The Role Of MicroRNAs In Ethanol-induced Rat Gastric Mucosa Injury And The Interference Of Capsinoids

Chapter1Role of miRNAs in ethanol-induced rat mucosa injuryBACKGROUNDLarge amount of alcohol consumption significantly increases acute cases of digestive diseases, such as gastric bleeding and gastric ulcer. Whereas chronic heavy drinking leads to gastrointestinal dysfunction, chronic atrophic gastritis etc. The mechanisms underlying ethanol induced gastric mucosa injury is complicated. In addition to direct chemical stimulation, it also involves cellular apoptosis and necrosis. There is a great deal of evidence that ethanol can stimulate gastric mucosa to synthesize and release various inflammatory cytokines and oxygen radicals, resulting in celluar apoptosis and necrosis. However, the exact mechanisms are not fully understood.MiRNAs are able to selectively regulate expression of target genes at post-transcriptional level. The regulation miRNAs itself expression involves in MAPKs/TRBP signal pathways. So far, at least more than30miRNAs are reported to participate in the regulation of cell apoptosis.In this study, by using the rat model of ethanol-induced mucosa injury, we explored whether the expression of apoptosis-related miRNAs were abnormal and which signaling pathways were involved. The cell model of ethanol-induced apoptosis was used to do further study. METHODS1. Animal experimentsThe SD rats were randomly divided into the control and the ethanol groups. Gastric mucosal injury was induced by oral administration of1ml60%acidified ethanol via intragastric administration.1h later, gastric tissue were collected for measurement of ulcer idex (UI, Modified Guth method), contents of MDA (TBA method) and TNF-a(ELISA), activities of catalase, SOD and caspase-3(Spectrophotometry method), apoptosis (Tunel), expression of TNF-a, miR145, miR21, miR181a, miR19a, miR17, miR200c (by real-time PCR) and protein level of TRBP, JNK/p-38/ERK (by Western blot).2. Cell experimentsThe cultured GES-1cells were pretreated by MG132(a proteasomes inhibitor), SP600125(a JNK inhibitor) or transfected by miR19a mimics, miR21mimics, miR200c mimics or miR145inhibitor, respectively. After incubation with5%ethanol for4h, the cells were collected for measurement of contents of TNF-a, activity of caspase-3, apoptosis percentage (cell flowmetry), expression of TNF-a, miR19a, miR21, miR200c, miR145and protein level of TRBP and JNK.RESULTS(1) In the rat model of ethanol-induced gastric mucosa injury, the levels of inflammatory reaction and oxidative stress were significantly increased due to the increased contents of TNF-a and MDA and the decreased activities of SOD and catalase; the ratio of apoptosis was elevated concomitantly with an increase in caspase-3acivity; the anti-apoptosis miRNAs (miR19a,miR21,miR17) were down-regualted while pro-apoptosis miRNAs (miR145) were up-regualted; the phosphorylation level of JNK was increased accompanied by downregulation of TRBP. Whereas there were no significant changes in phosphorylation levels of p38MAPK and ERK.(2) In the cell model of ethanol-induced apoptosis, ethanol-induced downregulation of TRBP was attenuated by JNK or proteasomes inhibitor; the effects of ethanol on expression of miR19a, miR21, miR17or miR145were reversed by JNK or proteasomes inhibitor; ethanol-induced cellular apoptosis was attenulated by JNK, proteasomes inhibitor or by transfection of miR19a mimics, miR21mimics or miR145inhibitorCONCLUSIONThe ethanol-induced mucosa injury is closely related to the increased levels of inflammatory reaction and oxidative stress, which is involved in abnormal expression of apoptosis-related miRNAs and JNK/TRBP pathway. Chapter2Protective effect of vanillyl nonanoate against ethanol-induced gastric mucosa injury in ratBACKGROUNDLots of drugs in treating digestive diseases depend on endogenous substances release by targeting certain receptors (such as propantheline targeting M receptor, tagamet targeting H2receptor) or depend on direct compensation of endogenous substances (such as prostaglandin E). The gastrointestinal system is rich in capsaicin-sensitive sensory nerves, which play an important role in regulation of gastrointestinal functions. Capsaicin is able to attenuate ethanol or NSAID-induced gastric mucosal injury through stimulating CGRP release by activation of capsaicin receptor (TRPV1). Due to its nerve toxicity and strong pungent, the clinical application of capsaicin (such as treating gastric ulcer) is limited.Recently, capsiate was found in CH-19Sweet peppers. It shares similar pharmacological properties with capsaicin. Since capsiate is less pungent and toxicity, it showes the tendency in replacing capsaicin for pain relief, weight loss and cancer prevention. However, due to the source monopoly and high price and lack of monomer, the research on capsiate is restricted.Vanillyl nonanoate is a simplified analogue of natural capsiate. It is usually refered as "synthetic capsite" due to its closely similar structure with capsiate. Vanillyl nonanoate maintains some properties of nature capsiate, such as less pungent and possessing anti-oxidant effect. However, it is unknown whether capsiate is able to activate TRPV1and stimulate CGRP release.In this study, by using a rat model of ethanol-induced gastric mucosal injury, we explored the questions as follow:1) whether vanillyl nonanoate was able to protect gastric mocusa against ethanol-induced injury;2) whether the beneficial effect of vanillyl nonanoate on gastric mocusa was related to stimulate CGRP release via activation of TRPV1;3) whether the apoptosis-related miRNAs and which signaling pathways were involved in this process.METHODSThe SD rats were randomly divided into8treatment groups:(i) The control group;(ii) the ethanol group;(iii) the low-dose vanillyl nonanoate group(0.3mg/kg, i.g.);(iv) the high-dose vanillyl nonanoate group(l mg/kg, i.g.);(v) the high-dose vanillyl nonanoate plus capsazepine group(5mg/kg, i.g.);(vi) the capsaicin group (0.3mg/kg, i.g.);(vii) the capsaicin plus capsazepine group and (viii) the vehicle group (0.5%carboxymethyl cellulose sodium, i.g.). All reagents were administered45min before ethanol treatment.1h after ethanol treatment, gastric tissue were collected for measurement of UI, contents of MDA, TNF-a and CGRP, activities of catalase, SOD and caspase-3, apoptosis, expression miR19a, miR21, miR17, miR145and protein level of TRBP, JNK/ERK.RESULTS(1) Administration of vanillyl nonanoate significantly reduced ethanol-induced gastric ulcer and apoptosis, these effects were attenuated by the antagonist of TRPV1.(2) Administration of vanillyl nonanoate significantly inhibited ethanol-induced production TNF-a and MDA and upregulated SOD activity, these effects were blocked by the antagonist of TRPV1.(3) Administration of vanillyl nonanoate significantly increased CGRP release, which was inhibited by the antagonist of TRPV1.(4) Administration of vanillyl nonanoate significantly reversed the effect of ethanol on expression of miR19a, miR21, miR17and miR145, these effects were attenuated by the antagonist of TRPV1.(5) Administration of vanillyl nonanoate significantly increased the phosphorylation level of ERK and reduced the phosphorylation level of JNK and the degradation of TRBP, these effects were attenuated by the antagonist of TRPV1.CONCLUSIONVanillyl nonanoate is able to protect gastric mucosa against ethanol-induced injury. The underlying mechanisms are related to stimulating the release of CGRP, which in turn reverse the abnormal expression of the apoptosis-related miRNAs and reduce the levels of inflammation and oxidative stress. Chapter3Protective effects of CGRP on ethanol-induced epithelial cells of gastric mucosa and the underlying mechanismsBACKGROUNDCGRP is a principal neurotransmitter in capsaicin-sensitive nerves. It is widely distributed in cardiovascular system and exerts beneficial effects on it. CGRP participates in mediation of cardioprotective effects afforded by heat stress, ischemia or drugs-induced myocardial preconditioning. CGRP possesses various functions such as promotion of endothelial cell proliferation, anti-apoptosis of endothelial cells and anti-senescence of endothelial progenitor cells.CGRP is also an important protective factor for gastric mucosa. The protective effects of capsaicin and vanillyl nonanoate on gastric mucosa are related to stimulation of CGRP release. In addition to increasing the blood flow in gastrointestinal tract by vascular dilation, the befeficial effects of CGRP on gastric mucosa are also involved in its action of anti-inflammation, anti-oxidant and anti-apoptosis. However, the exact mechanisms remain largely unknown.CGRP is able to inhibit isoprenaline-induced cardiomyocyte apoptosis through reduction of ROS production and reversing the effect of isoprenaline on miR-1and miR133expression. It is not known, however, that whether the protective effects of CGRP on gastric mucosa is also involved in regulation of the apoptosis-related miRNAs expression.In this study, by using the cell model of ethanol-induced apoptosis three goals were explored as follow:1) whether exogenously administration of CGRP is able to exert anti-apoptosis effect on GES-1cells;2) whether CGRP is able to regulate the apoptosis-related miRNAs expression;3) Which signal pathways are involved in the effect of CGRP.METHODS(1) To explore the effects of CGRP on ethanol-induced GES-1cell apoptosis and the possible pathways involved, the cultured GES-1cells were pretreated by CGRP, CGRP8-37or PD98059(a ERK inhibitor). After incubation with5%ethanol for4h, the cells were collected for measurement of contents of TNF-α, activity of caspase-3, apoptosis percentage, expression of miR19a, miR21, miR17, miR145.(2) To explore time-effect relationship between CGRP and expression of miR19a, miR21,miR17or miR145, the cultured GES-1cells were incubated with CGRP (10nM) for0.5,2and6h. The cells were collected for measurement of expression of miR19a, miR21, miR17or miR145.(3) To explore whether ERK/TRBP pathway is involved in the effects of CGRP on regulation of miRNAs expression, the cultured GES-1cells were incubated with CGRP for0.5or2h with or without the presence of CGRP8-37or PD98059. The cells were collected for measurement of protein levels of ERK and TRBP. RESULTS(1) Administration of CGRP significantly reduced ethanol-induced TNF-α prodution and apoptosis, these effects were attenuated in the presence of CGRP8-37and PD98059.(2) Administration of CGRP reversed the effects of ethanol on the expression of miR19a, miR21, miR17and miR145, which were blocked by CGRP8-37and PD98059.(3) Administration of CGRP significantly upregulated the expression of miR19a, miR17and miR21while downregulated the expression of miR145. The expression level of miR19a, miR17and miR21reached peak at time point of2h. The decrease of miR145expression showed a tendency in a time-dependent manner.(4) Within2h, administration of CGRP significantly increased phosphorylation level of ERK and reduce the degradation of TRBP, these effects were attenuated in the presence of CGRP8-37and PD98059.CONCLUSIONCGRP is able to protect GES-1cell against ethanol-induced apoptosis. The underlying mechanisms are involved in activation of ERK/TRBP pathway, which in turn reverse the abnormal expression of the apoptosis-related miRNAs and reduce the levels of inflammation.