Effects Of Qufeng Bushen Recipe Regulating AMPK-mTOR Signal On The Mechanism Of High Glucose-induced Podocyte Injury

Research BackgroundDiabetic nephropathy is the most common microvascular complication in diabetic complications,and its incidence in diabetic patients is 20% to40%,which is already the single most important cause of end-stage renal disease.The study found that changes in the glomerular filtration barrier,especially in which the podocyte damage due to autophagy play a key role in the pathological changes of DN.Several studies have shown that the regulation of related signal pathways such as AMPK,mTOR,p53,JNK,and SIRT1 are involved in autophagy.Changes in key proteins on these signaling pathways play an important role in the development of diabetic nephropathy.Based on the above studies,this project uses ex vivo cultured glomerular mouse podocytes as experimental subjects,and uses molecular biology methods to study the effect of Qufeng Bushen Recipe regulating AMPK-mTOR signaling on improving high glucose-induced podocyte injury,in order to discover The Effect of Feng Bushen Recipe on Diabetic Nephropathy.In order to better simulate the effect of Hufeng Bushen Fang in the body,the extraction of drug-containing animal serum was used as experimental drug.PurposeBy observing the effect of Qufeng Bushen Recipe on the growth of conditional immortalized podocytes and the expression of AMPK-mTOR signaling pathway and related proteins,the molecular mechanism of Qufeng Bushen Recipe in improving diabetic nephropathy was explored.Method1.Effect of hyperglycemia and hyperosmia on podocyte growth and related protein expressionThe podocytes were cultured in vitro and MTT was used to screen high glucose concentration and duration of action.The experiment was divided into three groups: the normal group(5.6 mmol/L glucose)as the control group and the mannitol group(5.6 mmol/L glucose+19.4 mmol/L mannitol)as Osmotic control group,high glucose group(25 mmol/L glucose).After 24 hours of podocyte culture,the apoptosis of three groups of cells was detected by flow cytometry,and the effect of hyperglycemia and hyperoxia on apoptosis was analyzed.Western blot was used to detect AMPK,P-AMPK,mTOR,P-mTOR and Beclin-1 in each group.,Nephrin,Podocin protein expression.2.The effect of high glucose on AMPK-mTOR signaling pathway on the expression of podocyte-related proteinsThe experiment was divided into five groups: high glucose group as the control group,AMPK agonist group,mTOR inhibitor group,AMPK inhibitor group,and AMPK agonist + mTOR inhibitor group.All the five groups were under high glucose conditions.After cultured conditional immortalized podocytes in vitro for 24 hours,the expression of Beclin-1,Nephrin and Podocin proteins in each group was detected by Western Blot.3.The Effect of Qufeng Bushen Recipe on the Growth and Injury of Podocytes Induced by High GlucoseThe podocytes were cultured in vitro and MTT was used to test the effect of different drug-containing serum on the survival rate of podocytes.Two groups were selected for experiments.The experiment was divided into four groups: normal group,high glucose group,low dose group of Qufeng Bushenfang,and high dose group of Qufeng Bushenfang.Flow cytometry was used to detect the apoptosis of the four groups of cells.The formation of autophagosomes in each group was observed under transmission electron microscope,and the autophagy of each group was observed.4.The Effect of Qufeng Bushen Recipe on High Glucose Induced Podocyte Protein ExpressionThe experiment was divided into five groups: normal group,high glucose group,low-dose group of Qufeng Bushenfang,low-dose + AMPKinhibitor group of Hufeng Bushenfang and AMPK inhibitor group.After podocytes were cultured in vitro for 24 h,the expression of AMPK,P-AMPK,mTOR,P-mTOR,Beclin-1,Nephrin and Podocin were detected by Western Blot.Result1.MTT results showed that when podocytes were added for 24 hours,glucose concentration was 25 mmol/L,high glucose had moderate damage to podocytes,and the model was stable.Flow cytometry showed that there was no significant apoptosis rate between normal group and mannitol group.The difference was that the high-glucose apoptotic rate was significantly increased;Western Blot results showed that the expression of Beclin-1 in the high-glucose group was significantly higher than that in the normal group,and the expression of Nephrin and Podocin was relatively decreased,and the ratio of P-AMPK/AMPK was decreased.P-mTOR/mTOR The ratio increased.There was no significant difference between the mannitol group and the normal group.2.Western Blot results showed that the expression of Beclin-1,Nephrin and Podocin in the higher glucose group was significantly increased in the AMPK agonist,mTOR inhibitor group and AMPK agonist +mTOR inhibitor group,especially in the AMPK agonist + mTOR inhibitor group.In the AMPK inhibitor group,the expression of Beclin-1,Nephrin and Podocin in the higher glucose group decreased.3.Flow cytometry results showed that the number of apoptotic cells in the low-dose and high-dose podocytes of Qufeng Bushenfang was higher in the sugar group,and the low dose was superior to the high dose.The results of transmission electron microscopy showed that the autophagosomes in the high-glucose group increased compared with the normal group,while the high-dose and high-dose autophagosomes in the high-dose group had a significant increase.4.Western Blot results showed that the high-dose group of Qifeng Bushenfang up-regulated the expression of Beclin-1,Nephrin,Podocin and P-mTOR/mTOR,and the expression of P-AMPK/AMPK decreased.In the low-dose +AMPK inhibitor group,the expression of Beclin-1,Nephrin,Podocin,and P-mTOR/mTOR in the low dose group of Qufeng Bushenfang was decreased,and the P-AMPK/AMPK ratio was decreased.Conclusion1.Podocyte can produce autophagy under the stimulation of high glucose,and through the AMPK-mTOR signaling pathway to enhance podocyte autophagy,can reduce the podocyte injury.2.Qufeng Bushen Recipe can enhance autophagy of podocytes under high-glucose and reduce podocyte injury.3.Qufeng Bushen Recipe may pass the AMPK-mTOR signaling pathway,which promotes podocyte autophagy to protect podocytes and improve high glucose-induced podocyte injury.