Effect And Mechanism Of Er-xian Decoction Onmyocardial Ischemia-Reperfusion Injury In Ovariectomized Rats

Aim:In this study,the chemical constituents of Er-xian Decoction were qualitatively and quantitatively analyzed by UHPLC/Q-TOF-MS and HPLC.Replication of myocardial ischemia-reperfusion injury?MIRI?model in ovariectomized rats by ovariectomy?Ovx?combined with left anterior descending coronary artery ligation and reperfusion?I/R?,The effect and mechanism of Er-xian Decoction on myocardial ischemia-reperfusion injury in ovariectomized rats were studied by intragastric administration of Er-xian Decoction?EXD?.The oxidative stress model of primary cardiomyocytes was activated by H₂O₂,and mangiferin was given to investigate the effect of mangiferin on oxidative stress injury of primary cardiomyocytes.Method:1?UHPLC/Q-TOF-MS and HPLC techniques were used to qualitatively and quantitatively analyze the active constituents in Er-xian Decoction.The data were collected by electrospray ionization?ESI?ion source positive and negative ion mode,and the chemical composition retention time,precise relative molecular weight and two level mass spectrometry fragment ion information provided by Peakview mass spectrometry software were combined with chemicalbook and chemspider chemical information retrieval to establish the chemical composition database analysis data.2?SPF female Sprague-Dawley rats were randomly divided into groups according to their body weight.Except for the sham group,the other groups were subjected to ovarian ablation.The rats were successfully screened by vaginal smearand divided into ovariectomized group?Ovx?and ovariectomized+Ischemia-reperfusion group?Ovx+I/R?,Estradiol Valerate group(E2V,0.8mg·kg^-1),Er-xian Decoction low dose group(EXD/L,4g·kg^-1),Er-xian Decoction Middle group(EXD/M,8g·kg^-1),Er-xian Decoction High group(EXD/H,12g·kg^-1).The ischemia-reperfusion model was established after 80 days of intragastric administration in the above groups.Except for the Sham group and the Ovx group,the left anterior descending coronary artery?LAD?was ligated and reperfused.After the ligation,the ST segment of the electrocardiogram was lifted up,the left ventricular wall was cyanotic,pale,and the ST segment was re-infused,and the ventricular wall was restored to rosy for the ischemia-reperfusion injury model.3?Evaluation of Er-xian Decoction in preventing and treating myocardial ischemia-reperfusion injury in ovariectomized rats:The electrocardiogram of normal ovariectomized rats and rats after ischemia-reperfusion were collected in lead II to compare the effects of Er-xian Decoction on electrocardiogram of rats at different stages.Evans Blue and TTC staining were used to detect the area of myocardial ischemia and infarction in rats.The serum levels of glutamic oxaloacetic transaminase?AST?,lactate dehydrogenase?LDH?,creatine kinase-MB?CK-MB?,estradiol?E2?and superoxide dismutase?SOD?and malondialdehyde?MDA?in rats were measured by biochemical method and enzyme-linked immunosorbent assay?Elisa?.Pathological section HE staining of rat myocardial tissue and uterus.Detection of Bcl2,Bax and estrogen receptor alpha?ER??protein expression in rat myocardial tissue by Western Blot.4?Effect of mangiferin on oxidative stress injury of cardiomyocytes:The cardiomyocytes were isolated and purified by enzymatic hydrolysis and differential adherence.They were divided into blank group,model group,resveratrol group?Res,1?M?and mangiferin low dose groups?75?M?,middle dose group?150?M?and high dose group?300?M?,after 24h treatment,except for the blank group,H₂O₂ 400?M was added for 1h to replicate the oxidative stress injury model of cardiomyocytes.The effect of mangiferin on the activity of cardiomyocytes was detected by MTT assay.The contents of lactate dehydrogenase?LDH?,aspartate aminotransferase?AST?and superoxide dismutase?SOD?and malondialdehyde?MDA?in cardiomyocytes were detected by kit.The levels of Ca²⁺and ROS in cardiomyocytes were detected by Fluo-3AM and DCFH-DA fluorescent probes.The apoptosis of each group was detected by AnnexinV-FITC/PI double staining flow cytometry.Western Blot was used to detect the expression of apoptosis-related protein Bax and Pten/Akt/Foxo1 pathway-related proteins in cardiomyocytes.Result:1?Through data analysis,72 compounds were identified in Er-xian decoction,including 28flavonoids,4 saponins,6 anthraquinones,11 Phenolic glycosides,3 alkaloids,5 organic acids and 15 others.2?The electrocardiogram of the rats showed that,compared with the Sham group,the amplitudes of P wave,R wave and T wave in the Ovx group and Ovx+I/R group were significantly decreased?P<0.01?,PR interval,QRS time course,The QT interval was shortened to varying degrees;compared with the Ovx+I/R group,the pathological changes of ECG in the EXD group and the E2V group were significantly improved.Compared with the electrocardiogram of rats after ischemia-reperfusion,there was no significant change in heart rate and ST segment amplitude in Ovx group and Sham group.Compared with Ovx group,Ovx+I/R group was electrocardiogram in LAD ligation and reperfusion stage,the ST segment was significantly uplifted?P<0.01?.Compared with the Ovx+I/R group,the extent of ST segment elevation in the EXD group and the E2V group was significantly decreased?P<0.05?.Evans Blue and TTC staining showed that there was no significant change in myocardial ischemic area and infarct area,Sham group and Ovx group in rats.Compared with Ovx group,myocardial ischemic area and infarct area in Ovx+I/R group The increase was significantly increased?P<0.01?.Compared with Ovx+I/R group,the area of myocardial ischemic area and infarct area in EXD group and E2V group was significantly decreased?P<0.05,P<0.01?.The results of kit test showed that the contents of AST,LDH,CK-MB in serum and SOD in myocardium of rats in Sham group had no significant change compared with Ovx group;compared with Ovx group,the contents of AST,LDH and CK-MB in serum of rats in Ovx+I/R group increased significantly?P<0.05,P<0.01?,the contents of MDA and SOD in myocardium of rats increased significantly?P<0.05?;compared with Ovx+I/R group,the contents of SOD in EXD group and E2 V group decreased significantly?P<0.05?.The levels of AST,LDH,CK-MB in serum and MDA in myocardium of rats decreased to different degrees,while the levels of SOD increased significantly?P<0.05,P<0.01?.Elisa test results showed that compared with Sham group,the serum E2 level in Ovx group and Ovx+I/R group decreased significantly?P<0.01?;compared with Ovx+I/R group,the E2 level in EXD/M and EXD/H group increased significantly?P<0.01?.Myocardial histopathological sections showed that the pathological changes of myocardial tissue in the EXD group were significantly improved,and the myocardial fibers and myocardial cells were arranged neatly,the muscle gap was significantly reduced,and the myocardial fibers were less broken,but some were wavy.Rat uterus pictures,pathological sections and Western Blot results showed that compared with the Sham group,the uterus of the Ovx group and the Ovx+I/R group was significantly dry and wrinkled,and the HE staining endometrial layer became thinner and the number of glands was obvious.The expression of ER?protein in myocardium was significantly decreased?P<0.01?.Compared with Ovx+I/R group,the EXD and E2V groups in each dose group could significantly reverse the pathological changes of the uterus and increase the myocardial tissue ER?.Protein.Western Blot results showed that there was no significant difference in Bcl2 and Bax protein expression between Sham group and Ovx group.Compared with Ovx group,Bcl2protein expression in Ovx+I/R group was significantly up-regulated?P<0.01?,Bax protein expression was significantly down-regulated?P<0.01?;compared with Ovx+I/R group,Bcl2protein expression in EXD group and E2V group was significantly up-regulated?P<0.01?,and Bax protein expression was significantly down-regulated?P<0.01?.3?Effect of mangiferin on oxidative stress injury in cardiomyocytes:MTT results showed that compared with the blank group,the model group significantly inhibited the viability of cardiomyocytes?P<0.01?;compared with the model group,the mangiferin group was able to the inhibitory effect of H₂O₂ on the activity of cardiomyocytes was improved to different extent?P<0.05?.The results of kit test showed that,compared with the blank group,the contents of AST,LDH and MDA in the cardiomyocytes supernatant of the model group significantly increased?P<0.05,P<0.01?,and the contents of SOD in the cells significantly decreased?P<0.01?;Compared with the model group,the contents of AST,LDH and MDA in cardiomyocytes decreased in different degrees in mangiferin group?P<0.05,P<0.01?,and the contents of SOD in cardiomyocytes significantly increased?P<0.05,P<0.01?.The Fluo-3AM and DCFH-DA fluorescent probes showed that,the levels of calcium ion and ROS in the model group were significantly increased compared with the blank group?P<0.01?.Compared with the model group,the contents of calcium ion and ROS in the cardiomyocytes of mangiferin group decreased in varying degrees?P<0.05,P<0.01?.The results of flow cytometry showed that the apoptotic rate of cardiomyocytes in the model group was significantly higher than that in the blank group?P<0.01?.Compared with the model group,the apoptosis of cardiomyocytes in the mangiferin-administered group significantly reduced?P<0.01?.The results of Western Blot showed that compared with the blank group,the expression of Bax,Aktand p-Pten in the primary cardiomyocytes of the model group was significantly up-regulated?P<0.01?,and the expression of p-Akt and p-Foxo1 was down-regulated?P<0.01?;Compared with the model group,the mangiferin group significantly down-regulated the expression of Bax,Akt and p-PTEN?P<0.05,P<0.01?,and the mangiferin group regulated the expression of p-Akt and p-Foxo1 to varying degrees?P<0.05,P<0.01?.There was no significant difference in the expression of total protein of PTEN and Foxo1 between the groups.Conclusion:1?Er-xian decoction contains more chemical components,a total of 72 compounds,mainly divided into flavonoids,saponins,terpenoids,alkaloids and so on,among which mangoside,icariin,ferulic acid and berberine hydrochloride are relatively high.2?Er-xian Decoction reverses the pathological changes ofrat ECG caused by the decrease of estrogen level by regulating the level of estrogen in rats,and maintains the protective effect of estrogen on the heart.Er-xian Decoction has anti-oxidation effect and reduces myocardial damage caused by ischemia-reperfusion by increasing the activity of antioxidant enzymes.3?Mangiferin reduces the adverse state of cardiomyocytes produced by oxidative stress,reduces the damage and apoptosis of cardiomyocytes,reduces the number of oxygen free radicals in cardiomyocytes,enhances the activity of antioxidant enzymes in cardiomyocytes,and reduces calcium overload,thereby alleviating oxidative stress damage in cardiomyocytes.