Study On The Effect And Mechanism Of Combined Application Of Traditional Chinese Medicine Tongxinluo, Loading Dose Atorvastatin And Nicorandil In Preventing Cardiac Rupture After Acute Myocardial Infarctio

Objective:The popularization of coronary intervention in the past two decades has significantly improved prognosis of patients with myocardial infarction,nonetheless,the occurrence of post-infarction cardiac rupture has not been fundamentally eliminated.Therefore,it is clinically meaningful to explore promising precautions to cardiac rupture in the treatment of cardiovascular diseases.Our group previously reported that Tongxinluo superfine powder mainly activated PKA/eNOS signaling pathway to protect cardiac microvascular endothelial cells after AMI and MIRI,and meanwhile reduced inflammatory cell infiltration,myocardial edema and intramyocardial hemorrhage.Statins,for example,atorvastatin,showed pleiotropic effects apart from lowering lipids.Previous experiments suggested that infiltration of inflammatory cells in infarcted myocardium and its surrounding area was significantly inhibited by the administration of statins,and that inflammatory factors in myocardium and circulation were also significantly down-regulated,which was conducive to myocardial repair and reduction of the infarction area.Nicorandil,a common drug in coronary heart diseases,has also been observed in vitro experiments to protect the cardiovascular endothelial barrier.The main objectives of this research are:(1)to discover and verify that the new drug combination(Tongxinluo,loading-dose atorvastatin,and nicorandil)can effectively alleviate post-infarction cardiac rupture;(2)To clarify the pathological characteristics and main development time of cardiac rupture;(3)To explore the target cells of the drug combination to prevent cardiac rupture;(4)To further elucidate the upstream regulatory mechanism of TRAF6/NF-κB p65/C/EBPβ pathway under AMI condition.Methods:SPF-class male 8-week-old C57 bl/6N mice were needed to construct AMI model,and echocardiography should be performed within 24 hours after establishment of model.Mice without AMI should be excluded from the experiment,and ones with AMI were randomly divided into different groups,and different interventions by gavage were performed for 14 days.The dead mice were taken out and autopsied.The diagnostic criteria for CR were(1)blood clots in the thoracic cavity;(2)myocardial wound in general;(3)ruptured myocardium indicated by hematoxylin-eosin staining.Kaplan-Meier survival curve and frequency of cardiac rupture within 14 days were collected and compared between intervention groups and AMI group.On the third day and the seventh day after AMI,mice serum was collected respectively.The expression levels of inflammatory factors,such as TNF-α,IL-6,IFN-y,and secreted metalloproteinase,for instance,MMP2 and MMP9,were measured by ELISA kits.Six mice were randomly selected from each group and sacrificed to harvest hearts on the fourth day after myocardial infarction.The expression level of proteins in infarcted myocardium,TRAF6,NF-κB p65,C/EBPβ,MMP2 and MMP9,was evaluated by western blot.Extracellular matrix proteins in myocardial tissue,such as collagen Ⅰ,collagen Ⅲ,laminin α2,and agrin,were also detected by western blot.To illustrate infiltration of macrophage in myocardium,the expression of Ml-macrophage biomarkers,F4/80,CD11b and iNOS,was analyzed by Western Blot,and the number and distribution of macrophages in myocardium around infarction area were confirmed by immunofluorescence at the same time.To explore upstream mechanism of TRAF6 up-regulation in cardiac rupture,miRNA sequencing was performed on infarcted myocardial tissue on the fourth day after AMI.Bone marrow-derived macrophages were isolated from 8-week-old male mice,and identified as F4/80 positive cells by flow cytometry.The macrophages were planted into 96-well plates,and cultured under different drug concentrations for 48 hours.Cell viability was determined by the CCK-8 method to determine the proper concentration of drug intervention.Mcp and M1 macrophages,stimulated with LPS and IFN-γ,were used as negative control and positive control respectively.The effects of different drugs and their combination on the invasive ability of macrophages were evaluated by transwell chamber and Giemsa staining.MMP9 and TRAF6 levels in macrophages in each group after transfection with miRNA inhibitors were analyzed to verify the regulatory effect of different miRNAs on TRAF6.Subsequently,Ml macrophages pretreated with drugs were analyzed after overexpression of TRAF6 and C/EBPβ or inhibition of p65 translocation to clarify that TRAF6/NF-κB p65/C/EBPβ signaling pathway is the core mechanism of three drugs.Co-immunoprecipitation and dual-luciferase reporter gene were introduced to detect and prove the synergistic effect of NF-κB p65 and C/EBPβ on MMP9 gene transcription.Results:The new drug combination can significantly prevent heart rupture and improve the survival rate of myocardial infarction.However,compared with AMI group,drug alone and the combination of two drugs failed to reduce the incidence of cardiac rupture and the mortality of AMI.The drug administration down-regulated circulatory metalloproteinases and inflammatory factors in early stage of myocardial infarction,and the drug combination developed the upmost inhibitory effect.The combination of Tongxinluo,loading atorvastatin and nicorandil can most effectively inhibit the TRAF6-mediated translocation of NF-κB p65 and C/EBPβ to hinder metalloproteinases expression and excessive dissolution of myocardial extracellular matrix,such as collagen Ⅰ,collagen Ⅲ,laminin and agrin,in injured myocardium,and ameliorate over-inflammation caused by infiltration and polarization of macrophages.The miRNA sequencing suggested that Tongxinluo,atorvastatin and nicorandil can down-regulate TRAF6 by up-regulating miR-299b-3p,miR215-5p and miR122-5p respectively.TRAF6-mediated signaling pathway is the core mechanism,through which three drugs reduced the breakdown of myocardial extracellular matrix and prevent heart rupture.Immunofluorescence staining illustrate that Tongxinluo,atorvastatin and nicorandil can decelerate the exudation of macrophages in the peripheral area of infarction and dilute MMP9 secreted from macrophages.In vitro experiments also showed that the new drug combination effectively inhibited the invasion of macrophages,which is similar to the results of in vivo experiments.The inhibitory effect on MMP9 expression conducted by the drug combination can be well blocked by TRAF6 overexpression,but translocation of C/EBPβ and NF-κB p65 can merely block MMP9 down-regulation partially.Via co-immunoprecipitation and dual-luciferase reporter gene,we verified the synergistic effect of NF-κB p65 and C/EBPβ in nucleus rather than NF-κB p65 or C/EBPβ alone could aggravate MMP9 expression.Conclusion:The new drug combination can effectively improve the short-term mortality and the incidence of cardiac rupture after AMI via inhibiting over-inflammation mediated by excessive macrophage infiltration and the decomposition of extracellular matrix posed by MMPs’ upregulation.The main mechanism is that drugs could upregulate microRNAs respectively and simultaneously inhibit TRAF6-mediated NF-κB p65 and C/EBPβ translocation in M1 macrophages,thereby down-regulate MMP9 gene transcription.Objective:Programmed cell death,such as apoptosis,pyroptosis,necrosis,ferroptosis,necrotic apoptosis in plays an important role in many physiological and pathological processes.Recently,as a new form of non-apoptotic cell death,ferroptosis has been confirmed to be involved in an array cardiovascular diseases,such as atherosclerosis,acute myocardial infarction,myocardial ischemia-reperfusion injury,cardiomyopathy,and heart failure.During acute myocardial infarction,many inflammatory immune cells,especially macrophages,break through endothelial barrier to clean up necrotic cells and cell debris,meanwhile,cardiac fibroblasts will be recruited into the injured myocardium and participate in myocardial tissue repair.Excessive inflammation and lipid peroxidation may trigger ferroptosis of cardiac fibroblast to hinder myocardial repair,resulting in the occurrence of cardiac rupture.Previously,it have been shown that Tongxinluo,atorvastatin and nicorandil can significantly improve the myocardial microenvironment in the infarcted area and reduce lipid peroxidation and oxidative stress.At the same time,excessive oxidative stress is also an important pathogenic mechanism of cardiac rupture after myocardial infarction.The first part of the study suggests that all three drugs can reduce the levels of interferon-y and interleukin-6,proved to induce ferroptosis,in serum.Generally,main purposes of this study is:(1)to prove the new drug combination can significantly inhibit ferroptosis and lipid peroxidation of infarcted myocardium and serum;(2)to explore the pharmacological effects and mechanisms of drugs to inhibit ferroptosis of cardiac fibroblasts co-cultured with macrophages.Methods:A mouse model of acute myocardial infarction was constructed,and ultrasonic tests were performed within 24 hours after AMI.Mice without myocardial infarction were excluded from the experiment,and mice with myocardial infarction were randomly divided into different groups.All mice were well-fed and well-observed for 14 days before sacrifice.Kaplan-Meier survival curve and the frequency of cardiac rupture in mice were recorded and analyzed;serum was collected from mice on the third and seventh days after acute myocardial infarction,and the level of indicators of oxidative stress,such as malondialdehyde and superoxide dismutasel,in serum was measured.Myocardium in the infarcted area were collected from mice on the fourth day after myocardial infarction.Ferroptosis-related proteins,such as glutathione peroxidase 4,ferroptosis suppressor protein l,Cystine/Glutamate Transporter,p53,in infarcted myocardium were detected with Western Blot,and the level of 4-hydroxynonenal,a metabolite of lipid peroxidation in infarcted myocardium was detected by immunofluorescence.Bone marrow-derived macrophages were isolated from 8-week-old male mice,and identified by flow cytometry,and mouse neonatal cardiac fibroblasts were extracted and identified by flow cytometry as well.Cardiac fibroblasts were planted into 96-well plates respectively,and cultured in complete medium containing different drug concentrations for 48 hours.Then the cell viability was measured by CCK-8 method to determine the proper doses of drugs.Macrophages and cardiac fibroblasts were transported into Transwell chambers and plates for cell culture respectively,and cultured in different medium under 3%oxygen for 48 hours.The levels of MDA in cell medium were detected;then,cardiac fibroblasts were collected,and the expression levels of ferroptosis-related proteins(GPX4,xCT,FSP1,p53)in the cells were detected by Western Blot.The levels of ferrous ions and lipid peroxidation in cardiac fibroblasts of each group were measured by flow cytometry,and the activity of cardiac fibroblasts was detected by CCK-8 assay.Results:The new drug cocktail therapy can significantly reduce the risk of cardiac rupture after acute myocardial infarction and improve the survival rate in the early stage of myocardial infarction.The levels of oxidative stress has been improved in treatment groups,and the drug combination was the most effective;The combination of Tongxinluo,atorvastatin and nicorandil can supremely inhibits ferroptosis and lipid peroxidation in infarcted myocardium on the fourth day after myocardial infarction;the in-vitro experiments have shown that atorvastatin inhibits ferroptosis of co-cultured cardiac fibroblasts mainly through GPX4 pathway,while Tongxinluo and Nicorandil can simultaneously activate GPX4 and FSP1 pathways to inhibit ferroptosis;the results of flow cytometry and CCK-8 assays have shown that the drug combination can most effectively reduce ferrous ions and lipid peroxidation of cardiac fibroblasts,thereby reduce ferroptosis.Conclusion:The new drug combination can improve the short-term mortality of acute myocardial infarction,and can also most significantly reduce myocardial lipid peroxidation and ferroptosis after infarction;drug combination effectively upregulated GPX4 and FSP1 signaling pathways to protect cardiac fibroblasts from ferroptosis and lipid peroxidation-induced and decrease cell viability in a inflammatory and hypoxic microenvironment.