European Journal of Pharmacology (v.770, #C)

Age-related macular degeneration (AMD) is a major cause of blindness worldwide. Oxidative stress plays a crucial role in the pathogenesis of dry AMD. Quercetin has potent anti-oxidative activities, but poor bioavailability limits its therapeutic application. Herein, we prepared the phospholipid complex of quercetin (quercetin-PC), characterized its structure by differential scanning calorimetry, infrared spectrum and x-ray diffraction. Quercetin-PC had equilibrium solubility of 38.36 and 1351.27 μg/ml in water and chloroform, respectively, which was remarkably higher than those of quercetin alone. Then we established hydrogen peroxide (H2O2)-induced oxidative injury model in human ARPE-19 cells to examine the effects of quercetin-PC. Quercetin-PC, stronger than quercetin, promoted cell proliferation, and the proliferation rate was increased to be 78.89% when treated with Quercetin-PC at 400 μM. Moreover, quercetin-PC effectively prevented ARPE-19 cells from apoptosis, and the apoptotic rate was reduced to be 3.1% when treated with Quercetin-PC at 200 μM. In addition, quercetin-PC at 200 μM significantly increased the activities of SOD, CAT and GSH-PX, and reduced the levels of reactive oxygen species and MDA in H2O2-treated ARPE-19 cells, but quercetin at 200 μM failed to do so. Molecular examinations revealed that quercetin-PC at 200 μM significantly activated Nrf2 nuclear translocation and significantly enhanced the expression of target genes HO-1, NQO-1 and GCL by different folds at both mRNA and protein levels. Our current data collectively indicated that quercetin-PC had stronger protective effects against oxidative-induced damages in ARPE-19 cells, which was associated with activation of Nrf2 pathway and its target genes implicated in antioxidant defense.
Keywords: Age-related macular degeneration; Quercetin; Phospholipid complex; Oxidative stress; Nrf2;

Notoginsenoside R1 (NG-R1), a unique and main active ingredient of Panax notoginseng, has been described to exhibit anti-inflammatory activity. However, its protective effects against oxidized low-density lipoprotein (oxLDL)-induced inflammatory injury in vascular endothelial cells have not been clarified. In the present study, we have evaluated the anti-inflammatory effects of NG-R1 on oxLDL-induced endothelial cells and its possible molecular mechanism of action. Our results showed that NG-R1 treatment significantly attenuated oxLDL-induced expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β. These effects were accompanied with suppression of oxLDL-induced activation of NF-κB and Mitogen-activated protein kinases (MAPK). Moreover, NG-R1 also increased in Peroxisome proliferator-activated receptor γ (PPARγ) protein expression and transcription levels, and attenuated oxLDL-induced suppression of PPARγ expression. The inhibition of NG-R1 on oxLDL-induced TNF-α and IL-1β productions can be reversed by PPARγ antagonist GW9662. In conclusion, these data suggested that NG-R1 could suppress oxLDL-induced inflammatory cytokines production via activating PPARγ, which subsequently inhibiting oxLDL-induced NF-κB and MAPK activation.
Keywords: Notoginsenoside R1; Inflammatory cytokines; Oxidized low-density lipoprotein; Peroxisome proliferator-activated receptor γ; Endothelial cell;

Epigallocatechin gallate attenuates amyloid β-induced inflammation and neurotoxicity in EOC 13.31 microglia by James Cheng-Chung Wei; Hsiu-Chen Huang; Wei-Jen Chen; Chien-Ning Huang; Chiung-Huei Peng; Chih-Li Lin (16-24).
Microglia are the primary immune cells that contribute to neuroinflammation by releasing various proinflammatory cytokines and neurotoxins in the brain. Microglia-mediated neuroinflammation is one of the key characteristics of Alzheimer's disease (AD). Therefore, inhibitory reagents that prevent microglial activation may be used as potential therapeutic agents for treating AD. Recently, many studies have been performed to determine the bioactivities of green tea polyphenol epigallocatechin-3-gallate (EGCG), an efficient antioxidant that prevents neuroinflammation. However, limited information is available on the effects of EGCG on microglia-mediated neuroinflammation. In this study, we investigated the inhibitory effects of EGCG on amyloid β (Aβ)-induced microglial activation and neurotoxicity. Our results indicated that EGCG significantly suppressed the expression of tumor necrosis factor α (TNFα), interleukin-1β, interleukin-6, and inducible nitric oxide synthase (iNOS) in Aβ-stimulated EOC 13.31 microglia. EGCG also restored the levels of intracellular antioxidants nuclear erythroid-2 related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), thus inhibiting reactive oxygen species-induced nuclear factor-κB (NF-κB) activation after Aβ treatment. Furthermore, EGCG effectively protected neuro-2a neuronal cells from Aβ-mediated, microglia-induced cytotoxicity by inhibiting mitogen-activated protein kinase-dependent, Aβ-induced release of TNFα. Taken together, our findings suggested that EGCG suppressed Aβ-induced neuroinflammatory response of microglia and protected against indirect neurotoxicity. These results suggest that EGCG is a possible therapeutic agent for preventing Aβ-induced inflammatory neurodegeneration.Display Omitted
Keywords: Microglia; Amyloid β; Epigallocatechin gallate; Tumor necrosis factor α; Neuroinflammation;

Honokiol abrogates chronic restraint stress-induced cognitive impairment and depressive-like behaviour by blocking endoplasmic reticulum stress in the hippocampus of mice by Ashok Jangra; Shubham Dwivedi; Chandra Shaker Sriram; Satendra Singh Gurjar; Mohit Kwatra; Kunjbihari Sulakhiya; Chandana C. Baruah; Mangala Lahkar (25-32).
The primary objective of our study is to investigate the neuroprotective efficacy of honokiol and imipramine against restraint stress (RS)-induced cognitive impairment and depressive-like behaviour in mice. We examined whether the neuroprotective activity of honokiol and imipramine mediates through the inhibition of endoplasmic reticulum stress. Adult Swiss albino mice were restrained for 6 h/day for 28 days. Honokiol (3 and 10 mg/kg) and Imipramine (10 and 30 mg/kg) were administered for last 7 days to the different groups. Cognitive function was assessed by Morris water maze and novel object recognition test. Forced swimming test and tail suspension test were performed to evaluate the restraint stress-induced depressive-like behaviour. Proinflammatory cytokines, brain-derived neurotrophic factor, and ER stress markers i.e. 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP) were quantified in the hippocampus. We observed cognitive impairment and depressive-like behaviour in RS-exposed animals. Honokiol (10 mg/kg) treated group depicted marked reduction in cognitive impairment and depressive-like behaviour. However, imipramine (10 and 30 mg/kg) prevented the depressive-like behaviour but failed to prevent RS-induced cognitive impairment. Moreover, proinflammatory cytokines, GRP78 and CHOP were elevated in the hippocampus of stressed mice as compared to unstressed mice. Honokiol (10 mg/kg) significantly prevented the RS-induced elevated levels of proinflammatory cytokines and endoplasmic reticulum stress markers. Our results clearly suggest the beneficial potential of honokiol in restraint stress through inhibition of proinflammatory cytokines and endoplasmic reticulum stress. Honokiol could be an intriguing therapeutic approach in endoplasmic reticulum stress related neuro-pathophysiological conditions.
Keywords: Endoplasmic reticulum stress; Cognitive impairment; Restraint stress; Depressive-like behaviour; Honokiol; Imipramine;

Studies on the reproductive effects of chronic treatment with agomelatine in the rat by Sinan Canpolat; Nazife Ulker; Ahmet Yardimci; Ozgur Bulmus; Gokcen Ozdemir; Zafer Sahin; Zubeyde Ercan; Ihsan Serhatlioglu; Emine Kacar; Mete Ozcan; Gaffari Turk; Yusuf Ozkan; Murad Atmaca; Bayram Yilmaz; Haluk Kelestimur (33-39).
Agomelatine is an antidepressant with a novel mechanism of action. It is a melatonergic agonist for MT1 and MT2 receptors and a serotonin (5-HT2C) receptor antagonist. Agomelatine has been suggested not to have adverse effects on sexual functions. However, the effects of chronic agomelatine administration on reproductive functions have not been sufficiently studied in animal models. We mainly aimed to explore the effects of agomelatine on reproductive functions in the male and female rats. For the experimental studies, Sprague Dawley rats were used. The animals started to receive daily oral agomelatine (10 mg/kg) on post-natal day 21. Agomelatine advanced vaginal opening in the female rats whereas it delayed puberty onset in the male rats. Agomelatine treatment significantly decreased intromission frequencies, which indicates a facilitator role of this antidepressant on male sexual behavior. In the forced swimming test (FST) used for assessing antidepressant efficacy, agomelatine induced a significant decrease in duration of immobility, and an increase in the swimming time, respectively, which confirms the antidepressant-like activity of agomelatine. The present findings suggest that agomelatine shows a strong antidepressant effect in the male rats without any adverse influences on sexual behavior, and its effects on pubertal maturation seem to show sex-dependent differences.
Keywords: Agomelatine; Antidepressant; Sexual behavior;

Pharmacological evidence that NaHS inhibits the vasopressor responses induced by stimulation of the preganglionic sympathetic outflow in pithed rats by David Centurión; Saúl Huerta De la Cruz; Erika J. Gutiérrez-Lara; Jesús H. Beltrán-Ornelas; Araceli Sánchez-López (40-45).
It has been reported that i.v. administration of NaHS, a donor of H2S, elicited dose-dependent hypotension although the mechanisms are not completely understood. In this regard, several mechanisms could be involved including the inhibition of the vasopressor sympathetic outflow. Thus, this study was designed to determine the potential capability of NaHS to mediate inhibition of the vasopressor responses induced by preganglionic sympathetic stimulation. For this purpose, Wistar rats were anaesthetised, pithed and cannulated for drug administration. In animals pre-treated with gallamine, the effect of i.v. infusion of NaHS (310 and 560 μg/kg min) or its vehicle (phosphate buffer) was determined on the vasopressor responses induced by: (1) sympathetic stimulation (0.03–10 Hz); (2) i.v. bolus injections of exogenous noradrenaline (0.03–3 μg/kg); or (3) methoxamine (1–100 μg/kg). The vasopressor responses induced by preganglionic sympathetic stimulation were dose-dependently inhibited by i.v. infusion of NaHS (310 and 560 μg/kg min), but not by vehicle, particularly at high frequencies. In marked contrast, the vasopressor responses to exogenous noradrenaline or methoxamine were not inhibited by the above doses of NaHS or its vehicle. The above results, taken together, demonstrate that NaHS inhibited the vasopressor responses induced by preganglionic sympathetic outflow by a prejunctional mechanism. This is the first evidence demonstrating this effect by NaHS that may contribute, at least in part, to the hypotension induced by NaHS.
Keywords: Blood pressure; Cardiovascular; Hypotension; H2S; Sympathetic outflow;

Paclitaxel-induced peripheral neuropathy increases substance P release in rat spinal cord by Terumasa Chiba; Yusuke Oka; Toshie Kambe; Naoya Koizumi; Kenji Abe; Kazuyoshi Kawakami; Iku Utsunomiya; Kyoji Taguchi (46-51).
Peripheral neuropathy is a common adverse effect of paclitaxel treatment. The major dose-limiting side effect of paclitaxel is peripheral sensory neuropathy, which is characterized by painful paresthesia of the hands and feet. To analyze the contribution of substance P to the development of paclitaxel-induced mechanical hyperalgesia, substance P expression in the superficial layers of the rat spinal dorsal horn was analyzed after paclitaxel treatment. Behavioral assessment using the von Frey test and the paw thermal test showed that intraperitoneal administration of 2 and 4 mg/kg paclitaxel induced mechanical allodynia/hyperalgesia and thermal hyperalgesia 7 and 14 days after treatment. Immunohistochemistry showed that paclitaxel (4 mg/kg) treatment significantly increased substance P expression (37.6±3.7% on day 7, 43.6±4.6% on day 14) in the superficial layers of the spinal dorsal horn, whereas calcitonin gene-related peptide (CGRP) expression was unchanged. Moreover, paclitaxel (2 and 4 mg/kg) treatment significantly increased substance P release in the spinal cord on day 14. These results suggest that paclitaxel treatment increases release of substance P, but not CGRP in the superficial layers of the spinal dorsal horn and may contribute to paclitaxel-induced painful peripheral neuropathy.
Keywords: Paclitaxel; Substance P; Calcitonin gene-related peptide; Spinal cord; Peripheral neuropathic pain;

Downstream modulation of extrinsic apoptotic pathway in streptozotocin-induced Alzheimer's dementia in rats: Erythropoietin versus curcumin by Doaa M. Samy; Cherine A. Ismail; Rasha A. Nassra; Teshreen M. Zeitoun; Azhar M. Nomair (52-60).
Erythropoietin and curcumin showed promising neuroprotective effects in various models of Alzheimer’s dementia. This study was designed to compare the beneficial effects of erythropoietin and/or curcumin in intracerebro-ventricular (ICV) streptozotocin-induced Alzheimer’s like disease in rats. Rats received ICV injection of either saline (control, n=8 rats), or streptozotocin. Three weeks following surgery, streptozotocin-injected rats were assigned into 4 groups (8 rats each); vehicle, curcumin (80 mg/kg/day, orally), erythropoietin (500 IU/kg every other day, intraperitoneally) and combined (curcumin and erythropoietin)-treated groups. After 3 months of treatment, rats were subjected to neurobehavioral testing, and then killed for biochemical and histological assessment of hippocampus. Fas ligand protein and caspase-8 activity as mediators of extrinsic apoptotic pathway, oxidative stress markers (malondialdehyde and reduced glutathione) and β-amyloid (1-40 and 1-42) peptides were measured. The results showed that administration of erythropoietin suppressed extrinsic apoptosis better than curcumin, while curcumin was more effective in combating oxidative stress in ICV-streptozotocin injected rats. Both erythropoietin and curcumin treatments (individually or combined) equally reduced the hippocampal β-amyloid accumulation and improved cognitive impairment in Morris water maze and passive avoidance tasks. The combined treatment was the most effective in ameliorating apoptosis and oxidative stress rather than behavioral responses or β-amyloid burden. In conclusion, ICV-streptozotocin-induced Alzheimer’s dementia activates hippocampal Fas ligand-mediated apoptosis, which could be reduced by erythropoietin and/or curcumin treatment. Curcumin supplementation alone could ameliorate cognitive deficits and reverse biochemical alterations in ICV-streptozotocin Alzheimer’s rat model without the hazardous polycythemic effect of long-term erythropoietin injection.
Keywords: Alzheimer’s; Streptozotocin; Erythropoietin; Curcumin; Extrinsic apoptosis; Beta-amyloid peptides;

In vitro and in vivo preclinical profile of abediterol (LAS100977), an inhaled long-acting β2-adrenoceptor agonist, compared with indacaterol, olodaterol and vilanterol by Mònica Aparici; Amadeu Gavaldà; Israel Ramos; Carla Carcasona; Raquel Otal; Joan Antoni Fernández-Blanco; Jose Luís Montero; Vicente Marco García; Rosa López; Jorge De Alba; Christopher Doe; Carlos Puig; Dolors Vilella; Montserrat Miralpeix (61-69).
Abediterol is a novel long-acting β2-adrenoceptor agonist (LABA) currently in development for once-daily combination maintenance therapy of asthma and COPD. This study investigated the preclinical profile of abediterol in terms of affinity, potency, selectivity, duration of action and cardiac effects in comparison to the marketed once-daily LABAs indacaterol, olodaterol and vilanterol. Abediterol was the compound with the highest in vitro potency for dog, guinea pig and human β2-adrenoceptors. In electrical field stimulated guinea pig trachea, abediterol demonstrated 5-, 44- and 77-fold greater potency than olodaterol, indacaterol and vilanterol, respectively. In anaesthetised guinea pigs, inhaled abediterol was also the most potent compound, with 5–20 times higher bronchoprotective potency than other once-daily LABAs against acetylcholine. The bronchoprotective half-life of abediterol in guinea pigs was 36 h compared with 51 h for indacaterol, 47 h for olodaterol, and 18 h for vilanterol. In anaesthetised dogs, abediterol also inhibited acetylcholine-induced bronchoconstriction, with higher potency than olodaterol and vilanterol [ID40 (dose inhibiting bronchoconstriction by 40%) of 0.059 µg/kg, 0.180 µg/kg and 2.870 µg/kg, respectively]. In parallel, effects on heart rate in dogs were also measured. Abediterol showed greater safety index (defined as the ratio of the maximal dose without effect on heart rate and the ID40) than olodaterol and vilanterol (10.5 versus 4.9 and 2.4, respectively). Taken together, these data suggest that abediterol offers potent bronchodilation and a sustained duration of action suited to once-daily dosing, plus a reduced potential for class-related cardiac side effects.
Keywords: COPD; bronchodilator; long-acting β2-adrenoceptor agonist;

In the present study we focused our attention on the family of hydroxycarboxylic acid (HCA) receptors, a GPCR family of three members, of which the HCA2 and HCA3 receptors share 95% high sequence identity but differ considerably in C-terminus length with HCA3 having the longest tail. The two receptors were expressed and analysed for their activation profile in Saccharomyces cerevisiae MMY yeast strains that have different G protein Gα subunits. The hHCA2 receptor was promiscuous in its G protein coupling preference. In the presence of nicotinic acid the hHCA2 receptor activated almost all G protein pathways except Gαq (MMY14). However, the Gα protein coupling profile of the hHCA3 receptor was less promiscuous, as the receptor only activated Gαi1 (MMY23) and Gαi3 (MMY24) pathways.We then constructed two mutant receptors by ‘swapping’ the short (HCA2) and long (HCA3) C-terminus. The differences in HCA2 and HCA3 receptor activation and G protein selectivity were not controlled, however, by their C-terminal tails, as we observed only minor differences between mutant and corresponding wild-type receptor. This study provides new insights into the G protein coupling profiles of the HCA receptors and the function of the receptor's C terminus, which may be extended to other GPCRs.
Keywords: G protein-coupled receptor; Gα-engineered yeast cells; Hydroxycarboxylic acid receptors; HCA2; HCA3; Nicotinic acid; G protein coupling;

Antioxidant effect of 1,3,4-thiadiazolium mesoionic derivatives on isolated mitochondria by Amanda do Rocio Andrade Pires; Gustavo Jabor Gozzi; Guilhermina Rodrigues Noleto; Aurea Echevarria; Camilla Moretto Reis; Maria Eliane Merlin Rocha; Glaucia Regina Martinez; Silvia Maria Suter Correia Cadena (78-84).
Mesoionic compounds have shown antitumor and citotoxic activity against different tumor cells lines, which has been attributed to their physical and chemical characteristics. Among these compounds, the 1,3,4-thiadiazolium-2-phenylamine derivatives have been highlighted due to their important anti-melanoma activity. In this work, the effects of three derivatives that belong this class, MI-J, MI-4F and MI-2,4diF, on the oxidative stress parameters were evaluated using rat liver mitochondria. All the derivatives prevented natural and calcium induced oxidation of pyridine nucleotides at lower concentrations (6.5 and 32.5 nmol/mg protein). The calcium uptake was inhibited by all the derivatives at higher concentrations (65 and 130 nmol/mg protein), whereas the cation efflux was inhibited only by the MI-J (52%) and MI-4F (50%), possibly by inhibiting the formation of the permeability transition pore (PTP) by 100% and 50%, respectively, as observed in the same experimental conditions. MI-2,4diF did not inhibit the mitochondrial permeability transition or calcium efflux. The enzymatic activity of glutathione reductase, glutathione peroxidase and catalase was not affected by any derivative, but superoxide dismutase was inhibited by all the derivatives. MI-J inhibited enzyme activity significantly (85%) at the highest concentration (130 nmol/mg protein); on the other hand, their activity was less affected by fluorine derivatives (MI-4F-20% and MI-2,4diF-32%). These results suggest that these derivatives exert antioxidant effects on isolated mitochondria.
Keywords: 1,3,4-Thiadiazolium mesoionic derivatives; Liver mitochondria; Oxidative stress;

Mangiferin alleviates lipopolysaccharide and D-galactosamine-induced acute liver injury by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation by Chen-wei Pan; Zhen-zhen Pan; Jian-jian Hu; Wei-lai Chen; Guang-yao Zhou; Wei Lin; Ling-xiang Jin; Chang-long Xu (85-91).
Mangiferin, a glucosylxanthone from Mangifera indica, has been reported to have anti-inflammatory effects. However, the protective effects and mechanisms of mangiferin on liver injury remain unclear. This study aimed to determine the protective effects and mechanisms of mangiferin on lipopolysaccharide (LPS) and D-galactosamine (D-GalN)-induced acute liver injury. Mangiferin was given 1 h after LPS and D-GalN treatment. The results showed that mangiferin inhibited the levels of serum ALT, AST, IL-1β, TNF-α, MCP-1, and RANTES, as well as hepatic malondialdehyde (MDA) and ROS levels. Moreover, mangiferin significantly inhibited IL-1β and TNF-α production in LPS-stimulated primary hepatocytes. Mangiferin was found to up-regulate the expression of Nrf2 and HO-1 in a dose-dependent manner. Furthermore, mangiferin inhibited LPS/d-GalN-induced hepatic NLRP3, ASC, caspase-1, IL-1β and TNF-α expression. In conclusion, mangiferin protected against LPS/GalN-induced liver injury by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation.
Keywords: Mangiferin; LPS; Nrf2; NLRP3;

The present study was designed to investigate the antinociception of spirocyclopiperazinium salt compound LXM-10-M (2,4-dimethyl-9-β-m-hydroxyphenylethyl-3-oxo-6, 9-diazaspiro [5.5] undecane chloride) in thermal and chemical pain models, and further to explore the molecular target and potential signal pathway. We assessed the antinociception of LXM-10-M in hot-plate test, formalin test and acetic acid writhing test in mice. The possible changes of calcium/calmodulin-dependent protein kinase IIα (CaMKIIα)/cAMP response element-binding protein (CREB)/calcitonin gene related peptide (CGRP) signaling pathway were detected by Western Blot in mice. Administration of LXM-10-M produced significant antinociception in hot-plate test, formalin test and acetic acid writhing test in mice, with no obvious toxicity. The antinociceptive effects were blocked by pretreatment with methyllycaconitine citrate (MLA, α7 nicotinic receptor antagonist) or tropicamide (TRO, M4 muscarinic receptor antagonist). Western blot analysis showed that the upregulations of p-CaMKIIα, p-CREB and CGRP in the spinal cord were reduced by LXM-10-M in chemical pain model in mice, and the effects were blocked by MLA or TRO pretreatment. This is the first paper to report that LXM-10-M exerted significant antinociception, which may be attributed to the activation of α7 nicotinic receptor and M4 muscarinic receptor and thereby triggering the inhibition of CaMKIIα/CREB/CGRP signaling pathway in mice.
Keywords: Spirocyclopiperazinium salt compound LXM-10-M; Antinociception; α7 nicotinic receptor; M4 muscarinic receptor; CaMKIIα/CREB/CGRP signaling pathway;

Emodin improves lipid and glucose metabolism in high fat diet-induced obese mice through regulating SREBP pathway by Jinmei Li; Lili Ding; Baoliang Song; Xu Xiao; Meng Qi; Qiaoling Yang; Qiming Yang; Xiaowen Tang; Zhengtao Wang; Li Yang (99-109).
Currently, obesity has become a worldwide epidemic associated with Type 2 diabetes, dyslipidemia, cardiovascular disease and chronic metabolic diseases. Emodin is one of the active anthraquinone derivatives from Rheum palmatum and some other Chinese herbs with anti-inflammatory, anticancer and hepatoprotective properties. In the present study, we investigated the anti-obesity effects of emodin in obese mice and explore its potential pharmacological mechanisms. Male C57BL/6 mice were fed with high-fat diet for 12 weeks to induce obesity. Then the obese mice were divided into four groups randomly, HFD or emodin (40 mg/kg/day and 80 mg/kg/day) or lovastatin (30 mg/kg/ day) for another 6 weeks. Body weight and food intake were recorded every week. At the end of the treatment, the fasting blood glucose, glucose and insulin tolerance test, serum and hepatic lipid levels were assayed. The gene expressions of liver and adipose tissues were analyzed with a quantitative PCR assay. Here, we found that emodin inhibited sterol regulatory element-binding proteins (SREBPs) transactivity in huh7 cell line. Furthermore, emodin (80 mg/kg/day) treatment blocked body weight gain, decreased blood lipids, hepatic cholesterol and triglyceride content, ameliorated insulin sensitivity, and reduced the size of white and brown adipocytes. Consistently, SREBP-1 and SREBP-2 mRNA levels were significantly reduced in the liver and adipose tissue after emodin treatment. These data demonstrated that emodin could improve high-fat diet-induced obesity and associated metabolic disturbances. The underlying mechanism is probably associated with regulating SREBP pathway.
Keywords: SREBPs; Obesity; Emodin; Insulin resistance; Type 2 diabetes;

We previously reported that nerve growth factor (NGF) facilitated perivascular sympathetic neuropeptide Y (NPY)- and calcitonin gene-related peptide (CGRP)-containing nerves injured by the topical application of phenol in the rat mesenteric artery. We also demonstrated that mesenteric arterial nerves were distributed into tyrosine hydroxylase (TH)-, substance P (SP)-, and neuronal nitric oxide synthase (nNOS)-containing nerves, which had axo-axonal interactions. In the present study, we examined the effects of NGF on phenol-injured perivascular nerves, including TH-, NPY-, nNOS-, CGRP-, and SP-containing nerves, in rat mesenteric arteries in more detail. Wistar rats underwent the in vivo topical application of 10% phenol to the superior mesenteric artery, proximal to the abdominal aorta, under pentobarbital-Na anesthesia. The distribution of perivascular nerves in the mesenteric arteries of the 2nd to 3rd-order branches isolated from 8-week-old Wistar rats was investigated immunohistochemically using antibodies against TH-, NPY-, nNOS-, CGRP-, and SP-containing nerves. The topical phenol treatment markedly reduced the density of all nerves in these arteries. The administration of NGF at a dose of 20 µg/kg/day with an osmotic pump for 7 days significantly increased the density of all perivascular nerves over that of sham control levels. These results suggest that NGF facilitates the reinnervation of all perivascular nerves injured by phenol in small resistance arteries.
Keywords: Nerve growth factor; Neurotrophic effect; Perivascular nerves; Reinnervation; Phenol-induced nerve injury, Rat mesenteric artery;

In vitro bidirectional permeability studies identify pharmacokinetic limitations of NKCC1 inhibitor bumetanide by Maria D. Donovan; Harriët Schellekens; Geraldine B. Boylan; John F. Cryan; Brendan T. Griffin (117-125).
Recently, it has been suggested that bumetanide, an inhibitor of the Na–K–2Cl co-transporter (NKCC1), may be useful in the treatment of central nervous system (CNS) disorders. However, from a physicochemical perspective, bumetanide may not cross the blood–brain barrier to the extent that is necessary for it to be an effective brain NKCC1 inhibitor in vivo. High plasma–protein binding, potentially high brain–tissue binding and putative efflux transporters including organic anion transporter 3 (OAT3) contribute to the poor pharmacokinetic profile of bumetanide. Bidirectional permeability assays are an in vitro method to determine the impact of plasma-protein/brain tissue binding, as well as efflux transport, on the permeability of a compound. We established and validated a cell line stably overexpressing human OAT3 using lentiviral cloning techniques for use in in vitro bidirectional permeability assays. Using efflux transport studies, we show that bumetanide is a transported substrate of human OAT3, exhibiting a transport ratio of ≥1.5, which is attenuated by OAT3 inhibitors. Bidirectional permeability assays were carried out in the presence and absence of either albumin or brain homogenate to elucidate the effect of plasma-protein/brain tissue binding. These tests confirmed the pharmacokinetic limitations for brain delivery of bumetanide. In this experiment, bumetanide is 53% bound to albumin, 77% bound to brain tissue and accumulates in brain cells. Moreover, we conclusively established that bumetanide is a transported substrate of OAT3. Taken together, these bidirectional permeability studies highlight the potential of efflux transporter inhibition as an augmentation strategy for enhanced delivery of bumetanide to the CNS.
Keywords: Bidirectional permeability assay; Efflux transport; Bumetanide; Neonatal seizures; Pharmacokinetics; Blood–brain barrier;

Protective role of autophagy in methionine–choline deficient diet-induced advanced nonalcoholic steatohepatitis in mice by Rui Chen; Quanxing Wang; Shaohua Song; Fang Liu; Bin He; Xiaogang Gao (126-133).
The methionine choline-deficient (MCD) diet leads to severe liver injury similar to human nonalcoholic steatohepatitis (NASH). Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. The goal of this study was to elucidate the role of autophagy in MCD-induced steatosis, fibrosis, inflammation, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress in mice. Mice were fed with MCD diet and treated with rapamycin (an autophagy enhancer) or chloroquine (an autophagy inhibitor) for 10 weeks. Liver injury was evaluated biochemically and histologically together with hepatic gene expression analysis. Autophagic flux was impaired in livers of mice fed with MCD diet, evidenced by reduced ratio of LC3-II/LC3-I and increased protein expression of p62. It was found that autophagy activation by rapamycin attenuated MCD-induced steatosis, fibrosis, inflammation, mitochondrial dysfunction, and ER stress. By contrast, MCD mice treated with chloroquine developed more liver injury. In conclusions, the autophagic pathway plays an important protective role in MCD-induced advanced NASH. Thus, pharmacological promotion of autophagy may provide a novel therapeutic strategy for treatment of NASH.
Keywords: Autophagy; Nonalcoholic steatohepatitis; Methionine choline-deficient diet; Rapamycin; Chloroquine;

Carvacrol induces the apoptosis of pulmonary artery smooth muscle cells under hypoxia by Qianlong Zhang; Kai Fan; Peng Wang; Juan Yu; Ruxia Liu; Hanping Qi; Hongli Sun; Yonggang Cao (134-146).
The abnormal apoptosis of pulmonary artery smooth muscle cells (PASMCs) is an important pathophysiological process in pulmonary vascular remodeling and pulmonary arterial hypertension (PAH). Carvacrol, an essential oil compound from oregano and thyme, has displayed antimicrobial, antitumor, and antioxidant properties. Although carvacrol has pro-apoptosis properties in tumor cells, the underlying mechanisms of carvacrol in PASMC apoptosis remain unclear. Thus, in this study, we aim to investigate the role of carvacrol in pulmonary vascular remodeling and PASMC apoptosis in hypoxia. Right Ventricular Hypertrophy Measurements and pulmonary pathomorphology data show that the ratio of the heart weight/tibia length (HW/TL), the right ventricle/left ventricle plus septum (RV/LV+S) and the medial width of the pulmonary artery increased in chronic hypoxia and were reversed by carvacrol treatment under hypoxia. Additionally, carvacrol inhibited PASMC viability, attenuated oxidative stress, induced mitochondria membrane depolarization, increased the percentage of apoptotic cells, suppressed Bcl-2 expression, decreased procaspase-3 expression, promoted caspase-3 activation, and inhibited the ERK1/2 and PI3K/Akt pathway. Taken together, these findings suggest that carvacrol attenuates the pulmonary vascular remodeling and promotes PASMC apoptosis by acting on, at least in part, the intrinsic apoptotic pathway. This process might provide us new insight into the development of hypoxic pulmonary hypertension.
Keywords: Pulmonary arterial hypertension; Pulmonary artery smooth muscle cells; Carvacrol; Hypoxia; Apoptosis; Pulmonary vascular remodeling;

MrgC receptors are selectively expressed on peripheral and central terminals of small calibre nociceptive fibres. Peptide agonists of the MrgC receptor were reported to modulate nociceptive transmission exerting either pro- or antinociceptive effects depending on site of action and pain model used. Here, we investigated the effect of intraplantar and intrathecal administration of the selective MrgC receptor agonist BAM6-22 on mechanically and electrically evoked nociceptive reflex activity as a uniform readout measure in naïve, monoarthritic and mononeuropathic rats. In naïve rats, intraplantar BAM6-22 enhanced, whereas intrathecal BAM6-22 did not modulate mechanically-evoked nociceptive reflex activity. In monoarthritic rats, intraplantar BAM6-22 had no effect, whereas intrathecal BAM6-22 inhibited mechanically evoked nociceptive reflex activity. In mononeuropathic rats, BAM6-22 reduced mechanically evoked nociceptive reflex activity after both intraplantar and intrathecal administration. BAM6-22 did not modulate electrically evoked nociceptive reflex activity in any condition. Thus, the results of the present investigation confirm and add to previous studies demonstrating that site of action, (patho)-physiological state and stimulus modality determine the effect quality of MrgC receptor agonists. It still needs to be explored how concurrent activation of peripheral and spinal MrgC receptors modulates nociceptive processing under conditions of both acute and chronic pain to evaluate the therapeutic potential of putative small molecule MrgC receptor agonists as innovative analgesics.
Keywords: MrgC receptor; BAM6-22; Acute pain; Chronic pain; Nociceptive reflex;

Betaine prevented fructose-induced NAFLD by regulating LXRα/PPARα pathway and alleviating ER stress in rats by Chen-Xu Ge; Rong Yu; Min-Xuan Xu; Pei-Qin Li; Chen-Yu Fan; Jian-Mei Li; Ling-Dong Kong (154-164).
Betaine has been proven effective in treating nonalcoholic fatty liver disease (NAFLD) in animal models, however, its molecular mechanisms remain elusive. The aims of this study were to explore the mechanisms mediating the anti-inflammatory and anti-lipogenic actions of betaine in fructose-fed rats. In this study, betaine improved insulin resistance, reduced body weight gain and serum lipid levels, and prevented hepatic lipid accumulation in fructose-fed rats. It up-regulated hepatic expression of liver X receptor-alpha (LXRα) and peroxisome proliferator-activated receptor-alpha (PPARα), with the attenuation of the changes of their target genes, including hepatic carnitine palmitoyl transferase (CPT) 1α, glycosylphosphatidylinositol anchored high density lipoprotein binding protein 1, apolipoprotein B, sterol regulatory element-binding protein 1c and adipocyte differentiation-related protein, involved in fatty acid oxidation and lipid storage in these model rats. Furthermore, betaine alleviated ER stress and inhibited acetyl-CoA carboxylase α, CPT II, stearoyl-CoA desaturase 1 and fatty acid synthase expression involved in fatty acid synthesis in the liver of fructose-fed rats. Betaine suppressed hepatic gluconeogenesis in fructose-fed rats by moderating protein kinase B -forkhead box protein O1 pathway, as well as p38 mitogen-activated protein kinase and mammalian target of rapamycin activity. Moreover, betaine inhibited hepatic nuclear factor kappa B /nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 inflammasome activation-mediated inflammation in this animal model. These results demonstrated that betaine ameliorated hepatic lipid accumulation, gluconeogenesis, and inflammation through restoring LXRα and PPARα expression and alleviating ER stress in fructose-fed rats. This study provides the potential mechanisms of betaine involved in the treatment of NAFLD.
Keywords: Dietary fructose; NAFLD; Betaine; LXRα and PPARα; Hepatic ER stress;

Involvement of nitric oxide in anticompulsive-like effect of agmatine on marble-burying behaviour in mice by Nitin B. Gawali; Amrita A. Chowdhury; Pankaj S. Kothavade; Vipin D. Bulani; Dnyaneshwar M. Nagmoti; Archana R. Juvekar (165-171).
In view of the reports that nitric oxide modulates the neurotransmitters implicated in obsessive–compulsive disorder (OCD), patients with OCD exhibit higher plasma nitrate levels, and drugs useful in OCD influence nitric oxide. Agmatine is a polyamine and widely distributed in mammalian brain which interacts with nitrergic systems. Hence, the present study was carried out to understand the involvement of nitrergic systems in the anticompulsive-like effect of agmatine. We used marble-burying behaviour (MBB) of mice as the animal model of OCD, and nitric oxide levels in hippocampus (HC) and cortex homogenate were measured. Results revealed that, agmatine (20 and 40 mg/kg, i.p) significantly inhibited the MBB. Intraperitoneal administration of nitric oxide enhancers viz. nitric oxide precursor – l-arginine (l-ARG) (400 mg/kg and 800 mg/kg) increased MBB as well as brain nitrites levels, whereas treatment with NG-nitro-l-arginine methyl ester (l-NAME) neuronal nitric oxide synthase inhibitor (30 mg/kg and 50 mg/kg, i.p.) and 7-nitroindazole (7-NI) (20 mg/kg and 40 mg/kg) attenuated MBB and nitrites levels in brain. Further, in combination studies, the anticompulsive-like effect of agmatine (20 mg/kg, ip) was exacerbated by prior administration of l-ARG (400 mg/kg) and conversely l-NAME (15 mg/kg) or 7-NI (10.0 mg/kg) attenuated OCD-like behaviour with HC and cortex changes in the levels of NO. None of the above treatment had any significant influence on locomotor activity. In conclusion, Agmatine is effective in ameliorating the compulsive-like behaviour in mice which appears to be related to nitric oxide in brain.
Keywords: Agmatine; Marble burring behaviour; Nitric oxide; Obsessive compulsive disorder;