Study On The Effect And Mechanism Of Baoyuan Huoluo Prescription On Myocardial Ischemia-reperfusion Injury

ObjectiveTo investigate the possible effect and mechanism of Baoyuan huoluo prescription(BYHL)in improving myocardial ischemia-reperfusion injury(MIRI).MethodsIn this study,to observe the protective effect of BYHL on MIRI,eligible patients with acute myocardial infarction were recruited to conduct a randomized controlled trial.Then,the possible mechanism of BYHL in the treatment of MIRI would be preliminarily predicted by network pharmacological methods.Finally,an in vitro experiment was designed to verify the most relevant possible pathway of improvement of MIRI by BYHL.1.Clinical study:Patients with acute myocardial infarction receiving percutaneous coronary intervention(PCI)were recruited and randomly divided into experimental group and control group.The control group was given conventional treatment,and the experimental group was given BYHL on the basis of conventional treatment.The treatment period was 2 months.Then the peak values of cardiac troponin I(cTnI),creatine kinase(CK)and creatine kinase-MB(CK-MB)before and 24 hours after surgery,as well as the left ventricular ejection fraction(LVEF)24 hours and 2 months after surgery in 2 groups were observed to explore the effect of BYHL on myocardial injury degree and cardiac function after MIRI.2.Network pharmacological research:The chemical components and targets of BYHL were obtained in the TCMSP database.Targets of MIRI were obtained from GeneCards and OMIM databases.The targets of BYHL and MIRI were intersected.The "chemical composition-target" network of BYHL in the treatment of MIRI was constructed by using Cytoscape software,and the key compounds were obtained by topological analysis.The protein interaction network was constructed by using STRING database and optimized by importing into Cytoscape software,and the key targets were obtained by cluster analysis.GO function analysis and KEGG pathway enrichment analysis were performed by using the plugin of "ClueGo" of Cytoscape.Finally,the most relevant pathway and mechanism of BYHL on MIRI would be predicted by combining with the results of network analysis and the current status of basic research on MIRI.3.Experimental study:Cardiomyocytes of rats(H9C2)were treated with hypoxia and reoxygenation(H/R)to establish MIRI models,which were divided into normal group(control group),model group(H/R group)and drug-containing serum groups with different concentrations of BYHL(H/R+LBYHL group,H/R+MBYHL group,H/R+HBYHL group).Cell activity was detected by CCK-8 assay.Cell apoptosis was detected by flow cytometry with Annexin V/PI staining.Caspase-3 and Caspase-9 activities were determined by colorimetry,and protein expression levels of NF-κB p65,IκBα,Bcl-2,caspase-3 and caspase-9 were detected by Western Blot.Result1.Clinical study:(1)A total of 76 people who met the criteria of inclusion and exclusion were recruited,among which 10 cases fell off midway.Finally,66 people were actually enrolled,including 34 people in the experimental group and 32 people in the control group.There was no significant difference in baseline data between the two groups,such as gender,age,smoking history,complicated basic diseases(hypertension,diabetes,dyslipidemia,etc.),diseased vessels,target vessels and their TIMI grade(P>0.05).There was no significant difference in the levels of cTnI,CK,CK-MB before surgery and LVEF 24 hours after surgery between 2 groups(P>0.05).(2)Compared with the preoperative level,the peak levels of cTnI,CK and CK-MB in serum 24 hours after operation were significantly decreased in 2 groups(P<0.01).Compared with the control group,the peak values of serum cTnI,CK and CK-MB in the experimental group 24 hours after surgery were significantly lower(P<0.01).(3)After 2 months of treatment,the LVEF of the control group was slightly decreased(P<0.05),while the LVEF of the experimental group was slightly increased(P<0.01).Moreover,compared with the control group,the LVEF of the experimental group was significantly higher after 2 months of BYHL treatment(P<0.05).2.Network pharmacological research:There were 103 active ingredients(65 of Salvia miltiorrhiza,8 of Panax notoginseng,6 of Hawthorn,20 of Astragalus membranaceus and 11 of American ginseng)in BYHL prescription,corresponding to 120 targets.There were 937 MIRI-related targets.57 targets from MIRI to BYHL,corresponding to 72 compounds,were obtained by the intersection of the above two results.11 key compounds such as quercetin,kaempferol and isorhamnetin were screened and 51 action targets were obtained.Two subnetworks,involving 16 targets such as CASP3,NOS3,IL6 and ICAM1,were obtained by clustering analysis of PPI network.239 biological functions including in vitro apoptotic signaling pathway,response to reactive oxygen species and migration of vascular endothelial cells were revealed by GO function analysis.63 pathways including TNF signaling pathway,NF-κB signaling pathway,VEGF signaling pathway and PPAR signaling pathway were enriched by KEGG pathway enrichment analysis.Finally,by combining the results of network pharmacological analysis and recent basic studies on MIRI,the possible way of BYHL in the treatment of MIRI were screened out,which might be to improve cell apoptosis by regulating NF-κB signaling pathway.3.Experimental study:(1)Compared with Control group,after H/R treatment,the cell viability of H9C2 cardiomyocytes were decreased,the apoptosis level were increased,the activity of Caspase-3 and Caspase-9 were increased,the protein expression levels of NF-κB p65,Caspase-3 and Caspase-9 were significantly increased,and the protein expression levels of IκBα and Bcl-2 were significantly decreased(P<0.01).(2)Compared with H/R group,the cell viability of H9C2 cardiomyocytes was slightly increased after treatment with low concentration of BYHL drug-containing serum(P>0.05),but the cell viability was significantly increased after treatment with medium and high concentration of BYHL drugcontaining serum(P<0.01).(3)Compared with H/R group,the apoptosis rate of H9C2 cardiomyocytes in all groups was decreased after treatment with different concentrations of BYHL drug-containing serum(P<0.01),and the apoptosis rate in medium and high concentrations of BYHL drug-containing serum was significantly lower than that in low concentrations of BYHL drug-containing serum(P<0.01).(4)Compared with H/R group,the Caspase-3 activity of H9C2 cardiomyocytes in all groups treated with different concentrations of BYHL drug-containing serum decreased(P<0.01),and the level of Caspase-3 activity in each group was negatively correlated with the concentration of BYHL drug-containing serum(P<0.05).(5)Compared with H/R group,the Caspase-9 activity of H9C2 cardiomyocytes treated with low concentration drug-containing serum was slightly decreased,but the difference was not significant(P>0.05),while the Caspase-9 activity of H9C2 cardiomyocytes treated with medium and high concentration BYHL drug-containing serum was significantly decreased(P<0.01).(6)Compared with H/R group,the expression levels of NF-κB p65 in H9C2 cardiomyocytes were significantly decreased(P<0.01),the expression levels of IκBα and Bcl-2 were significantly increased(P<0.01),and the expression levels of Caspase-3 and Caspase-9 were significantly decreased(P<0.05 in low concentration group and P<0.01 in medium and high concentration groups).The protein expression level of NF-κB p65 and caspase-3 was negatively correlated with the concentration of BYHL drug-containing serum(P<0.05),and the protein expression level of Bcl-2 was positively correlated with the concentration of BYHL drug-containing serum(P<0.05).The expression levels of IκBα and Caspase-9 in H9C2 cardiomyocytes treated with high concentration of BYHL-containing serum were significantly higher than those in low and medium concentration groups(P<0.01).Conclusion1.RCT studies have confirmed that the addition of BYHL on the basis of conventional treatment after PCI in patients with myocardial infarction can effectively reduce MIRIrelated myocardial injury,save myocardium to a greater extent,and improve cardiac function in patients with myocardial infarction.2.The network pharmacological study revealed that BYHL may improve MIRI by playing a role of anti-apoptosis,anti-oxidation,anti-inflammatory and improving coronary vascular endothelial function through the multi-component,target and pathway pathway.Among them,the regulation of NF-κB signaling pathway to inhibit cell apoptosis may be the key pathway to improve MIRI by BYHL.3.Further cell experiments in vitro confirmed that BYHL can reduce H/R-induced myocardial cell injury,and the mechanism may be related to the regulation of NF-κB pathway and the inhibition of H/R-induced apoptosis through the mitochondrial pathway.