Study On The Protective Of Astragaloside ? On Cadmium-Induced MPC5 Damage And Its Possible Mechanism

Podocytes are highly differentiated cells,distributed outside the glomerular basement membrane(GBM),and participate in the structure of the glomerular filtration barrier together with a variety of pore membrane proteins such as Nephrin,Podocin,etc.After the kidneys are stimulated,the functions of the fissure membrane protein and other functions are impaired,the number is missing,the filtration barrier is abnormal,and a large amount of proteinuria is produced.Based on this pathogenesis similar to diabetic nephropathy,more and more scholars have focused their research on the occurrence and development of DN on the mechanism of renal podocyte injury.The previous study of this research group found that exposure to heavy metal cadmium,an environmental pollutant,can accelerate the occurrence of diabetic nephropathy,but the specific pathogenesis has not been fully elucidated.DN in the field of traditional Chinese medicine is classified as edema,anti-thirst,etc.,and the traditional Chinese medicine Astragalus has the effects of diminishing water and reducing swelling,nourishing qi and promoting yang,promoting blood circulation and removing blood stasis,nourishing yin and nourishing the kidney.It is the main component of Astragalus to exert its medicinal effects.Based on the mechanism of DN oxidative stress-promoting disease,it has been proposed that Astragaloside ?protects the kidney and delays the progression of DN,but its mechanism of action has not yet been clarified.This subject intends to establish a model of cadmium-induced podocyte injury to simulate the clinical state of diabetic nephropathy,and explore the protective effect of astragaloside on it and its possible mechanism.By constructing a model of cadmium-induced damage to kidney podocytes(MPC5)in mice,to simulate podocyte damage when diabetic nephropathy occurs,comparing the number,morphology,mitochondrial status and protein of cadmium-damaged cells in normal cells,cadmium-damaged cells,and astragaloside treatment And changes in gene levels,to clarify the type of cadmium-induced MPC5 cell damage and the role of astragaloside A.The specific research contents are as follows:(1)MPC5 cell culture and establishment of cadmium-induced MPC5 cell damage model:MPC5 cells are induced with different temperatures and with or without y-interferon to make them highly differentiated,and screen differentiated and mature MPC5 cells;screen Md5 for CdCl2 to act on MPC5 Cell concentration and duration of action;Annexin V-FITC/PI combined with flow cytometry analysis of late apoptosis and necrosis of cadmium-exposed MPC5 cells;(2)Study on the mechanism of cadmium exposure-induced damage to MPC5 cells:detection of oxidative and anti-oxidation indexes of cells in the cadmium exposure group;detection of total ROS content in cells in the cadmium exposure group by flow cytometry;comparison of JC-1 labeling and flow cytometry Mitochondrial membrane potential changes in the group of cells;Fluorescence inverted microscope was used to observe the changes in the fluorescence intensity of the mitochondrial membrane potential labeled by JC-1;Western blot and RT-PCR were used to analyze the podocyte structure,functional protein,and mitochondrial autophagy pathway PINK1/Parkin in the cadmium-exposed group Expression of protein and mRNA;immunofluorescence detection of mitochondrial autophagy pathway protein by fluorescence inverted microscope;(3)Research on the protective effect of astragaloside on cadmium-induced MPC5 cells based on podocyte structure and function changes and PINK1/Parkin pathway:screening the concentration of astragaloside on cadmium-damaged cell model by MTT method;detection by flow cytometry Different concentrations of astragaloside ? acted on the mortality of cadmium-damaged cells;detection of cell oxidation and antioxidant indexes in the astragaloside group;detection of total ROS content in the cells of the astragaloside group;changes in mitochondrial membrane potential;analysis of podocyte structure in the astragaloside group,Functional protein,mitochondrial autophagy pathway PINK1/Parkin protein and mRNA expression;immunological detection to further study the mitochondrial autophagy pathway protein changes.The results showed that when MPC5 cells were exposed to CdCl2 medium containing 6.25 ?M concentration and samples were taken at 0h,12h,24h and 48h,the cell death rate under 6.25 ?M CdCl2 was lower,but with the prolongation of cadmium exposure time,MPC5 cell death rate increased.Study on the mechanism of cadmium exposure-induced damage to MPC5 cells:the MDA content of each group of cadmium exposure was significantly increased(P<0.01),and the antioxidant activity of SOD was significantly reduced(P<0.001);the ROS peak value of the cadmium exposure group was shifted,The increase level was not obvious;the mitochondrial membrane potential of each group exposed to cadmium showed a different degree of reduction;and the mitochondrial membrane potential fluorescent staining showed that cadmium exposure caused mitochondrial apoptosis;compared with group N,the expression of Nephrin protein in the cadmium exposure group decreased significantly(P<0.05),the expression of Desmin protein was significantly increased(P<0.05),the expression of autophagy protein LC3-?,the cadmium exposure group was significantly increased at 12h(P<0.001);compared with the N group,cadmium exposure was 12h and 24h The mitochondrial autophagy pathway protein PINK1 was significantly increased(P<0.05),and the 48h group was significantly higher(P<0.01).The Parkin protein decreased to a certain extent,but there was no significant difference in the degree of decline;for the mitochondrial autophagy pathway Detection of mRNA levels compared with group N,the mRNA content of PINK1 in the group exposed to cadmium for 48h was significantly increased(P<0.01);mRNA levels of Parkin were reduced;immunological detection of mitochondrial autophagy pathway proteins further showed that cadmium Induced damage to MPC5 cells may be related to dysregulation of mitochondrial autophagy.Based on podocyte structure and function changes and PINK1/Parkin pathway,we investigated the protective effect of astragaloside on cadmium-induced MPC5 cells:12.5 ?M,25 ?M,and 50 ?M concentrations of astragaloside on cadmium-damaged cells for 24 h;compared with the cadmium-exposed group,The content of MDA in the astragaloside group decreased significantly;the antioxidant index SOD activity decreased;the ROS intensity chart of the astragaloside group was closer to the blank group;the mitochondrial membrane potential of the astragaloside group increased significantly(P<0.001);compared with the cadmium exposure group Compared,the expression of podocyte structure,functional protein and mitochondrial autophagy pathway protein in the astragaloside group was significantly improved.This study shows that cadmium-induced damage to MPC5 cells is related to MPC5 cell death,mitochondrial autophagy disorder,and oxidative stress,while astragaloside ? regulates the level of mitochondrial autophagy,scavenges free radicals,and maintains the body's oxidation-antioxidation dynamic balance.Its protective effect.