Cytoprotection And Mechanism Of Tong Xin Formula Against On Hypoxia Injury In Rat Cardiomyocyte Line H9c2

Objective:To investigate the cytoprotection and mechanism of Tong Xin Formula（TXF）against on hypoxia injury induced by cocl₂ in rat cardiomyocyte line H9c2.Methods:The model of hypoxia injury of H9c2 cardiomyocytes was established by chemical reagent cocl₂.The experiment was randomly divided into normal control group（Control,cells without any treatment）,hypoxia group（cocl₂ hypoxic-treated cells）,Tong xin Formula group（TXF;different concentrations of TXF+cocl₂）and positive control trimetazidine groups（TMZ;trimetazidine+cocl₂）.The cell viability was tested by CCK8 method,the colorimetric method was used to detect the damage of LDH and the activity of caspase 3 was detected by spectrophotometry;Flow cytometry Annexin V-FITC/PI assay was used to detect the cell apoptosis;Loading of JC-1 probe to detect changes in mitochondrial membrane potential by immunofluorescence microscopy,and Western blot was used to detect the expression of apoptosis-related proteins such as Bcl-2,Bax,Cleaved caspase 3,and the expression of crucial proteins in the PI3K/Akt pathway.Results:1.Firstly,a reliable and relatively stable chemical hypoxia model was successfully established.The experimental results showed that the optimal simulation condition was that the cells were treated with 800μM cocl₂ for 24 h.2.The concentration of TXF in the range of 0.1-16 mg/ml had no obvious inhibitory effect on the survival rate of normal cardiomyocytes,and the TXF concentration in subsequent experiments was carried out within this range.3.Cocl₂ treatment significantly reduced the survival rate of cardiomyocytes（P<0.001）,increased the release of LDH（P<0.001）and the apoptotic rate of cells,and significantly increased the activity of Caspase 3 and the expression of Cleaved caspase3 protein（P<0.001）,suggesting that cocl₂ has a significant inhibitory effect on the viability of cardiomyocytes,increases the release of LDH and promotes cardiomyocyte apoptosis.Different concentrations of TXF（0.1,0.5,1,2 mg/ml,m/v）can significantly increase cell viability,decrease LDH release,reduce apoptosis rate,caspase 3 activity and Cleaved caspase 3 protein expression levels.The TXF at 1mg/ml had the best effect（P<0.05）.4.TXF（1mg/ml）can significantly reduce the decline of mitochondrial membrane potential in hypoxic-injured cells（P<0.001）,which is consistent with the treatment effect of positive control trimetazidine（50 M）;Both TXF and trimetazidine can up-regulate the expression of Bcl-2 protein（P<0.05）and p-Akt protein（P<0.05 or P<0.001）,and significantly reduce the expression of Cleaved caspase 3 protein（P<0.01or P<0.001）.In addition,both TXF and trimetazidine could reduce the protein expression level of Bax,but the difference was not statistically significant（P>0.05 vs.cocl₂ group）.The results showed that TXF could regulate the expression of apoptosis-related proteins,alleviate mitochondrial membrane damage and inhibit cell apoptosis,and up-regulate the expression of p-Akt protein,which is involved in the activation of PI3K/Akt signaling pathway to protect hypoxic injured cardiomyocytes.Conclusions:TXF has a protective effect on hypoxic injury of H9c2 cardiac cells induced by cocl₂.It can reduce the mitochondrial membrane potential,reduce the expression of Cleaved caspase 3 protein,and thereby reduce apoptosis of cells.The protective mechanism may be related to up-regulate the expression of Bcl-2 protein by activation of the PI3K/Akt signaling pathway.