| Objective:To explore the protective effects and mechanisms of Astragaloside?ASTs?on Arsenic trioxide?ATO?-induced myocardial injury.Methods:?1?Isolate and culture primary Wistar neonatal rat cardiomyocytes,use ATO to establish the cardiomyocyte injury model and use ASTs to intervene.The effect of ASTs on the morphology of cardiomyocytes was observed under an inverted microscope.The viability of cardiomyocytes was measured using the CCK-8 method;Detect the activity of Lactic Dehydrogenase?LDH?,Creatine Kinase?CK?and Glutamic oxaloacetic transaminase?GOT?in cell culture fluid using a fully automatic biochemical analyzer;use kit method to detect the activity of Superoxide Dismutase?SOD?,Glutathione peroxidase?GSH-Px?and Catalase?CAT?in cardiomyocytes;Hoechst 33342fluorescent probe was used to detect the degree of cardiomyocyte apoptosis;flow cytometry was used to determine the rate of cardiomyocyte apoptosis;Western Blotting was used to detect p-AKt,AKt,Nrf2,HO-1 Bcl-2,Bax,Caspase-9,Caspase-3 expression levels;real-time fluorescence quantitative-polymerase chain reaction?RT-q PCR?method to detect PI3K,Akt,Nrf2,HO-1,Bcl-2,Bax,Caspase-9,Caspase-3 m RNA expression levels.?2?Fifty Wistar rats were randomly divided into normal control group,ATO model group,ASTs low,medium and high dose groups,with 10 rats in each group.Rats in the model group were given intragastric administration of ATO solution to induce myocardial injury at a dose of 5mg·kg-1·d-1.The ASTs low,medium,and high dose groups were given intragastric administration of the ASTs solution at the same time as the model,and the doses were 10mg·kg-1·d-1,20mg·kg-1·d-1and40mg·kg-1·d-1.Rats in the normal control group were given the same volume of drug vehicle by gavage.Monitor the body weight of the rats during the period of drug administration,take blood from the abdominal aorta 14 days later,and measure the viability of LDH,CK,GOT in the rat serum with a fully automatic biochemical analyzer.The rats were sacrificed to weigh the heart and calculate the heart-body ratio;Observe the myocardial injury in rats at the pathological level by HE staining of myocardial tissue;Using kit method to detect the activity of SOD,GSH-Px,CAT in rat myocardium;Western Blotting method was used to detect the expression levels of p-AKt,AKt,Nrf2,HO-1,Bcl-2,Bax,Caspase-9,Caspase-3 in myocardial tissue;The expression levels of PI3K,AKt,Nrf2,HO-1,Bcl-2,Bax,Caspase-9,and Caspase-3m RNA in myocardial tissue were detected by RT-q PCR.Results:?1?Compared with the blank control group,the activity of cardiomyocytes in the model group was significantly reduced,and the activity of LDH,CK,and GOT in the cell culture fluid was significantly increased?P<0.05?,The activities of SOD,GSH-Px and CAT in cardiomyocytes were significantly reduced?P<0.05?,and the apoptosis rate was significantly increased?P<0.05?,Nrf2 and HO-1 protein expression levels were significantly reduced?P<0.05?,Caspase-9 and Caspase-3 protein expression levels were significantly increased?P<0.05?,and p-AKt/AKt and Bcl-2/Bax ratios were significantly reduced?P<0.05?,PI3K,Akt,Nrf2,HO-1,Bcl-2 m RNA expression levels decreased significantly?P<0.05?,Caspase-9,Caspase-3,Bax m RNA expression levels increased significantly?P<0.05?.Compared with the model group,the activity of cardiomyocytes in each concentration group of ASTs was enhanced,the activity of LDH,CK,and GOT in cell culture fluid was significantly reduced?P<0.05?,and the activity of SOD,GSH-Px,and CAT in cardiomyocytes was significantly increased?P<0.05?,the apoptosis rate is significantly reduced?P<0.05?,and has a certain concentration dependence,In addition,the expression levels of Nrf2 and HO-1 protein in the high-concentration ASTs group were significantly increased?P<0.05?,the expression levels of Caspase-9 and Caspase-3 protein were significantly reduced?P<0.05?,p-AKt/AKt,Bcl-2/Bax ratio increased significantly?P<0.05?,PI3K,Akt,Nrf2,HO-1,Bcl-2 m RNA expression levels increased significantly?P<0.05?,Caspase-9 Caspase-3,Bax m RNA The expression level was significantly reduced?P<0.05?;Compared with the ASTs high concentration group,the expression levels of Nrf2 and HO-1 protein in LY294002?PI3K inhibitor?group decreased significantly?P<0.05?,and the expression levels of Caspase-9 and Caspase-3 protein increased significantly?P<0.05?,AKt/AKt,Bcl-2/Bax ratio decreased significantly?P<0.05?,PI3K,AKt,Nrf2,HO-1,Bcl-2 m RNA expression levels decreased significantly?P<0.05?,Caspase-9,Caspase-3,Bax m RNA expression level increased significantly?P<0.05?.?2?ATO model group showed large necrosis of myocardial tissue,severe destruction of muscle fibers,body weight and heart-body ratio decreased significantly?P<0.05?,serum LDH,CK,GOT vitality increased significantly?P<0.05?,myocardium The vitality of SOD,GSH-Px and CAT in the tissue decreased significantly?P<0.05?,The expression levels of Nrf2 and HO-1 protein in myocardial tissue decreased significantly?P<0.05?,the ratios of p-AKt/AKt and Bcl-2/Bax decreased significantly?P<0.05?,and the expression levels of Caspase-9 and Caspase-3protein were significant Increased?P<0.05?,PI3K,AKt,Nrf2,HO-1,Bcl-2 m RNA expression levels decreased significantly?P<0.05?,Caspase-9,Caspase-3,Bax m RNA expression levels increased significantly?P<0.05?;Compared with the model group,each dose group of ASTs significantly improved the state of myocardial tissue lesions,body weight and heart-to-body ratio were significantly increased?P<0.05?,serum LDH,CK,GOT activity was significantly reduced?P<0.05?,myocardial tissue The activity of SOD,GSH-Px,CAT was significantly increased?P<0.05?,and there was a certain dose dependence,The expression levels of Nrf2and HO-1 protein in myocardial tissue were significantly increased?P<0.05?,the ratios of p-AKt/AKt and Bcl-2/Bax were significantly increased?P<0.05?,and the expression of Caspase-9 and Caspase-3 protein significantly decreased?P<0.05?,PI3K,AKT,Nrf2,HO-1,Bcl-2 m RNA expression levels increased significantly?P<0.05?,Bax,Caspase-9,Caspase-3m RNA expression levels decreased significantly?P<0.05?.Conclusion:ASTs have a certain protective effect on ATO-induced myocardial injury,and this effect is related to its regulation of PI3K/AKt/Nrf2 signaling pathway and mitochondrial apoptosis signaling pathway. |
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