| When AMI occurs,some of the myocardial tissue surrounding the core area of the infarction still retains partial collateral blood supply.This area is called the infarct border zone.Although cardiomyocytes in the infarct border zone do not die rapidly due to initial ischemic injury,oxidative stress induced by ischemia can activate apoptotic signal transduction pathways and induce apoptosis of cardiomyocytes.At the same time,due to the necrosis of myocardial tissue in the core area of infarction and the release of a variety of inflammatory mediators,acute inflammatory reaction is triggered and irreversible damage is caused to myocardial cells.Myocardiums in the boundary area of infarction still retain some functions and can fight against the damage caused by acute ischemia and hypoxia.Therefore,it is of great significance to save the myocardial cells in the infarct border zone to reduce the size of MI and improve the prognosis.Lingbao Huxin Pills have definite clinical effect on the treatment of coronary heart disease.Previous pharmacological studies have proved that Lingbao Huxin Pills have the effects of increasing coronary flow,reducing tension-time index and myocardial oxygen consumption,etc.,and can significantly reduce angina symptoms and improve cardiac function in patients with coronary heart disease in clinical practice.Based on previous research,we came up with the hypothesis that "Lingbao Huxin Pills can protect the myocardial tissue in the infarct border zone",and conducted two studies around the hypothesis:(1)Elucidation of the potential mechanism of Lingbao Huxin pills in the treatment of acute myocardial infarction based on network pharmacology;(2)Lingbao huxin pills alleviated apoptosis and inflammation via Sirtl mediating FoxO1 and NF-κB signaling pathway in infarct border zone of AMI rats.Part1 Elucidation of the potential mechanism of Lingbao Huxin Pills in the treatment of acute myocardial infarction based on network pharmacologyObjective:A bioinformatics/topology based strategy was proposed for identification of the drug targets,therapeutic agents and molecular mechanisms of Lingbaohuxin pills against acute myocardial infarction.Methods:The AMI-related targets were screened through GeneCard databases.The active components and their targets related to the nine herbs of LBHX were searched through the TCMSP and TCMID database.The compound-target network was constructed using Cytoseape3.7.2 software;based on the STRING database,a target interaction network for LBHXagainst AMI was constructed,and the core target was selected based on topologicalparameters.GO(gene ontology)biological process enrichment analysis and KEGG(KEGG pathway analysis)pathway annotation analysis were performed on targets using the Cytoscape plugin ClueGO+Cluepedia.Results:57 protein targets and 104 active components of LBHX were identified.Bioinformatics analysis revealedthat 111 disease pathways associated with AMI therapy could be classified into the 16 statistically enrichedfunctional sub-groups.The multi-functional co-synergism of LBHX against AMI were predicted,including regulation of oxidative stress-induced intrinsic apoptotic signaling pathway,regulation of apoptotic mitochondrial changes and regulation of oxidative stress-induced cell death.The hub-bottleneckgenes of the protein networks including FOXO1,SIRT1,CASP3,IL-6,AKT1,SOD2,BCL2,BAX,STAT3,MMP9,MAPK1,SLC2A4 couldbecome potential drug targets and Quecetin,tanshinone IIA and β-sitosterol may be candidate agents.Conclusion:This study confirmed the multi-channel and synergistic effect of LBHX in the treatment of AMI and laid the foundation for experimental research of LBHX.The results showed that Lingbao Huxin Pills may inhibit intrinsic apoptosis induced by oxidative stress through FoxO1 signaling pathway,and reduce acute inflammatory response after AMI through NF-κB signaling pathway.Part 2 Lingbao huxin pills alleviated apoptosis and inflammation via Sirtl mediating FoxO1 and NF-κB signaling pathway in infarct border zone of AMI ratsObjective:To observe the intervention effect of Lingbao Huxin Pills on apoptosis and inflammation related indexes of myocardial cells in the infarct border zone of rats through Sirt1-mediated FoxO1/NF-κB signaling pathway,and to explore its mechanism.Methods:91 male Wistar rats were randomly divided into 7 groups(13 rats for each gruop):Sham,Model,Betaloc,LBHX-L,LBHX-M,LBHX-H and LBHX+EX527.Different drugs were given daily by gavage:equivalent normal saline water for Sham and Model group,0.9mg/kg betaloc for Betaloc group,0.45mg/kg LBHX for LBHX-L group,0.9mg/kg LBHX for LBHX-M group,1.8mg/kg LBHX for LBHX-H group,0.9mg/kg LBHX for LBHX+EX527 group.3 weeks later,the acute myocardial infarction model was established by ligation of the anterior descending branch of the left coronary artery.At the 7th day,5th day,1st day and 1h before model establishment,rats in LBHX+EX527 group were injeced 5 mg/kg EX527 intraperitoneally.The myocardial infarction area was observed by TTC staining 24 hours after modeling.TUNEL staining was used to observe the apoptosis of myocardial cells in the infarct border zone.The myocardial tissue structure and inflammatory infiltration were observed by HE staining.The blood markers of inflammation(IL-6,IL-1,TNF-α,ICAM-1)were measured by ELISA method.The expression of Sirt1 mRNA and FoxO1 mRNA in myocardium were evaluated by RT-qPCR test.The expression of Sirtl protein,FoxO1 protein,SOD2 protein,Bax protein and NF-κB p65 protein in myocardium were evaluated by Western Blotting.Results:Compared with Sham group,high infarct size(P<0.01),high number of apoptotic cells(P<0.01),high circulating levels of IL-1β,IL-6,TNF-α,ICAM-1(P<0.01)in plasma,high expression of Sirtl mRNA and FoxO1 mRNA(P<0.01),decreased Sirtl,FoxO1,SOD2 protein levels and high Bax,NF-κB p65 protein levels were observed in the rats of Model group.Compared with Model group,decreased infarct size(P<0.01),decreased number of apoptotic cells(P<0.01),low circulating levels of IL-1β,IL-6,TNF-α,ICAM-1(P<0.01 or P<0.05)in plasma,high expression of Sirt1 mRNA and FoxO1 mRNA(P<0.05,P<0.01)in myocardium,high Sirtl,FoxO1,SOD2 protein levels(P<0.01 or P<0.05),decreased Bax protein levels(P<0.01)were observed in the rats of LBHX-H group.Compared with Model group,decreased infarct size(P<0.01),decreased number of apoptotic cells(P<0.05),low circulating levels of IL-6 and TNF-α(P<0.05,P<0.05)in plasma,high expression of Sirtl mRNA and FoxO1 mRNA(P<0.05,P<0.05)in myocardium,high Sirtl,FoxO1,SOD2 protein levels(P<0.01 or P<0.05)in myocardium,decreased NF-κB p65 protein level(P<0.01)in myocardium were observed in the rats of LBHX-M group.Compared with Model group,there was no significant difference in infarct size,the number of apoptotic cells,inflammatory cytokines,the expression of Sirtl mRNA and FoxO1 mRNA in LBHX-L group.However,inhibition of Sirtl activity could partly offset the intervention effect of Lingbao Huxin Pills on apoptosis and inflammation.Compared with LBHX-M group,high number of apoptotic cells(P<0.05),high circulating levels of TNF-α,ICAM-1(P<0.01)in plasma,low FoxO1 and Bax protein levels(P<0.05,P<0.01),high NF-κB p65 protein level(P<0.05)were observed in the rats of LBHX+EX527 group.Conclusion:There were obvious apoptosis,inflammatory response,down-regulated Sirtl expression and myocardial injury in the myocardial tissue of the infarct border zone of acute myocardial infarction rats.Pretreatment of Lingbao Huxin Pills could inhibited apoptosis,inflammation,reduced myocardial infarction size and hence improve cardiac function in acute myocardial infarction rats.The protective Lingbao Huxin Pills is partly relevant with its activating effect on Sirtl activity,which could increase FoxO1 protein expression and decrease NF-κB p65 protein expression. |
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