The Mechanism Of MTOR Pathway In Cardiac Insulin Resistance Induced By Ethanol

ObjectivesUnder the background of urbanization and aging process in China,the prevalence of cardiovascular disease(CVD)has been increasing and it has become the top of the ten major cause of death in the world.In recent years,studies have shown that the change in dietary structure,including the change of drinking behavior,is one of the important cause of high incidence of carfiovascular disease.In addition to the role of high fat and high sugar diet in the development of cardiovascular disease,long term excessive alcohol intake is closely related to cardiovascular disease.Come so far,whether in the world or in China,non-rational drinking has become a prominent social problem,and it is difficult to overcome in the short term.Irrational drinking will cause alcoholic cardiomyopathy(ACM),with the main pathological features of myocardial hypertrophy and myocardial contractility dysfunction,accounts for 23%?40%of the total cardiomyopathy.At the same time,the relationship between alcohol and myocardial insulin resistance as well as alcoholic cardiomyopathy is getting more and more concerned by the domestic and overseas scholars.Studies have shown that insulin resistance is associated with early cardiac dysfunction,and the detection of insulin sensitivity can be used as a screening indicator.It is still unclear about the correlation between ethanol and myocardial insulin sensitivity as well as the mechanism of related pathway response.The purpose of this study is to explore the mechanism of mTOR signaling pathway in the myocardial insulin resistance induced by ethanol,in order to provide the theoretical basis for the prevention and treatment of myocardial insulin resistance.Methods1.Cell culture and treatment:H9c2 rat cardiomyocytes were cultured in DMEM culture medium containing 10%fetal bovine serum,and add 1%penicillin-streptomycin.The cells were cultured in the culture box at 37?.Cells in ethanol stimulated group were treated with ethanol at 100mM concentration for 24 hours,before the subsequent experiments.2.The effect of ethanol treatment on the relative vitality of H9c2 cells was detected by MTT test.The glucose uptake ability of H9c2 cells was detected by 2-NBDG uptake test,fluorescence intensity was observed using the fluorescence microscope,the difference of fluorescence intensity in each group was quantitatively analyzed by flow cytometry.The protein level of p-IRS-1(serine 307)was detected by Western blot.3.Real-time quantitative PCR and Western blot were used to detece the mRNA and protein level of the upstream molecules of mTOR signaling pathway(Rictor and Rheb)as well as the downstream molecule of mTOR signaling pathway(S6k2).4.Lentivirus mediated RNA interference was used to silence S6k2 expression at the gene level,the effect of S6k2 silence on glucose metabolism in H9c2 cells was detected by glucose oxidase method.The protein level of p-IRS-1(serine 307)was detected by Western blot.Results1.Effects of different concentrations of ethanol on the activity of H9c2 cells.Compared with the control group,the relative activity of H9c2 cells that treated with 50mM and 100mM concentration of ethanol were not significantly inhibited,while the relative activity of H9c2 cells that treated with 200mM,400mM and 800mM concentration of ethanol were reduced by 33.1%(P<0.01)for 200mM concentration,63.2%(P<0.01)for 400mM concentration and 75.7%(P<0.01)for 800mM concentration.2.Ethanol stimulation reduced glucose uptake and increased p-IRS-1(Ser307)expression in H9c2 rat cardiomyocytes.H9c2 cells cultured with 2-NBDG for 30 min,observed in fluorescence microscopy and found that compared with the blank control group,the 2-NBDG group could observe the obvious green fluorescence under the microscope,and the spontaneous fluorescence of H9c2 cardiomyocytes was weak,but the fluorescence intensity of the myocardial cells after ethanol stimulation was weaker than that of the non ethyl alcohol stimulation group.After the quantitative analysis of mean fluorescence intensity,it was found that the average fluorescence intensity of the group without ethanol stimulation was 46.5%higher than that of the ethanol stimulation group(P<0.05).Western blot results showed that p-IRS-1(Ser307)level increased by 14.4%(P<0.05)after ethanol stimulation compared with the control group without ethanol stimulation.3.Ethanol stimulates up-grading Rictor,Rheb and S6k2 expression in mTOR signaling pathway.Real-time quantitative PCR results showed that compared with the control group without ethanol stimulation,the mRNA expression levels of Rictor,Rheb and S6k2 in the ethanol stimulation group increased significantly,and increased by 42.4%(P<0.05),49.5%(P<0.05),and 16.8%(P<0.01),respectively.Western blot results showed that ethanol stimulation significantly increased the protein expression levels of Rictor,Rheb and S6k2 by 23.2%(P<0.05),20.4%(P<0.05)and 23.5%(P<0.05),respectively.4.S6k2 silencing inhibited the reduced of glucose uptake and the up-regulated expression of p-IRS-1(Ser307)induced by ethanol.After transfected with lentivirus mediated RNA interference in H9c2 cardiomyocytes,It was found that glucose uptake in LV-CON group decreased by 8.08%(P<0.05)after ethanol stimulation.Ethanol stimulation did not show a significant difference in glucose uptake compared with that of alcohol in LV-S6k2-RNAi group(P>0.05).Western blot results showed that the protein expression of p-IRS-1(Ser307)decreased by 33.2%(P<0.05)in group of LV-S6k2-RNAi compared with that in LV-CON group.After S6k2 silencing of H9c2 cardiac myocytes,stimulated the cells with 100mM concentration of ethanol for 24h,the protein expression of p-IRS-1(Ser307)was slightly higher,but there was no statistical significance(P>0.05).Conclusions1.High dose of ethanol stimulation reduced the relative activity of H9c2 cells.2.Ethanol stimulation reduced the glucose uptake of H9c2 cells and induced myocardial insulin resistance.3.Ethanol stimulation increased the expression of Rictor,Rheb and S6k2 in mTOR signaling pathway of myocardial.4.S6k2 silencing alleviated the glucose metabolism disturbance and myocardial insulin resistance induced by ethanol.