Effect And Mechanism Of GLP-1,7-36a And Its Metabolite 9-36a On Autophagy And Apoptosis Of Renal Podocytes

Objective: Glucagon-like peptide(GLP-1)is a gut peptide hormone.GLP-1 is a new drug for the treatment of diabetes in recent years.7-36 a is its naturally occurring form,9-36 a is its major metabolically active product.The GLP-1 receptor can be expressed in the kidney,and GLP-1 can protect the kidney by activating the receptor,and it is not clear whether the metabolite has a protective effect on the kidney and how it works.The aim of this study was to investigate whether GLP-1,7-36 a and its metabolite 9-36 a are sufficient for podocytes by observing whether autophagy and apoptosis levels are affected by high glucose and inflammatory factors in podocytes.The role and mechanism of phagocytosis and apoptosis provide new ideas for the prevention and treatment of diabetic nephropathy.Method: Human renal podocytes cultured in vitro were divided into NC group,HG group,HG+9-36 a group,HG+7-36 a group,TNF-? group,TNF-?+9-36 a group and TNF-?+7-36 a group.The first part of the study was to extract protein from each cell group twenty-four hours after adding treatment factors and determine the concentration.The autophagyrelated proteins mTOR,Phospho-mTOR,Beclin-1,LC3,ATG 5 and ATG 16L1 were detected by Western Blotting.The differences of protein expression were compared among the groups.SPSS 23.0 was used for statistical analysis.The second part: Forty-eight hours after the addition of treatment factors,the apoptotic cells of each group were detected by TUNEL method.Five visual fields were taken from each sample.The percentage of red fluorescent labeled cells in the total number of cells in the visual field was counted as the percentage of apoptotic cells.SPSS 23.0 was used for statistical analysis.Results:The first part of studies:1.Effects of GLP-1,7-36 a and 9-36 a on podocytes treated with high glucose(1)Compared with NC group,the expression of LC3,Beclin1 and Phospho-mTOR increased in HG group(P < 0.05),while the expression of LC3,Beclin1 and PhosphomTOR did not change significantly in HG + 9-36 a group and HG + 7-36 a group(P > 0.05);compared with HG group,the expression of LC3,Beclin1 and Phospho-mTOR decreased in HG + 9-36 a group and HG + 7-36 a group(P < 0.05);the expression of LC3,Beclin1 and Phospho-mTOR did not change significantly in HG + 9-36 a group and HG + 7-36 a group(P > 0.05).(2)Compared with NC group,the expression of ATG 5 increased in HG group,HG+9-36 a group and HG+7-36 a group(P<0.05);compared with HG group,the expression of ATG 5 did not change significantly in HG+9-36 a group and HG+7-36 a group(P>0.05).(3)Compared with NC group,the expression of ATG 16L1 in HG group,HG + 9-36 a group and HG + 7-36 a group had no significant change(P > 0.05).2.Effects of GLP-1,7-36 a and 9-36 a on podocytes treated with TNF-?(1)Compared with NC group,the expression of LC3,Beclin1 and Phospho-mTOR increased in TNF-? group(P < 0.05),while the expression of LC3,Beclin1 and PhosphomTOR did not change significantly in TNF-?+ 9-36 a group and TNF-?+7-36 a group(P > 0.05);compared with TNF-? group,the expression of LC3,Beclin1 and Phospho-mTOR decreased in TNF-?+ 9-36 a group and TNF-?+ 7-36 a group(P < 0.05);the expression of LC3,Beclin1 and Phospho-mTOR did not change significantly in TNF-?+ 9-36 a group and TNF-?+ 7-36 a group(P > 0.05).(2)Compared with NC group,the expression of ATG 5 increased in TNF-?group,TNF-?+9-36 a group and TNF-?+7-36 a group(P<0.05);compared with TNF-? group,the expression of ATG-5 did not change significantly in TNF-?+9-36 a group and TNF-?+7-36 a group(P>0.05).(3)Compared with NC group,the expression of ATG 16L1 in TNF-? group,TNF-?+ 9-36 a group and TNF-?+ 7-36 a group had no significant change(P > 0.05).The second part of studies:(1)Compared with NC group,the percentage of apoptotic cells in NC(+)group and HG group increased(P < 0.05).Compared with NC(+),the percentage of apoptotic cells in NC group,HG group,HG + 9-36 a group and HG + 7-36 a group decreased(P < 0.05);compared with HG group,the percentage of apoptotic cells in HG + 9-36 a group and HG + 7-36 a group decreased(P < 0.05).(2)Compared with NC group,the percentage of apoptotic cells in NC(+)group and TNF-? group increased(P < 0.05).Compared with NC(+),the percentage of apoptotic cells in NC group,TNF-? group,TNF-? + 9-36 a group and TNF-? + 7-36 a group decreased(P < 0.05);compared with TNF-? group,the percentage of apoptotic cells in TNF-? + 9-36 a group and TNF-? + 7-36 a group decreased(P < 0.05).Conclusion:(1)High glucose and TNF-? can activate podocyte autophagy and induce apoptosis.(2)GLP-1,7-36 a and its metabolite 9-36 a can inhibit podocyte autophagy induced by high glucose and inflammatory factors and reduce podocyte apoptosis.