The Intervention And Mechanism Of The Guben Huayu Tongluo Decoction On Autophagy In Podocytes Under High Glucose

Research background:There are more than one hundred people in our country suffer from diabetes,and nearly 1/3 of them will have diabetic nephropathy.But there are no specific drugs and treatments for the disease.The usual treatment can only relieve symptoms and delay the progression to end-stage kidney disease,but can not fundamentally prevent and reverse the course of the disease.At present,it has been confirmed that diabetic nephropathy is a kind of podocyte disease.Podocyte injury runs through the development and progression of diabetic nephropathy.Maintaining the morphology of podocyte and restoring its function is an effective target for treating diabetic nephropathy and reducing proteinuria.The Guben Huayu Tongluo Decoction is effective prescription in treating diabetic nephropathy,clinical studies have proved that it can effectively improve the clinical symptoms of patients with diabetic nephropathy,reduce urinary albumin excretion and improve renal function;in vitro experiment also suggested that the party can improve the morphology of podocytes induced by high glucose abnormalities,reduce the rate of podocyte apoptosis.However,the mechanism of the Guben Huayu Tongluo Decoction to prevent and cure diabetic nephropathy is not clear.Combined with related reports in recent years that autophagy plays an important role in podocyte injury,we speculate that autophagy is an important way for Guben Huayu Tongluo Decoction to play a therapeutic role.Therefore,this study intends to use the high glucose induced podocyte cell in vitro,and use normal rat serum and the Guben Huayu Tongluo Decoction serum to intervene,to observe the effects of Guben Huayutongluo Decoction on podocyte injury and autophagy in high glucose and provides subcellular microscopic pathological evidence for its efficacy.Research methods:1.Prepare the serum of normal rat and the Guben Huayu Tongluo Decoction by using CL grade male SD rats2.Cultured podocytes conditionally immortalized mouse podocytes,and divided into normal control group(group N),high glucose group(group H)and traditional Chinese medicine intervention group(group Z).The first two groups were intervened by normal rat serum,the third groups were treated with the serum of Guben Huayu Tongluo Decoction,and each groups were intervened 24,48,72 hours.3.Inverted phase contrast microscope was used to observe the morphological changes of the podocytes in each group.4.mRFP-GFP-LC3 fluorescence double standard adenovirus transfection was used to detect the autophagic flow in each group.5.Immunofluorescence detection of P62 protein expression in each group of podocytes6.Western-Blot the expression of autophagy related proteins LC3,P62,Atg12-Atg5,Atg7 and Sirt1.7.RT-PCR was used to detect the expression of autophagy related genes lc3 b,p62,atg5,atg7,atg12 and sirt1.Results:1.Morphological observation: Podocytes in group H showed obvious abnormal morphology in three time points,such as cell shrinkage,mutation,short foot fusion or disappeared,accompanied by a decrease in the number of cells.The cell morphology of group Z improved compared with group H,and more close to the normal group.2.mRFP-GFP-LC3 fluorescent double label: compared with group N,the number of red fluorescent spots and yellow fluorescent spots in the podocyte in group H decreased significantly in three time points.The number of two color spots in the podocyte in group Z was higher than that in the high sugar group.3.P62 immunofluorescence detection: compared with normal control group,the number of green fluorescent spots in podocytes in high glucose group increased significantly in three time points.The number of green fluorescent spots in podocyte in group Z was significantly lower than that in high glucose group.4.Western-Blot assay: Compared with group N in three time points,the expression of LC3-II,Atg12-Atg5,Atg7,Sirt1 and p-Sirt1 protein in the podocytes of group H decreased significantly.The ratio of LC3-II /LC3-I was also significantly decreased,and the expression of P62 protein increased significantly.The expression of LC3-II,Atg12-Atg5,Atg7,Sirt1 and p-Sirt1 protein in podocyte in group Z increased significantly compared with that in group H,and the ratio of LC3-II /LC3-I increased significantly,and the expression of P62 protein decreased significantly.5.RT-PCR assay: Compared with group N in three time points,the expression of lc3b?atg5?atg7?atg12 and sirt1 mRNA in the podocytes of group H decreased significantly,and the expression of p62 mRNA increased significantly.The expression of lc3b?atg5?atg7?atg12 and sirt1 mRNA in podocyte in group Z increased significantly compared with that in group H,and the expression of p62 mRNA decreased significantly.Conclusion:1.The Guben huayu tongluo decoction can improve the morphologic abnormalities of podocytes under high glucose environment,and reduce the damage of podocytes induced by high glucose.2.The Guben huayu tongluo decoction can improve the abnormal expression of LC3,P62,Atg5,Atg7 and Atg12 and promote autophagy in podocytes under high glucose.3.The Guben huayu tongluo decoction can improve the abnormal expression of Sirt1 in podocytes under high glucose.