Study On The Mechanism Of Fufang Danqi Tablets To Improve Energy Metabolism And Prevent Myocardial Ischemia

Objective:Therapeutic angiogenesis and improvement of myocardial energy metabolism have been the focus of research in the treatment of ischemic heart disease in recent years.Danshen Pills consists of Panax notoginseng and Salvia miltiorrhiza,which can significantly improve the cardiac function of rats with ischemic heart disease,is commonly used in the clinical treatment of ischemic heart disease.However,its mechanisms remain unclear.In this experiment,a myocardial ischemia model in rats,an oxygen-glucose deprivation/recovery injury model in H9C2 and a hydrogen peroxide injury model inhuman umbilical vein endothelial cell(HUVEC)were successfully established.Hematoxylin-eosin staining,immunohistochemistry,Western blots Cell Counting Kit-8,and Matrigel formation experiments were used to investigate the effects of Danqi Pills on PGC-1?,which is a major factor in energy metabolism process and other angiogenesis-related proteins.Methods:1?Sixty Sprague-Dawley male rats of SPF grade were randomly divided into two group,namely,50 in the model group and 10 in the sham operation group.Rats with left anterior descending coronary artery ligation were then divided into model group,Danqi Pills group and trimetazidine group.28 days after surgery,echocardiography was used to detect cardiac function ofrats.2?The effect of Danqi Tablet on myocardial cell alignment in rats with myocardial ischemia was evaluated by HE staining.Immunohistochemistry and Western blots was used to detect the expression of PGC-1? and angiogenesis-related proteins such as VEGF in rats with myocardial ischemia.3?Oxygen-glucose deprivation/recovery induced H9C2 was treated with Danqi Pills and trimetazidine solution.The ATP kit was used to detect the production of ATP in the cells.The ROS kit was used to detect the ROS production in the cells.Western Blots were used to detect the expression PGC-1?.4.H2O2 induced HUVEC were treated with Danqi Pills and CPT1A inhibitor Etomoxir.The effects of Danqi Pills and Etomoxir on angiogenesis of endothelial cells in vitro were examined.The expression of energy metabolism related proteins CD36 and CPT1A was detected by Western Blots.Result:1?After 28 days of LAD ligation,the EF and FS values in the model group were significantly lower than those in the sham operation group(P<0.01,P<0.01).At the same time,LVEDd and LVEDs also increased by 55.7%and 296%respectively(P<0.01,P<0.01)compared with the rat in the model group.These suggest that Danqi Pills can significantly increase the EF and FS values(P<0.01),down-regulate LVEDs(P<0.01),and LVEDd compared with the model groupthereby effectively improving the heart function.2?HE staining showed that the cardiomyocytes =of rats in Danqi Pills group was more arranged than that of the model group.Both immunohistochemistry and Western Blots showed that the expression of PGC-la in ischemic myocardium of Danqi Pills group was significantly increased compared with the model group(P<0.0015,P<0.01).The results of Western Blots showed that the expression of Adenosine 5'-monophosphate(AMP)-activated protein kinase ?1,Sirtuin 1,mitofusinl,mitofusin2,and nuclear respiratory factor 2 in the Danqi Pills group was significantly higher than that of the model group(P<0.05,P<0.05,P<0.05,P<0.05,P<0.05).Similarly,in H9C2 rat cardiomyocytes,Danqi Pills significantly promoted the expression of AMPK-al,SIRT1,PGC-la,Mfnl,Mfn2,and NRF-2 compared with the model group(P<0.05,P<0.05,P>0.05,P>0.05,P<0.05,P<0.01).At the same time,Danqi Pills significantly increased ATP levels in cells(P<0.001)and removed excess ROS(P<0.05).Combined the results of animal experiments with cell experiments,Danqi Pills may regulate mitochondrial fusion and division,and promote energy production of cardiomyocytes through regulate the PGC-la expression,thereby maintaining the function of cardiomyocytes.3?The results of immunohistochemical staining showed that the expression of PGC-1? and CD31 in Danqi Pills group was significantly increased compared with the model group(P<0.01).The results of Western Blots showed that the expression of e PGC-1?was significantly decreased in the model group compared with the sham operation group.The expression of AMPK-?,SIRT1,PGC-1?,Mfnl,Mfn2 and NRF-2 was significantly decreased,and the expression of PPAR-a,CD36,CPT1A,HIF-1? and VEGF was significantly decreased.Compared with the model group,Danqi Pills can significantly increase the expression of these proteins:AMPK-?,SIRT1,PGC-1?,Mfn1,Mfn2,NRF-2,and the expression of PPAR-?,CD36,CPT1A,HIF-1?,VEGF protein apparently increased4?In animal experiments,the results of immunohistochemistry showed that the expression of CD31 protein in ischemic myocardium of Danqi Pills group was significantly increased compared with the model of coronary artery LAD ligation group(P<0.001).The results of Western Blots showed that the expression of PPAR-?,CD36,CPT1A,HIF-1?and VEGFR-2 in Danqi Pills group was significantly higher than that in model group(P<0.05,P<0.001,P<0.01,P<0.05,P<0.05).In the modelof hydrogen peroxide-induced HUVEC injury,Western Blots showed that the expression of CPT1A was significantly increased in Danqi Pills group compared with the model group(P<0.05),and PPAR-a and CD36 protein expression were also increased,however,there was no statistical difference.The results of Matrigel endothelial cell tube formation experiments showed that Danqi Pills can significantly promote the vascularization of endothelial cells,and the inhibitor of CPT1A can attenuate the effect of Danqi Pills on angiogenesis.In conclusion,Danqi Pills may promote angiogenesis of ischemic myocardium by regulating endothelial cell PPAR-?-CD36-CPT1A signaling pathway.Conclusion:Danqi Pills may improve the energy supply inischemic myocardium by regulating the energy metabolism-related PGC-1? signaling pathway and promoted the angiogenesis by regulating the PPAR-?-CD36-CPT1A signaling pathway inendothelial cells.This study preliminarily revealed the pharmacological mechanism of Danqi Pills on energy metabolism and angiogenesis,and provided a basic experimental basis for the clinical application of Danqi Pills.