Experimental Study Of Aconitum Chishizhi Pills Against Myocardial Ischemia-reperfusion Injury In Rats

Objective:In this study,a model of myocardial ischemia-reperfusion injury in rats was established to observe the related indexes of ECG,coagulation function,endothelia cells,serum kinase,energy metabolism,oxidative stress and Keap1-Nrf2 signaling pathway in MIRI rats.To investigate the protective effect of Aconitum Chishihzi Pill Recipe on myocardial ischemia-reperfusion injury（myocardialischemia/reperfusion,MIRI）in rats with arrhythmias,with a view to providing a good service for clinical treatment of myocardial ischemia-reperfusion injury.METHODS:40 SD rats were randomly divided into normal group,model group,positive drug group（urine kinase group）and Aconitum Chishizhi Pill Recipe group.The MIRI model was established except for the normal group.The experiment observes the changes of ECG after reperfusion in rats.The pathological changes of cardiomyocytes in rats were observed by HE staining.Serum CK and LDH levels,the contents of endothelin-1（ET-1）,thromboxane A2（TXA₂）,prostacyclin（PGI₂）,myocardium superoxide dismutase（SOD）,malondialdehyde（MDA）and glutathione peroxidase（GSH-Px）in endothelial cells were determined by ELISA.The changes of four indexes of coagulation（Prothrombin time PT,activated partial thromboplastin time APTT,thrombin time TT and fibrinogen FIB）were determined by coagulometer instrument.The effects of Na⁺-K⁺ATPase and Ca²⁺-Mg²⁺-ATPase levels in myocardial muscle tissues were determined by spectrophotometry.Western blot assay for the detection of oxidative stress protein Keap1 and Nrf2 expression levels in rat cardiomyocytes.Result:1.Effect of Aconitum Chishizhi Pill Recipe on ECG Score of Arrhythmia in MIRI RatsThe results showed that compared with the model group,the arrhythmia score of the urokinase group was slightly decreased,but there was no statistical difference,the arrhythmia score of the Aconitum Chishizhi Pill Recipe group was significantly decreased（P<0.05）.Compared with the model group,the arrhythmia score of the urokinase group was decreased during the reperfusion period,which was statistically significant（P<0.05）.The arrhythmia score of the Aconitum Chishizhi Pill Recipe group was significantly reduced during the reperfusion period,and the difference was significant（P<0.01）.The result confirmed that the Aconitum Chishizhi Pill Recipe group showed a better trend of arrhythmia protection in MIRI rats than in the urokinase group.2.Effect of Aconitum Chishizhi Pills Recipe on Pathological Changes in MIRI RatsIn the normal group,the myocardial cells were arranged neatly,the fibers were intact,the cells were aplastic and necrotic.In the MIRI group,the cardiomyocytes were disordered,the fibers were incomplete and the cell volume increased.The cardiomyocytes were irregularly arranged in the urokinase group and the Aconitum chishizhi Pill Recipe group,but significantly improved compared with the MIRI group,the cardiomyocytes were partially broken,the cell volume was reduced,the interstitial swelling was reduced,and the inflammatory cells had less exudation.3.Effect of Aconitum Chishizhi Pills Recipeon on four clotting items（PT、TT、APTT、FIB）in MIRI ratsCompared with the normal group,the PT,APTT,TT in the model group shortened significantly,and the FIB content increased significantly（P<0.01）.Compared with the model group,the PT,APTT and TT in urokinase group were prolonged,and the FIB was significantly decreased（P<0.05,P<0.01）,the difference is statistically significant.Aconitum chishizhi Pill Recipe group prolonged the PT,TT,and the FIB content decreased significantly（P<0.05,P<0.01）.And the APTT was slightly extended,but the difference was not statistically significant.4.Effects of Aconitum Chishizhi Pills Recipe on Serum PGI₂、TXA₂、ET-1 content inMIRI ratsCompared with the normal group,the model group results showed that serum PGI₂ were significantly decreased,TXA₂ and ET-1 were significantly increased after myocardial ischemia（P<0.01）.Compared with the model group,the PGI₂ were significantly increased,and TXA₂ and ET-1 were significantly decreased in the urokinase group and Aconitum chishizhi Pill Recipe group（P<0.05,P<0.01）.5.Effect of Aconitum Chishi Pills Recipe on Serum CK,LDH and Na⁺-K⁺ATPase,Ca²⁺-Mg²⁺-ATPase Activity of myocardial tissue in MIRI ratsCompared with the normal group,the model group results showed that the levels of CK and LDH in serum of MIRI rats were significantly increased,the activity of Na⁺-K⁺ATPase and Ca²⁺-Mg²⁺-ATPase in myocardial tissue were significantly decreased（P<0.01）.Compared with the model group,Aconitum Chishizhi Pill Recipe group and urokinase group significantly reduced the levels of CK and LDH in serum of MIRI rats,and elevated Na⁺-K⁺ATPase and Ca²⁺-Mg²⁺-ATPase activity in myocardial tissues（P<0.05,P<0.01）.6.The effect of Aconitum Chishizhi Pill Recipe on SOD,MDA,GSH-Px and theexpression of Keap1 and Nrf2 of myocardial tissue in MIRI ratsCompared with normal group,the myocardial SOD and GSH-PX activity decreased significantly,MDA content increased significantly（P<0.01）,Keap1 protein expression increased significantly and Nrf2 protein expression decreased significantly in model group.The Aconitum Chishizhi Pill Recipe group and the urokinase group were able to significantly reduce the MDA content and increase the activity of SOD and GSH-Px in the myocardial of rats（P<0.01）.Meanwhile,the Aconitum Chishizhi Pill Recipe group was able to activate the Keap1/Nrf2 signaling pathway,which significantly increased nrf2 expression and significantly decreased keap1 expression（P<0.05,P<0.01）.Conclusion:Aconitum Chishizhi Pill Recipe can protect myocardial injury and reduce the incidence of arrhythmias in myocardial ischemia-reperfusion rats.The specific mechanism may be to protect MIRI by modulating vascular endothelial cell homeostasis,reducing serum CK,LDH and increasing Na⁺-K⁺ATPase and Ca²⁺-Mg²⁺-ATPase activity in myocardium,regulating oxidative stress levels and activating Keap1/Nrf2 signaling pathway.