Alteration Of PI3K/Akt/mTOR Signaling And Autophagy During The Development Of Diabetic Cardiomyopathy And The Regulation Of Sirt1

Objective Diabetes(diabetes mellitus,DM)is easy to cause a variety of complications,diabetic cardiomyopathy(diabetic cardiomyopathy DCM)is a kind of complications,is an independent,specific cardiomyopathy.Diabetic hyperglycemia to cardiovascular disease,and the prognosis of patients with cardiovascular disease diabetes ability is poor,the mortality rate is very high.This topic through detection Sirtl,the expression of PI3K/Akt/m TOR signaling pathway and autophagy related protein in the myocardium in DCM rats,and by resveratrol(Res)and Nicotinamide(Nam)specific activation and inhibition of Sirt1 expression,the effect of Sirt1 on H9C2 cells cultured in high glucose medium in PI3K/Akt/m TOR signaling pathway and autophagy related protein,provide a new direction for the clinical research of DCM.Methods1.In vivo experiments1.1.DM model established40 SD rats were randomly divided into two groups: control group(Control group)and model group(DM group),the model group 30.Control normal feeding group,DM group and high fat diet,and in the 5 week after fasting 12 h in STZ injection by intraperitoneal injection of 30 mg/kg the ice bath under the condition of the current allocation,while the same volume of buffer.48 h Control rats were injected with DM rats after all rats were fasted for 24 h,with a syringe needle punctured the tail vein blood glucose strips and connect to blood glucose,fasting blood glucose detection values of all subjects(FBG).The animal model of success:compared with the control group,ISI decreased significantly,while FBG is more than 11.1mmol/L.1.2.Research process Group DM rats were randomly divided into model group of 2 weeks and 4 weeks model group,Control group and normal feeding for 4 weeks,DM group with high fat diet for 4weeks.Echocardiographic changes of heart function of rats.To evaluate anesthesia laparotomy,5m L syringe aortic blood in a centrifuge tube,separation of serum.Detection of serum FBG,TG,TC,HDL-C and LDL-C levels,ELISA rats were detected the fasting plasma insulin(FINS).Remove the heart,left heart,immunohistochemistry(immunohistochemical,IHC)detection Myocardial Sirt1 of rats,PI3 K,Akt,m TOR,p-m TOR,expression of S6K1 and LC3-II,by Western blotting(WB)to detect rat myocardial samples PI3 K,Akt,p-Akt,m TOR,p-m TOR,S6K1,LC3-II,Beclin-1 and Sirt1 protein expression changes.2.In vitro experiments2.1.Cell culture and treatment The growth of rat myocardial cells were inoculated with H9C2 in good condition in 96 Kong Banzhong,divided into normal control group(0.1%),DMSO group(HG,33 mmol/L),high glucose group,high glucose + resveratrol(Res,20 mol/L)group,high glucose +nicotinamide(Nam,40 mmol/L)group,48 h.2.2.Research process MTT method to detect cell proliferation rate of.Western blotting(WB)were detected in PI3 K,Akt,p-Akt,m TOR,p-m TOR,S6K1,LC3-II,.QRT-PCR changes were detected in PI3 K,Beclin-1 and Sirt1 protein expression of Akt,m TOR,S6K1,LC3-II,Beclin-1 and Sirt1 m RNA expression.Results1.The general characteristics of DM rats and the pathological changes Compared with the control group,DM group rats gradually depressed symptoms,and accompanied by xingsao odor and in the breeding process and found that the rats polydipsia,polyphagia,polyuria,weight loss,these are the hallmark of diabetes symptoms.The experimental rats 18 rats in group.DM FBG FINS,significantly increased,ISI decreased,CI and LVWI were higher than the control group.Using a small animal imaging system of LVIDd,LVIDs,FS,LVEF and LV Mass were detected,results show that the above value had obvious changes,the first two were significantly increased after.The three values decreased significantly.Serum TG,TC,LDL-C were significantly increased.2.Expression of Sirt1,PI3K/Akt/m TOR signaling and autophagy in DM rat myocardium WB results showed that compared with control group rats,2 weeks of DM the expression of Sirt1 in rats was significantly increased(P<0.05),PI3 K,Akt,p-Akt,m TOR,p-m TOR and S6K1 expression increased significantly(P<0.05).Compared with 2wk DM rats,DM rats Sirt1 myocardial 4wk expression increased PI3 K,Akt,m TOR and S6K1 showed no significant change,but p-Akt and p-m TOR in the expression of 4wk decreased significantly(P<0.05).Autophagy related protein II and LC3-The expression of Beclin-1 in 2 weeks and 4weeks of DM in myocardium of rats was significantly increased compared with the control group(P<0.05).3.The expression of PI3K/Akt/m TOR signaling,Sirt1 and autography-related protein in H9C2 cells MTT results showed that compared with the control group,DMSO treatment group had no significant change in cell proliferation rate(P>0.05)and high glucose group;cell proliferation rate increased significantly(P<0.05);but compared with the high glucose group,Res treatment group and Nam treatment group had no obvious changes in cell proliferation rate(P>0.05.WB).The results show that compared with the the control group,high glucose group H9C2 cells Sirt1 expression was significantly increased,Akt and m TOR showed no significant change,but p-Akt and p-m TOR expression decreased significantly.Compared with the high glucose group,Nam treatment group Sirt1 expression was significantly reduced Akt,and the expression of m TOR had no obvious change,while p-Akt and p-m TOR expression increased significantly.Compared with the high glucose group,Res treatment group significantly increased expression of Sirt1,Akt and m TOR showed no significant change,but p-Akt and p-m TOR expression decreased significantly.Compared with the control group,high glucose group and Beclin-1 LC3-II was significantly increased,compared with with the high glucose group,Nam treatment group and LC3-II Beclin-1 expression significantly decreased in Res treatment group,Beclin-1 and LC3-II.QRT-PCR expression increased significantly.To detect PI3 K,Akt,m TOR,S6K1,LC3-II,consistent with the results of WB and Beclin-1 expression of Sirt1 m RNA.Conclusion1.The rats were induced by high-fat diet joint STZ accompanied by insulin resistance, and cardiac function had a tendency to reduce gradually,along with significant myocardial hypertrophy.2.In 4wk DM group,Sirt1 and autophagy was activated,but PI3K/Akt/m TOR signaling pathway was was inhibited.These resules indicated that Sirt1,PI3K/Akt/m TOR signaling pathway and autophagy participated in the development of DCM,and we speculated that Sirt1 could regulate PI3K/Akt/m TOR signaling pathway in the development of DCM.3.In H9C2 cells,the expression of PI3K/Akt/m TOR signaling pathway was increased and the level of autophagy was decreased by inhibiting the expression of Sirt1.However,the expression of PI3K/Akt/m TOR signaling pathway was decreased and the level of autophagy was increased by activating the expression of Sirt1.These resules indicated that Sirt1 could negatively regulate PI3K/Akt/m TOR signaling in DCM and this also provides a specific inhibitor for targets on the signaling pathways.