Study On Protective Mechanism Of Serum Containing Yishen Jiangzhuo Granule On H₂O₂ Damage To HK-2 Cells

Objective:to elucidate the microbiological basis and therapeutic mechanism of Yishen Jiangzhuo Granules?YSJZG?for the protection of renal tubular epithelial cells from oxidative damage,so as to provide a basis for clinical administration.Methods:?1?Preparation of YSJZG drug-containing serum:30 rats in the drug-containing serum group and 30 rats in the blank serum group were given YSJZG and the same amount of normal saline respectively by gavage,and blood was taken after continuous gavage for 1 week,and the serum was separated for use.?2?Screening of H₂O₂ cell modeling concentration by MTT assay:H₂O₂ at four concentrations of 120?mol/L,250?mol/L,500?mol/L and 1,000?mol/L were used to pretreatment HK-2 cells,and the optimal modeling concentration was selected according to the relative activity of the cells.MTT assay cell Viability screening YSJZG drug-containing serum intervention conditions:YSJZG drug-containing serum?5%,10%,20%?and 10%blank serum were used to intervene model cells for 24 h,48 h,and 72 h,respectively.MTT was used to detect the relative viability of cells in each group to determine the optimal drug concentration and time.Follow-up experiments were conducted according to the optimal drug concentration,and the experimental cells were divided into:normal control group,H₂O₂ group,H₂O₂+10%blank serum group,and H₂O₂+YSJZG drug-containing serum group.?3?LDH release was used to detect cytotoxicity in each group.?4?Intracellular ROS content in each group was detected;?5?MDA content of lipid peroxidation products in each group was detected.?6?The content of SOD,an antioxidant enzyme,was detected in each group.?7?Flow cytometry was used to detect the changes of mitochondrial membrane potential and apoptosis rate in each group.?8?Relative mRNA expression levels of apoptosis-related Cyt C,Caspase-9,Caspase-3,Bcl-2and Bax in each group were detected by RT-PCR.?9?Western Blot was used to detect the protein expression levels of apoptosis-related Cyt C,Caspase-9,Caspase-3,Bcl-2 and Bax in each group.Results:?1?MTT assay results showed that the cell activity decreased with the increase of H₂O₂ concentration in an obvious dose-dependent manner.According to the relative cell activity,H₂O₂ was used to intervene HK-2 cells with 500?mol/L as the oxidative stress cell model.The cell activity of different concentrations of drug serum was significantly increased in the 3 periods.According to the relative cell activity,10%YSJZG drug serum was used for48 h as the follow-up intervention condition.?2?LDH toxicity test results showed that YSJZG serum could reduce cytotoxicity?p<0.05?.?3?The intracellular ROS content detection results showed that YSJZG drug-containing serum could reduce the production of ROS in cells?p<0.05?.?4?The detection results of intracellular MDA content showed that YSJZG drug-containing serum could reduce the intracellular MDA content?p<0.05?.?5?The results of SOD detection of antioxidant enzyme showed that YSJZG drug-containing serum could increase the content of SOD in cells?p<0.05?.?6?The test results of mitochondrial membrane potential showed that YSJZG serum could improve mitochondrial membrane potential?p<0.05?.?7?The detection results of apoptosis rate showed that YSJZG serum could reduce the apoptosis rate?p<0.05?.?8?Real-time screening Compared with the normal control group,the levels of Cyt C,Bax,Caspase-3 and Caspase-9 mRNA in the H₂O₂ group were significantly increased,while the levels of Bcl-2 mRNA were significantly decreased?p<0.05?.Compared with the H2O2 group,the levels of Cyt C,Bax,Caspase-3 and Caspase-9 mRNA in YSJZG drug-containing serum+H₂O₂ group were significantly decreased,while the levels of Bcl-2mRNA were significantly increased?p<0.05?.?9?Western Blot analysis of apoptosis-related proteins showed that compared with the normal control group,the expression levels of Cyt C,Bax,Caspase-3 and Caspase-9 proteins in the H₂O₂ group were significantly increased,while the expression levels of Bcl-2 proteins were significantly decreased?p<0.05?.Compared with the H₂O₂ group,the YSJZG drug-containing serum+H₂O₂ group showed that the protein expression levels of Cyt C,Bax,Caspase-3 and Caspase-9 were significantly decreased,while the protein expression levels of Bcl-2 were significantly increased?p<0.05?.Conclusion:?1?YSJZG drug-containing serum has a protective effect on H₂O₂ damaged HK-2 cells.The mechanism may be that ROS production in HK-2 cells is reduced and antioxidant capacity of cells is enhanced,thereby reducing oxidative damage of ROS to mitochondria and thereby reducing apoptosis rate of mitochondrial pathways.At the same time,YSJZG-containing serum also reduces the apoptosis of HK-2 caused by oxidative stress by regulating the expression levels of proteins and their mRNA related to mitochondrial apoptosis pathways.?2?The clinical efficacy of YSJZG in protecting kidney of CKD and delaying the progression of CKD may be realized through the above pharmacological mechanism.