The Role Of RIP 1 Mediating Autophagy Flux In Myocardial Fibrosis With Type 2 Diabetic Rats

Objective To observe the role of RIP 1 mediating autophagy flux in myocardial fibrosis with type 2 diabetic rats.Methods In vivo,4 weeks old SD male rats were used to prepare the model of myocardial fibrosis in type 2 diabetic rats.The experiment was divided into 2 groups randomly: CON group and DM group,n = 20.The DM group was fed with high fat and high glucose diet for eighth weeks,and was injected intraperitoneally with streptozotocin(STZ)solution at a dose of 50 mg/kg.At the end of twenty-fourth weeks,the rats in the two group were anesthetized and extracted from the heart.ELISA was used to detect the content of collagen I and III;immunohistochemistry was used to analyze myocardial fibrosis;RIP 1,P62,LC3 II/I and Cathepsin D protein expression were determined by western blot and immunohistochemistry.In vitro,primary cardiac fibroblasts were cultured with normal glucose(5.5 m M)or high glucose(25 m M)for different time.And according to the expression peak of RIP1 in high glucose(48 h),RIP 1 gene was silenced by lentivirus transfection.The experiment was divided into 6 groups: NG group,NG + scr sh RNA group,NG + sh RNA RIP 1 group,HG group,HG+ scr sh RNA group,HG + sh RNA RIP 1 group.ELISA was used to detect the content of collagen I and III;RIP 1,P62,LC3 II/I and Cathepsin D protein expression were determined by western blot;The changes of autophagosome and lysosome in autophagy flux were detected by double fluorescence m RFP-GFP-LC3 plasmid transfection and acridine orange(AO)fluorescence staining.Results In vivo,compared with the CON group,fasting blood glucose was increased(P < 0.05),the content of serum insulin had no change(P > 0.05),but its sensitivity was decreased(P < 0.05);Heart Weight/Body Weight was increased(P < 0.05);HE staining indicated myocardial hypertrophy and Masson staining prompted collagen deposition(P < 0.05);the expression of RIP 1,LC3 II/I,P62 and Cathepsin D were up-regulated in DM group(P < 0.05).In vitro,compared with the NG group,the expression of collagen I and III was decreased(P < 0.05);the expression of RIP 1,LC3 II/I,P62 and Cathepsin D were up-regulated in HG group;double fluorescence m RFP-GFP-LC3 plasmid transfection and acridine orange(AO)fluorescence staining suggested that the autophagosome accumulated and the lysosome integrity was destroyed in the process of autophagy flux.Compared with the HG group,the expression of collagen I and III and the expression of RIP 1,LC3 II/I,P62 and Cathepsin D were down-regulated in HG + sh RNA RIP 1 group(P < 0.05);impaired autophagy flux was decresed(P < 0.05).Conclusion Upregulation of RIP 1 can induce myocardial fibrosis in type 2 diabetic rats,and its mechanism may be through mediating autophagy flux in cardiac fibroblasts.