The Protective Effect And Mechanism Of HSP22 On The Kidney In Type 2 Diabetic Mice

Objective:To investigate the effect and mechanism of HSP22 on kidney fibrosis,oxidative stress and tissue damage in diabetic kidney injury.Methods:Animal:1.The diabetic mouse model was established by STZ induced type 2 diabetes,and randomly divided into 4 groups: wild control group(WT-control group),wild diabetes group(WT-diabetes group),HSP22 knockout control group(KO-control group)and HSP22 knockout diabetes group(KO-diabetes group).2.H&E staining and PAS staining were used to observe the pathological changes of kidney tissues in each group.3.Massson staining was used to observe the changes of kidney fibrosis in each group.4.The expression level of HSP22,COL3 in kidney tissues of mice in each group were detected using Western blot.5.The expression level of HSP22,COL3,TGFB1,STAT3,p-STAT3,Smad3 and p-Smad3 in kidney tissues of mice in each group were detected using immunohistochemistry.6.DHE staining,SOD activity and MDA content were used to detect the levels of oxidative stress in kidney tissues of mice in each group.7.The expression of STAT3 and Smad3 in kidney tissues of diabetic mice was detected by immunofluorescence assay.8.The interaction between STAT3 and Smad3 in kidney tissue of diabetic mice was detected by immunoprecipitation.Cell:1.HK-2 cells were stimulated by 33.3 mmol/L glucose for 24 h to establish the hyperglycemic cell model.Meanwhile,33.3 mmol/L mannitol was used to set the hyperosmolar cell model and 1 mmol/L glucose was used as the control.The cells were divided into 3 groups: Control group,Osmotic control(OC)group and High glucose(HG)group,respectively.In addition,si RNA was used to interfere the gene expression of HSP22 in HK-2 cells,which was set to the following 5 groups: Control group,Scramble si RNA group,Scramble si RNA+HG group,HSP22 si RNA group and HSP22 si RNA+HG group.2.Immunofluorescent staining were used to observe the expression level of HSP22 in HK-2 cells.3.The expression level of HSP22,COL3,TGFB1,STAT3,p-STAT3,Smad3 and p-Smad3 in kidney tissues of mice in each group were detected using Western blot.4.The changes of oxidative stress level in HK-2 were detected by SOD enzyme activity and MDA content.Results:Animal:1.The results of H&E staining showed that,compared with the control group,the glomerular structure of the diabetic group was disordered and the renal tubule was atrophy;After the knockout of HSP22 gene,renal tissue structure damage in the KO-diabetes group was more serious than that in the WT-diabetes group.The results of PAS staining and glomerular sclerosis index showed that the glomerular sclerosis index was significantly increased in the WT-diabetes group compared with the WT-control group.After the knockout of HSP22 gene,the glomerular sclerosis index was further increased in the KO-diabetes group compared with the WT-diabetes group.2.The results of Masson staining showed that collagen was deposited between kidney tissues in the WT-diabetes group compared with the WT-control group.After the knockout of HSP22 gene,collagen fiber deposition was more obvious in KO-diabetes group.3.The results of immunohistochemical and Western blot of renal tissue: 1)The expression of COL3 and TGFB1: the expression levels of COL3 and TGFB1 in the kidney of the diabetic group were significantly increased compared with that of the control group,and the protein expression levels of COL3 and TGFB1 in the diabetic kidney after the HSP22 gene knockout were higher between the two diabetic groups.2)The expression of STAT3,p-STAT3,Smad3 and p-Smad3: the phosphorylation levels of STAT3 and Smad3 were significantly increased in the kidney of the diabetic group compared with the control group,and phosphorylation levels of STAT3 and Smad3 were significantly increased in the kidney of the diabetic group after the HSP22 knockout.4.The results of DHE staining showed that ROS accumulation in the kidney of diabetic mice significantly increased after the gene HSP22 knockout.The results of SOD enzyme activity and MDA content showed that,compared with WT-diabetes group,the activity of SOD in the kidney of diabetic mice after HSP22 knockout was significantly decreased;nevertheless,the content of MDA was significantly increased.5.The results of STAT3 and Smad3 immunofluorescence staining showed that,compared with the WT-diabetes group,the KO-diabetes group presented more co-expression in the same renal tissue region,mainly in the renal tubule region.6.The co-immunoprecipitation results of STAT3 and Smad3 showed that the interaction between STAT3 and Smad3 was more obvious after the gene HSP22 knockout.Cell:1.The expression levels of HSP22,COL and TGFB1 were increased after high glucose intervention in HK-2 cells.2.The results of Western blot of high glucose HK-2 cell: compared with the Scramble si RNA+HG group,the expression levels of HSP22 and fibrosis factors(COL3 and TGFB1)were significantly increased in the HSP22 si RNA+HG group.3.The results of SOD activity and MDA content in the high-glucose HK-2 cell model showed that,compared with the control group and the Scramble si RNA group,the SOD activity in HK-2 cells after high glucose intervention showed a decreasing trend,while the SOD activity further decreased after HSP22 si RNA interference in the HK-2 cell.Conclusion:1.HSP22 played an endogenous protective role in the pathogenesis of renal injury caused by diabetes.2.HSP22 deficiency promoted fibrosis and oxidative stress in diabetic kidney.3.The deficiency of HSP22 may promote the interaction of STAT3 and SAMD3 signals and play a role in the occurrence and progression of diabetic nephropathy.