Relationships among alcoholic liver disease,antioxidants,and antioxidant enzymes

Excessive consumption of alcoholic beverages is a serious cause of liver disease worldwide.The metabolism of ethanol generates reactive oxygen species,which play a significant role in the deterio-ration of alcoholic liver disease(ALD).Antioxidant phytochemicals,such as polyphenols,regulate the expression of ALD-associated proteins and peptides,namely,catalase,superoxide dismutase,glutathione,glutathione peroxidase,and glutathione reductase.These plant antioxidants have electrophilic activity and may induce antioxidant enzymes via the Kelch-like ECH-associated protein 1--NF--E2--related factor--2 pathway and antioxidant responsive elements.Furthermore,these antioxidants are reported to alleviate cell injury caused by oxidants or inflammatory cytokines.These phenomena are likely induced via the regulation of mitogen--activating protein kinase(MAPK)pathways by plant antioxidants,similar to preconditioning in ischemia-reperfusion models.Although the relationship between plant antioxidants and ALD has not been adequately investigated,plant antioxidants may be preventive for ALD because of their electrophilic and regulatory activities in the MAPK pathway.

Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode

AIM： To explore the effect of Echinococcusmultilocularis on the activation of mitogen-activated protein kinase （MAPK） signaling pathways and on livercell proliferation.METHODS： Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen （PCNA）expression were measured in the liver of patients withalveolar echinococcosis （AE）. MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase （ERK）kinase] and ribosomal S6 kinase （RSK） phosphorylationwere detected in primary cultures of rat hepatocytesin contact in vitro with （1） E. multilocu/aris vesicle fluid（EmF）, （2）E. multilocularis-conditioned medium （EmCM）.RESULTS： In the liver of AE patients, ERK 1/2 andp38 MAPK were activated and PCNA expression wasincreased, especially in the vicinity of the metacestode.Upon exposure to EmF, p38, c-Jun N-terminal kinase（JNK） and ERK1/2 were also activated in hepatocytesin vitro, as well as MEK1/2 and RSK, in the absenceof any toxic effect. Upon exposure to EmCM, only JNKwas up-regulated.CONCLUSION： Previous studies have demonstratedan influence of the host on the MAPK cascade inE. multilocularis. Our data suggest that the reverse,i.e. parasite-derived signals efficiently acting onMAPK signaling pathways in host liver ceils, is actuallyoperating.

Regulation of extracellular signal-regulated kinase 1/2 influences hippocampal neuronal survival in a rat model of diabetic cerebral ischemia

Activation of extracellular signal-regulated kinase 1/2 has been demonstrated in acute brain ischemia. We hypothesized that activated extracellular signal-regulated kinase 1/2 can protect hippocampal neurons from injury in a diabetic model after cerebral ischemia/reperfusion. In this study, transient whole-brain ischemia was induced by four-vessel occlusion in normal and diabetic rats, and extracellular signal-regulated kinase 1/2 inhibitor （U0126） was administered into diabetic rats 30 minutes before ischemia as a pretreatment. Results showed that the number of surviving neurons in the hippocampal CA1 region was reduced, extracellular signal-regulated kinase 1/2 phosphorylation and KuT0 activity were decreased, and pro-apoptotic Bax expression was upregulated after intervention using U0126. These findings demonstrate that inhibition of extracellular signal-regulated kinase 1/2 activity aggravated neuronal loss in the hippocampus in a diabetic rat after cerebral ischemia/reperfusion, further decreased DNA repairing ability and ac- celerated apoptosis in hippocampal neurons. Extracellular signal-regulated kinase 1/2 activation plays a neuroprotective role in hippocampal neurons in a diabetic rat after cerebral ischemia/ reperfusion.

Metabolic derivatives of alcohol and the molecular culpritsof fibro-hepatocarcinogenesis:Allies or enemies?

Chronic intake of alcohol undoubtedly overwhelms the structural and functional capacity of the liver by initiating complex pathological events characterized by steatosis,steatohepatitis,hepatic fibrosis and cirrhosis.Subsequently,these initial pathological events are sustained and ushered into a more complex and progressive liver disease,increasing the risk of fibrohepatocarcinogenesis.These coordinated pathological events mainly result from buildup of toxic metabolic derivatives of alcohol including but not limited to acetaldehyde(AA),malondialdehyde(MDA),CYP2E1-generated reactive oxygen species,alcohol-induced gut-derived lipopolysaccharide,AA/MDA protein and DNA adducts.The metabolic derivatives of alcohol together with other comorbidity factors,including hepatitis B and C viral infections,dysregulated iron metabolism,abuse of antibiotics,schistosomiasis,toxic drug metabolites,autoimmune disease and other non-specific factors,have been shown to underlie liver diseases.In view of the multiple etiology of liver diseases,attempts to delineate the mechanism by which each etiological factor causes liver disease has always proved cumbersome if not impossible.In the case of alcoholic liver disease(ALD),it is even more cumbersome and complicated as a result of the many toxic metabolic derivatives of alcohol with their varying liver-specific toxicities.In spite of all these hurdles,researchers and experts in hepatology have strived to expand knowledge and scientific discourse,particularly on ALD and its associated complications through the medium of scientific research,reviews and commentaries.Nonetheless,the molecularmechanisms underpinning ALD,particularly those underlying toxic effects of metabolic derivatives of alcohol on parenchymal and non-parenchymal hepatic cells leading to increased risk of alcohol-induced fibrohepatocarcinogenesis,are still incompletely elucidated.In this review,we examined published scientific findings on how alcohol and its metabolic derivatives mount cellular attack on each hepatic cell and the underlying molecular mechanisms leading to disruption of core hepatic homeostatic functions which probably set the stage for the initiation and progression of ALD to fibro-hepatocarcinogenesis.We also brought to sharp focus,the complex and integrative role of transforming growth factor beta/small mothers against decapentaplegic/plasminogen activator inhibitor-1 and the mitogen activated protein kinase signaling nexus as well as their cross-signaling with toll-like receptormediated gut-dependent signaling pathways implicated in ALD and fibro-hepatocarcinogenesis.Looking into the future,it is hoped that these deliberations may stimulate new research directions on this topic and shape not only therapeutic approaches but also models for studying ALD and fibro-hepatocarcinogenesis.

Constraint-induced movement therapy promotes motor function recovery and downregulates phosphorylated extracellular regulated protein kinase expression in ischemic brain tissue of rats

Motor function impairment is a common outcome of stroke.Constraint-induced movement therapy（CIMT）involving intensive use of the impaired limb while restraining the unaffected limb is widely used to overcome the effects of＇learned non-use＇and improve limb function after stroke.However,the underlying mechanism of CIMT remains unclear.In the present study,rats were randomly divided into a middle cerebral artery occlusion（model）group,a CIMT＋model（CIMT）group,or a sham group.Restriction of the affected limb by plaster cast was performed in the CIMT and sham groups.Compared with the model group,CIMT significantly improved the forelimb functional performance in rats.By western blot assay,the expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi of cerebral ischemic rats in the CIMT group was significantly lower than that in the model group,and was similar to sham group levels.These data suggest that functional recovery after CIMT may be related to decreased expression of phosphorylated extracellular regulated protein kinase in the bilateral cortex and hippocampi.

Dendroaspis natriuretic peptide relaxes gastric antral circular smooth muscle of guinea-pig through thecGMP/cGMP-dependent protein kinase pathway

AIM: To systematically investigate if cGMP/cGMP- dependent protein kinase G (PKG) signaling pathway may participate in dendroaspis natriuretic peptide (DNP)-induced relaxation of gastric circular smooth muscle. METHODS: The content of cGMP in guinea pig gastric antral smooth muscle tissue and perfusion solution were measured using radioimmunoassay; spontaneous contraction of gastric antral circular muscles recorded using a 4-channel physiograph; and Ca2+-activated K+ currents (IK(Ca)) and spontaneous transient outward currents (STOCs) in isolated gastric antral myocytes were recorded using the whole-cell patch clamp technique. RESULTS: DNP markedly enhanced cGMP levels in gastric antral smooth muscle tissue and in the perfusion medium. DNP induced relaxation in gastricantral circular smooth muscle, which was inhibited by KT5823, a cGMP-dependent PKG inhibitor. DNP increased IK(Ca). This effect was almost completely blocked by KT5823, and partially blocked by LY83583, an inhibitor of guanylate cyclase to change the production of cGMP. DNP also increased STOCs. The effect of DNP on STOCs was abolished in the presence of KT5823, but not affected by KT-5720, a PKA-specific inhibitor. CONCLUSION: DNP activates IK(Ca) and relaxes guinea-pig gastric antral circular smooth muscle via the cGMP/PKG-dependent singling axis instead of cAMP/ PKA pathway.

Expression of DNA-dependent protein kinase in the nasopharyngeal tissue during carcinogenesis

Objective： To observe the expression of DNA-dependent protein kinase （DNA-PKcs） in the nasopharyngeal tissues during carcinogenesis. Methods： Patients including 30 cases of nasopharyngitis, 42 cases of nasopharyngeal atypical hyperplasia and 34 cases of nasopharyngeal carcinoma （NPC） were chosen from Zhongshan area in which there is high incidence rate of NPC. The expression of DNA-PKcs in the nasopharyngeal tissue was examined by immunohistochemistry. Results： The expression rates of DNA-PKcs in the nasopharyngitis tissue, the nasopharyngeal precancerous lesion and the NPC were 6.7%, 64.3% and 55.9%, respectively （P〈 0.001）. Conclusion： The expression of DNA-dependent protein kinase may play an important role in nasopharyngeal tissue carcinogenesis.

Banxia Xiexin Decoction Alleviated Cerebral Glucose Metabolism Disorder by Regulating Intestinal Microbiota in APP/PS1 Mice

Objective To identify whether Banxia Xiexin Decoction(BXD)alleviates cerebral glucose metabolism disorder by intestinal microbiota regulation in APP/PS1 mice.Methods Forty-five 3-month-old male APP/PS1 mice were divided into 3 groups using a random number table(n=15 per group),including a model group(MG),a liraglutide group(LG)and a BXD group(BG).Fifteen 3-month-old male C57BL/6J wild-type mice were used as the control group(CG).Mice in the BG were administered BXD granules by gavage at a dose of 6 g/(kg·d)for 3 months,while mice in the LG were injected intraperitoneally once daily with Liraglutide Injection(25 nmol/kg)for 3 months.Firstly,liquid chromatography with tandem-mass spectrometry was used to analyze the active components of BXD granules and the medicated serum of BXD.Then,the cognitive deficits,Aβpathological change and synaptic plasticity markers,including synaptophysin(SYP)and postsynaptic density protein 95(PSD95),were measured in APP/PS1 mice.Brain glucose uptake was detected by micropositron emission tomography.Intestinal microbial constituents were detected by 16S rRNA sequencing.The levels of intestinal glucagon-like peptide 1(GLP-1)and cerebral GLP-1 receptor(GLP-1R),as well as the phosphoinositide-3-kinase/protein kinase B/glycogen synthase kinase-3β(PI3K/Akt/GSK3β)insulin signaling pathway were determined by immunohistochemical(IHC)staining and Western blot analysis,respectively.Results BXD ameliorated cognitive deficits and Aβpathological features(P<0.01).The expressions of SYP and PSD95 in the BG were higher than those in the MG(P<0.01).Brain glucose uptake in the BG was higher than that in the MG(P<0.01).The intestinal microbial composition in the BG was partially reversed.The levels of intestinal GLP-1 in the BG were higher than those in the MG(P<0.01).Compared with the MG,the expression levels of hippocampal GLP-1R,Akt,PI3K and p-PI3K in the BG were significantly increased(P<0.01),while the levels of GSK3βwere reduced(P<0.01).Conclusion BXD exhibited protective effects against Alzheimer’s disease by regulating the gut microbiota/GLP-1/GLP-1R,enhancing PI3K/Akt/GSK3βinsulin signaling pathway,and improving brain glucose metabolism.

通便汤对STC大鼠模型结肠组织中PKA/MPKA信号通路的影响

目的:探讨通便汤对慢传输型便秘(slow transit constipation,STC)大鼠模型结肠组织中蛋白激酶A(protein kinase A,PKA)/丝裂原活化激酶(mitogen-activated protein kinase,MAPK)信号通路的影响及相关机制。方法:80只SD大鼠随机分为正常组和造模组,正常组20只,造模组60只,雌雄各半;正常组给予普通饲料喂养,模型组给予混有复方苯乙哌啶的饲料,造模时间120 d后,随机选取雌雄对半大鼠正常组10只,造模组20只,测定大鼠24 h排便量、含水量及小肠炭末推进率,观察结肠留存粪便粒数,评价STC大鼠造模是否成功;停药1周后,将造模组40只大鼠随机分为模型组,通便汤组(33 g·kg-1),通便汤+H89组(PKA信号通路阻滞剂,5 mg·kg-1),通便汤+U0126组(MAPK信号通路阻滞剂,0. 1 mg·kg-1)各10只,雌雄各半,药物通便汤干预4周后,测定大鼠24 h排便量、含水量及小肠炭末推进率,观察结肠留存粪便粒数;采用免疫组化(IHC),蛋白免疫印迹法(Western blot),实时荧光定量聚合酶链式反应(Real-time PCR)测定结肠内水通道蛋白3(AQP3),AQP4,PKA及MAPKs信号通路的蛋白及mRNA表达情况。结果:与正常组比较,造模组大鼠24 h排便量、粪便含水量、小肠炭末推进率及结肠存留粪便粒数均显著降低(P <0. 01);与模型组比较,通便汤组排便量、含水量及小肠炭末推进率均增加,结肠留存粪便粒数减少(P <0. 01),AQP3,AQP4显著降低(P <0. 01);与通便汤组比较,通便汤+H89组和通便汤+U0126组AQP3,AQP4,PKA蛋白与mRNA表达降低(P <0. 01);与通便汤+H89组比较,通便汤+U0126组排便量、含水量、小肠炭末推进率及结肠留存粪便粒数,AQP3,AQP4,PKA,MAPK蛋白表达量与mRNA含量无明显差异。结论:采用复方苯乙哌啶成功复制出慢性传输型便秘模型,通便汤可以抑制PKA和MAPK信号通路,从而下调AQP3,AQP4表达,增加肠道蠕动和肠道水分,有效治疗STC。

Salvianolate Reduces Murine Myocardial Ischemia and Reperfusion Injury via ERK1/2 Signaling Pathways in vivo

Objective： To analyze the effects of salvianolate on myocardial infarction in a murine in vivo model of ischemia and reperfusion （I/R） injury. Metheds： Myocardial I/R injury model was constructed in mice by 30 min of coronary occlusion followed by 24 h of reperfusion and pretreated with salvianolate 30 min before I/R （SAL group）. The SAL group was compared with SHAM （no I/R and no salvianolate）, I/R （no salvianolate）, and ischemia preconditioning （IPC） groups. Furthermore, an ERK1/2 inhibitor PD98059 （1 mg/kg）, and a phosphatidylinositol-3-kinase （PI3-K） inhibitor, LY294002 （7.5 mg/kg）, were administered intraperitoneal injection （i.p） for 30 min prior to salvianolate, followed by I/R surgery in LY and PD groups. By using a double staining method, the ratio of the infarct size （IS） to left ventricle （LV） and of risk region （RR） to LV were compared among the groups. Correlations between IS and RR were analyzed. Western-blot was used to detect the extracellular signal-regulated kinase 1/2 （ERK1/2） and protein kinase B （AKT） phosphorylation changes. Results： There were no significant differences between RR to LV ratio among the SHAM, I/R, IPC and SAL groups （P〉0.05）. The SAL and IPC groups had IS of 26.1% ± 1.4% and 22.3% ±2.9% of RR, respectively, both of which were significantly smaller than the I/R group （38.5% ± 2.9% of RR, P〈0.05, P〈0.01, respectively）. Moreover, the phosphorylation of ERK1/2 was increased in SAL group （P〈0.05）, while AKT had no significant change. LY294002 further reduced IS, whereas the protective role of salvianolate could be attenuated by PD98059, which increased the IS. Additionally, the IS was not linearly related to the RR （r=0.23, 0.45, 0.62, 0.17, and 0.52 in the SHAM, I/R, SAL, LY and PD groups, respectively）. Conclusion： Salvianolate could reduce myocardial I/R injury in mice in vivo, which involves an ERK1/2 pathway, but not a PI3-K signaling pathway.

Mitogen-activated protein kinase-dependent apoptosis in norcan-tharidin-treated A375-S2 cells is proceeded by the activation of protein kinase C

We have reported that norcantharidin (NCTD) induces human melanoma A375-S2cell apoptosis and that the activation of caspase and the mitochondrial pathway are involved in theapoptotic process. This study aimed at investigating the roles of mitogen-activated protein kinase(MAPK) and protein kinase C (PKC) in A375-S2 cell apoptosis induced by NCTD. We assessed theeffects of NCTD on cell growth inhibition using the 3-(4,5-dimethylthiazol-2-yl)-2 ,5-dipheyltetrazolium bromide ( MTT) assay, DNA fragmentation ( DNA agarose gel electrophoresis ) ,and MAPK protein levels (Western blot analysis) in A375-S2 cells. Photomicroscopic data were alsocollected. The NCTD inhibitory effect on A375-S2 cells was partially reversed by MAPK and PKCinhibitors. The expression of phosphorylated JNK and p38 also increased after the treatment withNCTD, and inhibitors of c-Jun NH2 - terminal kinase (JNK) and p38 ( SP600125 and SB203580,respectively) had significant inhibitory effects on the upregulation of phosphorylated JNK and p38expression. Simultaneously, the PKC inhibitor staurosporine blocked the upregulation ofphosphorylated JNK and phosphorylated p_(38), but had little effect on extracellularsignal-regulated kinase (ERK) expression. These results suggest that the activation of JNK andp_(38) MAPK promotes the process of NCTD-induced A375-S2 cell apoptosis and that PKC plays animportant regulation role in the activation of MAPKs.

Isoflurane induces expression of vascular endothelial growth factor through activating protein kinase C in myocardial cells

Objective： Vascular endothelial growth factor （VEGF） plays important roles in establishing collateral circulation of ischemic myocardium. This study aimed to investigate the effect of isoflurane on VEGF expression and the potential intracellular signal transduction pathway in cultured rat myocardial cells in order to further reveal the molecular mechanism of myocardial preservation of isoflurane. Methods： Primary myocardial cells of Sprague-Dawley rats were isolated and cultured. They were divided randomly into control group, isoflurane group, protein kinase C （PKC） inhibitor group and PKC inhibitor＋isoflurane group where cells were respectively incubated without any treatment, treated by 0.5, 1.0 and 1.5 minimum alveolar concentration （MAC） of isoflurane for 6 hours, by PKC inhibitor calphostin C at a final concentration of 50 nmol/L and by 50 nmol/L calphosfin C＋ 1.0 MAC isoflurane for 6 hours. VEGF expression was detected by enzyme-linked immunosorbent assay （ELISA） and the expression levels of PKC isoforms were determined by Western immunoblotting method. Results： Isoflurane increased the VEGF expression in myocardial cells in a dose-dependent way. VEGF levels were significantly higher in 1.0 and 1.5 MAC isoflurane groups than in the control group （both P〈0.01）. The effect of isoflurane on upregulating VEGF expression was blocked by PKC inhibitor calphostin C （P〈0.01）, but calphostin C did not alter VEGF expression （P〉0.05）. Isoflurane induced the activation and translocation of PKC Immunoblotting analysis revealed that the immunoreactivity of PKC ε increased significantly in the membrane fractions and deceased significantly in the kytoplasm fractions for cells treated with 1.0 MAC isoflurane as compared with the untreated cells, but not of PKC a, PKCα and PKCζ （P〈0.01）. Conclusion： Isoflurane induces myocardial cells to release VEGF through activating PKCε from the endochylema to the cytomembrane, suggesting a possible novel mechanism of isoflurane protecting myocardial cells.