The Regulation And Mechanism Of Vaspin In Treatment Of Diabetic Cardiomyopathy

Part I The regulation and mechanism of TNF-?-induced cardiomyocytes injury Objective1.To investigate the effects and mechanisms of TNF-?-induced H9C2 cells injury.2.To explore the effects and mechanisms of vaspin on TNF-?-induced H9C2 cells injury.Methods1.A TNF-?-induced H9C2 cells injury model was established to explore the effects of TNF-? on H9C2 cells at different time pionts.Cells were divided into the following four groups:(1)Control group: cells were cultured for 24 h;(2)TNF-? 4 h group: cells were cultured with 20 ng/ml TNF-? for 4 h;(3)TNF-? 12 h group: cells were cultured with 20 ng/ml TNF-? for 12 h;(4)TNF-? 24 h group: cells were cultured with 20 ng/ml TNF-? for 24 h;At the end of the experiment,the morphological changes of the cells were observed by microscopy,and the expression of LC3,Beclin-1,Bax and Bcl-2 protein were examined by western blot.2.To explore the causal relationship between apoptosis and autophagy induced by TNF-? in H9C2 cells.Cells were divided into the following four groups:(1)Control group: cells were cultured for 24 h;(2)TNF-? group: cells were cultured with 20 ng/ml TNF-? for 24 h;(3)TNF-? + rapamycin(Rap)group: cells were pretreated with Rap(100 nm)for 2 h and then co-incubated with Rap and TNF-? for24 h;(4)TNF-? + 3-MA group: cells were pretreated with 3-MA(5 mm)for 2 h and then co-incubated with 3-MA and TNF-? for 24 h.At the end of the experiment,the morphological changes of the cells were observed by microscopy,the expression of LC3,Beclin-1,Bax and Bcl-2 protein were examined by western blot,and the apoptosis of H9C2 cells measured with flow cytometry.3.To study the effect of vaspin on TNF-?-induced H9C2 cells injury.Cells were divided into the following five groups:(1)Control group: cells were cultured for 24 h;(2)Vaspin group: cells were cultured with 100 ng/ml vaspin for 24 h;(3)TNF-?group: cells were cultured with 20 ng/ml TNF-? for 24 h;(4)TNF-? + vaspin group:cells were pretreated with vaspin(100 ng/ml)for 2 h and then co-incubated with vaspin and TNF-? for 24 h;(5)TNF-? + vaspin + IGF-1 group: cells were pretreated with IGF-1(100 ng/ml)for 3 h and pretreated with vaspin for 2 h,and then co-incubated with IGF-1,vaspin and TNF-? for 24 h.Then perform next experiments:(1)Cell survival measured by MTT assay and assessment of ATP content;(2)The apoptosis of H9C2 cells measured with flow cytometry;(3)The expression of LC3,Beclin-1,Bax,Bcl-2,p-AKT,p-mTOR,AKT and mTOR protein were examined by western blot;(4)The autophagosomes were observed by transmission electron microscopy and MDC staining.Results1.TNF-? promoted cell apoptosis and inhibited autophagy in H9C2 cells.Compared with those in the control group,Bax expression increased and Bcl-2expression decreased in the treatment group at almost all time points.The ratio of Bax/Bcl-2 increased in a time-dependent manner.The LC3-II/LC3-I ratio and Beclin-1 expression decreased while P62 expression increased in each treatment group of different time points.2.Autophagy inhibition aggravated TNF-?-induced apoptosis while higher autophagy level mitigated TNF-?-induced apoptosis.Western blotting analysis showed that 3-MA downregulated the protein expression of Beclin-1,lowered the LC3-II/LC3-I ratio,and increased Bax/Bcl-2 ratio.On the other side,Rap treatment upregulated the protein expression of Beclin-1,increased the LC3-II/LC3-I ratio,and lowered the Bax/Bcl-2 ratio(P<0.01).Flow cytometry results showed that the number of early apoptotic cells in TNF-? group increased(P<0.05).In general,Rap treatment reduced the number of early apoptotic cells,while 3-MA treatment led to more apoptotic cells(P<0.05).3.Vaspin improved cell viability after the cells were treated with vaspin with concentration higher than 100 ng/ml,but 10 to 50 ng/ml vaspin didn't have significant impact.The intracellular ATP content measurement confirmed that ATP content dropped significantly in the TNF-? group and increased in the vaspin group(more than 100 ng/ml),but the difference of ATP content was not statisticallysignificant when the vaspin concentration was between10 and 50 ng/ml.Western blotting analysis suggested that the Bax/Bcl-2 ratio in vaspin group was not different from that of the control group while the ratio in the vaspin pretreated group declined compared with that in the TNF-? group.AnnexinV-FITC/PI double staining assay further confirmed that apoptosis cells showed no difference between the control group and the vaspin group while vaspin treatment after TNF-? stimulation reduced the number of early apoptotic cells.4.As shown in western blotting,the LC3-II/LC3-I ratio and Beclin-1 expression increased in the vaspin group,decreased in the TNF-? group and increased in the vaspin-pretreated group(P<0.01).TEM showed that,compared with that of the control group,the number of autophagosomes was significant higher in the vaspin group and lower in the TNF-? group(P<0.05).The number of autophagosomes in the vaspin-pretreated group was even higher than that in the TNF-? group(P<0.05).MDC staining suggested that the content of intracellular autophagic vesicles showed the same trend as TEM did.5.Compared with those of control group,the ratio of LC3-II/LC3-I increased and ratios of p-AKT/AKT and p-mTOR/mTOR decreased in vaspin group(P<0.01).Moreover,compared with those of TNF-? group,vaspin pretreatment increased the LC3-II/LC3-I ratio and decreased the ratios of p-AKT/AKT and p-m TOR/mTOR(P<0.01).However,the PI3K/AKT pathway agonist IGF-1 improved the expressions of p-AKT,p-mTOR and the ratio of Bax/Bcl-2 but lowered the ratio of LC3-II/LC3-I(P<0.01).Conclusions1.TNF-? induced H9C2 cells apoptosis through inhibiting autophagy.2.Vaspin attenuated the TNF-?-induced apoptosis by promoting autophagy through inhibiting the PI3K/AKT/mTOR pathway.Part II The effect of vaspin on myocardial fibrosis and cardiac function in diabetic cardiomyopathy rats ObjectiveTo investigate the effects of vaspin on myocardial fibrosis and cardiac function in diabetic cardiomyopathy rats.Methods:Male SD rats(body weight around 200 g and age about 10 weeks)were kept in clean cages for 1 week to adapt to the environment and then used in the experiments.Diabetic cardiomyopathy(DCM)was induced by a single intraperitoneal injection with Streptozotocin(60 mg/kg body weight)solution in 0.1 mol/l citrate buffer(pH adjusted to 4.2).Two weeks later,blood samples were collected from tail vein in both groups and were analyzed for blood glucose using a glucometer.Rats were considered to have diabetes if their fasting blood glucose levels were higher than 16.7mmol/l 2 weeks after STZ injection.SD rats were divided randomly into 3 groups,control group(n=12),DCM group(n=12),and vaspin group(n=12).Rats in control group were simply treated with normal saline and in vaspin group were injected intraperitoneally with vaspin(320 ng/kg/day)solution in saline daily for 12 weeks after diabetes was developed.Then perform next experiments:(1)The day before the experiment,echocardiography was performed to assess LVEF,LVFS,LVIDd,LVIDs and Em/Am;(2)Serum TNF-? concentration was measured by ELISA;(3)Masson's trichrome was performed to assess cardiac fibrosis in each group at end of experiment.(4)HE staining was used to detect the size and arrangement of myocardial cells in each group.(5)The expressions of LC3,Beclin-1,P62,Bax,Bcl-2,TGF-? and collagen I in the myocardium were examined by Western blot at end of experiment.Results1.Compared with those of control group,the fasting blood glucose(BFS)increased while the body weight decreased in DCM group(P<0.01),which was partially reversed by vaspin treatment(P<0.01).2.As for TNF-? levels,there was no statistically significant difference between the DCM group and vaspin group(P>0.05),but showing statistically significantdifference between the DCM and control group(P<0.01).3.Echocardiography was performed to assess the effect of vaspin on cardiac function and showed that,LVEF and FS decreased in DCM group while vaspin improved LVEF and FS levels(P<0.01),the ratio of Em/Am decreased in DCM group while vaspin did improve this ratio(P<0.01).4.Western blot analysis showed that the LC3-II/LC3-I ratio and Beclin-1expression decreased and P62 expression increased in DCM group and vaspin treatment reversed these changes(P<0.01).5.Diabetes disordered the arrangement of myocardial cells and rescued by vaspin.Compared with DCM group,collagen volume fraction(CVF)in the vaspin group decreased(P<0.05).Western blot suggested that vaspin could reduce the expression of collagen I(P<0.01),but had no significant effect on the expression of TGF-?1(P>0.05).ConclusionsVaspin could increase the level of autophagy,inhibit myocardial fibrosis and improve cardiac function in diabetic cardiomyopathy rats.