-mediated contractions in NGF-induced airway inflammation. Contractions induced by electrical field stimulation (EFS) were examined in tracheal segments isolated from male BALB/c mice. Tissues were both used fresh or after four days of culture with NGF to induce airway inflammation, and further exposed to cannabinoid receptor agonists. In order to evaluate nerve density, tracheal segments were also examined by immunohistochemistry after in vitro treatments. The CB1 receptor agonists ACEA and ACPA inhibited the constant train EFS-induced contractions in both fresh and NGF-exposed tracheas, an effect that could be blocked by the CB1 receptor antagonist AM251. Culturing the tissues with NGF up-regulated the frequency-dependent EFS-contractions in isolated tracheas. This up-regulation could be inhibited by concomitant treatment with ACEA or ACPA. The treatment with NGF and/or ACEA did not affect the potency or the maximum response to carbachol. In histological sections, it was recognized that the enhanced effect induced by NGF was associated with an increase in nerve density, which, similarly, could be prevented by ACEA treatment. This study shows that stimulation of cannabinoid CB1 receptors modifies the increase of neuronal activity and density in NGF-induced airway inflammation and directly inhibits cholinergic contractions in the airways by a presynaptic mechanism. These findings indicate a protective role of CB1 receptors in airway inflammation.
Keywords: Cannabinoid; Airway; Airway inflammation; Airway hyperreactivity; Nerve growth factor;

Effect of dexmedetomidine and cold stress in a rat model of neuropathic pain: Role of interleukin-6 and tumor necrosis factor-α by Hanan Sayed M. Farghaly; Ahmed Mostafa Mahmoud; Khaled A. Abdel-Sater (139-145).
Dexmedetomidine (Dex) is a novel Alpha 2-adrenoceptor agonist. It decreases sympathetic tone and attenuates the stress responses to anesthesia and surgery. People exposed to cold suffer unpleasant thermal pain, which is experienced as stress and causes the release of noradrenaline from the sympathetic terminals. The present study investigated the effects of cold stress and dexmedetomidine on chronic constriction injury (CCI) model of the sciatic nerve in rats. Sixty four male Wistar rats were divided into seven groups of eight rats each: repeated cold stress (RCS) group, sham RCS group, CCI group, sham CCI group, Dex-treated group received a single dose of Dex (5 μg/kg), CCI+Dex group, CCI+RCS group. Interleukin-6 (IL-6) and tumor necrosis factor- alpha (TNF-α) levels in the serum were measured by enzyme-linked immunosorbent assay. The mean body weight of CCI, RCS, CCI+RCS, CCI+Dex and RCS+Dex groups decreased significantly compared with pre-values. Dexmedetomidine and CCI caused significant changes of the systolic, diastolic and mean blood pressure. Both RCS and CCI groups showed significant decreased of reaction time in the hot plate test. The RCS and CCI groups demonstrated a significant mechanical hyperalgesia, while pain threshold was increased in the RCS+Dex group. A significant decrease of serum IL-6 and TNF-α was demonstrated in CCI+RCS and CCI+Dex groups. The therapeutic effectiveness of dexmedetomidine in neuropathic pain may be through inhibition of proinflammatory cytokines, primarily IL-6 and TNF-α. Moreover, cold stress may result in increased resistance to neuropathic pain.
Keywords: Cold stress; Dexmedetomidine; IL-6; Neuropathic pain; TNF-α;

Pyrrolidin-2-one derivatives may reduce body weight in rats with diet-induced obesity by Magdalena Dudek; Joanna Knutelska; Marek Bednarski; Leszek Nowiński; Małgorzata Zygmunt; Grzegorz Kazek; Barbara Mordyl; Monika Głuch-Lutwin; Paula Zaręba; Katarzyna Kulig; Jacek Sapa (146-155).
Obesity affects an increasing number of individuals in the human population and significant importance is attached to research leading to the discovery of drug which would effectively reduce weight. The search for new drugs with anorectic activity and acting within the adrenergic system has attracted the interest of researchers. This study concerns the experimental effects on body weight of α2-adrenoceptor antagonists from the group of pyrrolidin-2-one derivatives in rats with diet-induced obesity. Methods: The intrinsic activity of the test compounds at the α-adrenoreceptors was tested. Obesity in rats was obtained by the use of fatty diet and then the influence of the test compounds on body weight, food and water intakes, lipid and glucose profiles and glycerol and cortisol levels were determinated. The effects of the compounds on locomotor activity, body temperature, blood pressure and heart rate were tested. Results: One of the test compounds (1-(3-(4-phenylpiperazin-1-yl)propyl)pyrrolidin-2-one) reduces the animal's body weight and the amount of peritoneal adipose tissue during chronic administration, at the same time it does not cause significant adverse effects on the cardiovascular system. This compound decreases temperature and elevates glycerol levels and does not change the locomotor activity and cortisol level at anti-obese dose. Conclusions: Some derivatives of pyrrolidin-2-one that act as antagonists of the α2-adrenoreceptor may reduce body weight. Reducing body weight for 1-(3-(4-phenylpiperazin-1-yl)propyl)pyrrolidin-2-one can be associated with decrease in food intake, body fat reduction, reduction of blood glucose, and increased thermogenesis and lipolysis. This effect cannot be the result of changes in spontaneous activity or stress.
Keywords: Anti-obesity action; Obesity; Diet-induced obese rats; α2-Adrenoceptor antagonist; Pyrrolidin-2-one derivatives;

FMS-like tyrosine kinase 3 (FLT3) inhibitors: Molecular docking and experimental studies by Baratali Mashkani; Mohammad Hossein Tanipour; Mohammad Saadatmandzadeh; Leonie K. Ashman; Renate Griffith (156-166).
Activating mutations in FMS-like tyrosine kinase 3 (FLT3) occur in 25% of acute lymphoid and 30% of acute myeloid leukaemia cases. Therefore, FLT3 is a potential therapeutic target for small molecule kinase inhibitors. In this study, protein-ligand interactions between FLT3 and kinase inhibitors (CEP701, PKC412, sunitinib, imatinib and dasatinib) were obtained through homology modelling and molecular docking. A cellular system for experimental testing of the inhibitors was also established by expressing wildtype and internal tandem duplication mutant FLT3 (FLT3-WT and FLT3-ITD) in FDC-P1 cells. Imatinib and dasatinib could not be docked into any of the FLT3 models, consistent with their lack of activity in the experimental assays. CEP701, PKC412 and sunitinib interacted with the ATP-binding pocket of FLT3, forming H-bonds with Cys694 and Glu692. Based on the EC50 values in the cell proliferation assay, CEP701 was the most potent inhibitor; sunitinib and PKC412 were ranked second and third, respectively. Sunitinib was the most selective inhibitor, followed by PKC421 and CEP701. The potency of sunitinib and to a lesser extent CEP701 in inhibition of FLT3 autophosphorylation was lower than the cell proliferation inhibition, indicating that inhibition of FLT3 downstream proteins may contribute to the cellular effects. It was shown in this study that the docking procedure was able to differentiate FLT3 inhibitors from ineffective compounds. Additionally, interaction with the phosphate binding region in the ATP-binding pocket increased potency at the cost of selectivity. These findings can be applied in designing highly effective and selective inhibitors for FLT3 and other related kinases.Display Omitted
Keywords: FLT3; Docking; GOLD; Cell proliferation inhibition; Autophosphorylation inhibition; Small molecule kinase inhibitors;

Epigenetic modifications influence DNA damage response (DDR). In this study we explored the role of DNA methylation and histone acetylation in DDR in cells challenged with acute or chronic oxidative stress. We used retinal pigment epithelial cells (ARPE-19), which natively are exposed to oxidative stress due to permanent exposure to light and high blood flow. We employed a DNA methyltransferase inhibitor – RG108 (RG), or a histone deacetylase inhibitor – valproic acid (VA). ARPE-19 cells were exposed to tert-butyl hydroperoxide, an acute oxidative stress inducer, or glucose oxidase, which slowly liberates low-doses of hydrogen peroxide in the presence of glucose, creating chronic conditions. VA and RG reduced level of intracellular reactive oxygen species and DNA damage in ARPE-19 cells in normal condition and in oxidative stress. This protective effect of VA and RG was associated with the up-regulated expression of antioxidant enzyme genes: CAT, GPx1, GPx4, SOD1 and SOD2. RG decreased the number of cells in G2/M checkpoint in response to chronic oxidative stress. Neither RG nor VA changed the DNA repair or apoptosis induced by oxidative stress. Therefore, certain epigenetic manipulations may protect ARPE-19 cells from detrimental effects of oxidative stress by modulation of antioxidative enzyme gene expression, which may be further explored in pharmacological studies on oxidative stress-related eye diseases.
Keywords: ARPE-19; DNA damage response; Epigenetic inhibitors; Oxidative stress; Reactive oxygen species;

Proteases affect immune response by activating PI3K, ERK1/2 and p38 kinase. In present study, therapeutic effect of PI3K, ERK1/2 and p38 kinase inhibitor in combination with serine protease inhibitor was evaluated in cockroach extract (CE) induced airway inflammatory disease. Mice were sensitized on day 0, 7 and 14 and challenged on day 27, 28 and 29 with CE. Mice were given PI3K, ERK1/2 and the p38 kinase inhibitor (iPI3K, iERK1/2 and the ip38) alone or with serine protease inhibitor 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 1 h before challenge. On day 30 airway resistance of mice were determined and euthanized to collect blood, BAL fluid and lung for analysis. CE immunized mice showed PI3K, ERK1/2 and p38 kinase activation, increased airway resistance, cellular infiltration, Th2 cytokines IgE and IgG1. AEBSF given to mice reduced the CE induced allergic response. AEBSF given in combination of iPI3K/iERK1/2 reduced cellular infiltration in lungs. Furthermore, iPI3K/iERK1/2 with AEBSF significantly reduced the CE induced Th2 cytokines in comparison to monotherapy of kinase inhibitor and AEBSF (P<0.05). The combination of iPI3K/iERK1/2 with AEBSF enhanced IL-12 level that could further provide a mean of Th2 reduction. Best effect in reduction of allergic response in mice was observed on administration of AEBSF with iPI3K. Conclusively, the combination of PI3K kinase inhibitor with AEBSF reduced allergen induced airway response and has therapeutic potential for add-on therapy in allergic airway disease.
Keywords: Allergy; Protease inhibitor; Kinase inhibitor; Cytokine;

Divergent electrophysiologic profile of fluconazole and voriconazole in an experimental whole-heart model of proarrhythmia by Gerrit Frommeyer; Christina Fischer; Philipp S. Lange; Patrick Leitz; Michael Fehr; Harilaos Bogossian; Peter Milberg; Lars Eckardt (185-190).
In several case reports a prolongation of the QT-interval and even proarrhythmic effects of fluconazole and voriconazole were reported. The aim of the present study was to investigate if application of fluconazole or voriconazole has the potential to provoke polymorphic ventricular tachycardia in a sensitive model of proarrhythmia. In female rabbits, fluconazole (10, 30 and 50 µM, n=6) and voriconazole (10, 30 and 50 µM, n=6) were infused after obtaining baseline data. Eight endocardial and epicardial monophasic action potentials and a simultaneously recorded 12-lead ECG showed a significant QT prolongation after application of fluconazole as compared with baseline (10 µM:+12 ms, 30 µM:+22 ms, 50 µM:+37 ms; P<0.05) accompanied by an increase of action potential duration (APD90). Administration of voriconazole also induced QT prolongation (30 µM:+10 ms, 50 µM:+20 ms, P<0.05). Spatial dispersion of repolarization remained stable in voriconazole-treated hearts while fluconazole induced a significant increase (30 µM:+15 ms, 50 µM:+16 ms; P<0.05). Lowering of potassium concentration in bradycardic AV-blocked hearts did not provoke any early afterdepolarizations (EADs) or polymorphic ventricular tachycardia in voriconazole-treated hearts. Application of fluconazole led to the reproducible induction of EADs in 4 of 6 hearts and polymorphic ventricular tachycardia in 3 of 6 hearts (36 episodes). In the present study, voriconazole demonstrated a safe electrophysiologic profile despite significant QT prolongation. In contrast, fluconazole led to a more marked prolongation of myocardial repolarization combined with a more marked increase of dispersion of repolarization. These results imply that application of fluconazole might be torsadogenic and the QT-interval should be closely monitored.
Keywords: Fluconazole; Voriconazole; Dispersion of repolarization; Proarrhythmia; Sudden cardiac death;