The Effects And Mechanism Of Medicin-Containing Serum Of Chinese Herbal Medicinal Compound Guben Huayu Tongluo On Podocytes Injury, Apoptosis And Autophagy Induced By High Glucose

Objective: based on the view of "the podocyte damage is the key pathological change of diabetic nephropathy,abnormal autophagy is the pathological basis of podocyte damage",this study observed the situations of podocyte damage,apoptosis and autophagy in high glucose,and discuss that medicin-containing serum of chinese herbal medicinal compound Guben Huayu Tongluo whether and how to regulate podocytes abnormal autophagy by high glucose mediated,maintain the morphology and number of podocytes and reduce podocyte injury.We expect to find new prevention and treatment targets for DN.Method: According to the animal dose conversion formula of ?Pharmacological Research Methodology of Traditional Chinese Medicine?,SD male rats were given by gavage normal saline and different concentrations of Gubenhuatongtong complexing agent.The blank rat serum,twice and four times equivalent dose of medicin-containing serum of chinese herbal medicinal Compound Guben Huayu Tongluo was obtained.The podocytes in logarithmic growth phase were planted in cell culture plates,and sufficient podocyte complete medium was added.The podocytes were incubated at 37°C in a 5% CO 2 incubator and differentiated until maturity.Part 1: The podocytes were randomly divided into the control group(5.5 mmol/L glucose + normal rat serum),high glucose group(30 mmol/L glucose + normal rat serum),low-concentration medicin-containing serum group(30 mmol/ L glucose + 2 times the equivalent dose medicin-containing serum)and high-concentration medicin-containing serum group(30 mmol/L glucose + 4 times the equivalent dose medicin-containing serum).In this study,we observed the morphological changes of podocytes at 24 h,48 h,and 72 h,combined with CFSE/PI double staining experiments to detect cell mortality to determine the most suitable concentration of medicin-containing serum of chinese herbal medicinal compound Guben Huayu Tongluo to the podocyte in high glucose.Part 2: The podocytes were randomly divided into the control group(5.5 mmol/L glucose + normal rat serum),high glucose group(30 mmol/L glucose + normal rat serum)and chinese medication group(30 mmol/L glucose + medicin-containing serum).On this basis,the survival rate of cells was determined by CFSE/PI staining.The expressions of Nephrin mRNA and protein were detected with reverse transcription-polymerase chain reaction(RT-PCR)and Western Blotting method.The levels of podocytes injury and apoptosis were observed.Part 3: the expression of P62/SQSTM1 in podocytes in different culture conditions was detected by immunofluorescence.The expression of LC3 was detected by transfection of pGMAD-CMV-RFP-GFP-hLC3 adenovirus.The relative expression of mRNA of LC3 B,P62,atg5,atg7,atg12,AMPK and mTOR was detected by RT-qPCR.The protein expression of LC3-I,LC3-II,P62,Atg7,Atg12-Atg5,AMPK,p-AMPK,mTOR and p-mTOR was detected by Western Blotting.Result:Part 1: In control group,the podocytes are adhered to the bottom of the cultivated orifice,the cells are dendritic or star shaped.In high glucose group,the podocytes showed different degrees of shrinkage,Compared with the high sugar group,there was a significant improvement in the morphology of the podocytes in the low-concentration medicin-containing serum group.The protrusion of the high-concentration medicin-containing serum group was shorter,and the abscission number of podocytes was more.CFSE/PI: Low-concentration medicin-containing serum could reduce podocyte mortality caused by high glucose,while the death rate of podocyte in high concentration medicin-containing serum group was significantly higher than that in high glucose group.Therefore,we chose low concentration(2 times equivalent dose)medicin-containing serum for follow-up experiments.Part 2: At 48 h and 72 h,the apoptosis rate of podocytes in high glucose group was increased compared with the control group,while the apoptosis rate in Chinese medicine group decreased significantly compared with the high sugar group.RT-PCR and Western blotting: At each time point,the expression of nephrin mRNA and protein in podocytes of high glucose group decreased significantly compared with control group,while compared with high glucose group,the expression of nephrin mRNA and protein in podocytes of Chinese medicine group increased significantly.Part 3:(1)Immunofluorescence detection: At 24 h,the green fluorescent particles in high glucose group increased significantly compared with control group.while the green fluorescent particles in Chinese medicine group decreased compared with high glucose group(Figure 3-1).This trend became more and more obvious at 48 h and 72 h.(2)Adenovirus transfection detection: Compared with the control group,the number of yellow and red fluorescent particles in the high-sugar group was significantly decreased,and the yellow and red spots in the Chinese medicine group were significantly increased compared with the high-sugar group.(3)RT-PCR and Western Blotting detection: At three time points,the expression of mTOR mRNA and protein of LC3B?atg5?atg7?atg12?P62 and mTOR was significantly different within group comparison.But no significant changes were observed in the expression of AMPK mRNA and protein and the expression of p-mTOR protein.Conclusion:(1)The morphologica change and death of podocyte can be induced by high glucose.The using of low-concentration medicin-containing serum can improve the morphology of podocytes and reduce mortality in high-sugar environments.High-concentration medicin-containing serum showed no protective effect on podocytes in high-sugar environment,but on the contrary,resulted in an increased podocyte mortality.(2)The medicin-containing serum of chinese herbal medicinal compound Guben Huayu Tongluo can inhibit high glucose-induced podocyte injury and apoptosis,and maintain the podocyte morphology and function to a certain extent.It may be related to its regulation of podocyte abnormal autophagy.(3)The autophagic activity of podocytes were inhibited by high glucose.The medicin-containing serum of chinese herbal medicinal compound Guben Huayu Tongluo can improve abnormal autophagy in podocytes under high glucose conditions.It may regulate AMPK and mTOR signaling pathway through increasing the level of p-AMPK and decreasing the expression of mTOR,to promote the formation of autophagosome and degradation of autophagic substrates.It further inhibits podocyte injury and apoptosis,and plays a role in protecting podocytes.