The Research Of GushenXiezhuoHeluo Decoction Ameliorating Podocyte Mitochondrial Dysfunction Induced By High Glucose By Reducing The Expression Of Calpain 10

Objective:To explore the protective effect of Gushen Xiezhuo and Heluo Recipe(GSXZ)by observing the influence of GSXZ on the expression of calpain 10 in mitochondria of podocyte induced by high glucose,for providing the basis for the clinical application and further research of this prescription.Methods:1.Cell Grouping:The cells were divided into control group(Control):adding 5 mmol/1 glucose,high glucose group(HG):adding 30 mmol/1 glucose,mannite group(Mannite):adding 5 mmol/1 glucose+24.5 mmol/l mannite,low dose of GSXZ:adding 30 mmol/l glucose+0.5 mg/ml GSXZ,medium dose of GSXZ:adding 30 mmol/1 glucose+lmg/ml GSXZ,high dose of GSXZ:adding 30 mmol/1 glucose+2 mg/ml GSXZ.2.Detection of cell general function:The proliferation of cells was detected by CCK-8 method,the migration of cells was detected by scratch test,and the apoptosis was detected by Annexin V-EGFP fluorescence staining.3.Detection of mitochondria function:JC-1 probe was used to detect cell transmembrane potential,and flow cytometry was used to detect ROS.4.Real time PCR was used to detect expressions of calpain 10,nephrin,podocin,CD2AP,.5.Western Blot was used to detect the expression of calpain 10,nephrin,podocin,CD2AP.Results:1.The results of cell function test:①HG decreased the proliferation,migration of podocytes,and increased the apoptosis rate:compared with Control group and Mannite group,the proliferation of podocytes in HG group was significantly inhibited(P<0.01),the degree of closure decreased(P<0.01),and the apoptosis rate increased(P<0.01).②GSXZ protected podocytes injured by HG:After intervented by GSXZ,compared with HG group,the proliferation activity of podocytes was improved(P<0.05,P<0.01),the migration ability and the degree of closure was increased significantly(P<0.01);And the apoptptic rate decreased(P<0.05,P<0.01).And the effect of 2mg/ml was significant,which was concentration-dependent.2.Results of mitochondria function:①HG caused mitochondrial dysfunction in podocytes:compared with Control group and Mannite group,ROS level in podocytes of HG group was significantly increased(P<0.01),and transmembrane potential was significantly decreased(P<0.01).②GSXZ could repair the mitochondrial dysfunction of podocytes damaged by HG:compared with HG,the level of ROS in podocytes decreased(P<0.05,P<0.01)and MMP increased(P<0.05,P<0.01),and it’s concentration-dependent.3.PCR results:①podocyte injuried induced by HG:compared with Control group and Mannite group,calpain 10 mRNA expression was significantly increased in Hg group(P<0.01),nephrin,podocin mRNA expression were significantly decreased(P<0.01);②GSXZ had protective effect on the podocytes injured by HG:compared with HG,the expression of calpain 10mRNA in the podocytes of GSXZ group decreased significantly(P<0.01),and the expression of nephrin mRNA increased significantly(P<0.01).4.Western Blot Results:①podocyte injuried induced by HG:compared with Control group and Mannite group,calpain 10 protein expression was significantly increased(P<0.01),nephrin,podocin,CD2AP protein expression was decreased(P<0.01).②GSXZ had protective effect on podocytes induced by HG:compared with HG,the expression of calpain 10 protein in GSXZ group was decreased(P<0.05,P<0.01),the protein expression of nephrin,podocin and CD2AP increased(P<0.05,P<0.01).And the effect of 2mg/ml was significant,which was concentration-dependent.Conclusion:1.The proliferation activity and migration ability of podocytes can be improved and the apoptosis rate of podocytes induced by HG can be reduced by GSXZ.2.GSXZ can repair the cytoskeleton structure of podocytes,and its specific mechanism may be through enhancing the mRNA expression of nephrin and the protein expression of nephrin,podocin and CD2AP in podocytes.3.GSXZ can decrease the expression of calpain 10 mRNA and protein,reduce the level of ROS and enhancing the level of MMP in podocytes and improve the mitochondrial dysfunction of podocytes induced by HG.The mechanism may be through reducing the content and expression of calpain 10 in podocytes.4.The effect of high dose of GSXZ group is better than medium dose of GSXZ group.