Study On The Mechanism Of Qishen Granule Inhibiting Inflammatory Injury-mediated Cardiomyocyte Apoptosis After AMI

Aim:Aim at treating coronary heart disease especially the acute myocardial infarction(AMI),which present serious harm to human life and health,ranks first in all kinds of causes of death.This paper aims to regulate from arachidonic acid(AA)metabolism thereby inhibiting the inflammation damage as a starting point,the use of animal models of myocardial infarction and inflammatory cytokine-induced apoptosis in myocardial cell model as a tool to explore the mechanism of herbal compound Qishen Keli(QSKL)on inhibiting myocardial apoptosis in preventing AMI.This study not only provides new interventions and targets for the prevention and treatment of coronary heart disease,but also provides new research ideas on prevention and treatment of other diseases based on inflammation and apoptosis.Methods:1.The AMI model was produced by ligating of left anterior descending(LAD)of artery in C57BL/6 mice.Mice were orally administered one week with QSKL(3.33 g/kg)and the positive control drug Celecoxib(33.3 mg/kg).cardiac function measured by ultrasonic after 24 h(acute phase)and 7 d(restoration phase)in each group respectively.plasma creatine kinase MB(CK-MB),lactate dehydrogenase(LDH)content were detected by blood biochemical tests within each group of mice;The expression of tumor necrosis factor(TNF-a),prostaglandin E2(PGE2)were detected by ELISA assay;The expression of plasma ET,NO and myocardial tissue protein iNOS and eNOS were detected by radioimmunoassay and western blotting(WB)methods;Using HE and TUNEL staining to detect the expression of inflammatory cells and apoptotic cells in myocardial tissue in each group;The pharmacodynamics of QSKL in treating AMI were comprehensive evaluated based on the above results.2.The LPS-induced RAW264.7 macrophages inflammation model binding with ELISA,WB and other methods were used to explore the anti-inflammatory mechanism of QSKL.Cell viability were detected by CCK-8 assay;Griess method to detect the NO levels of cell supernatant;ELISA assay to detect the TNF-α,interleukin-6(IL-6),monocyte chemoattractant protein(MCP-1)content of cell supernatant.WB assay to detect the expression of phosphorylated nuclear transcription factors(NF-κB)and cyclooxygenase-2(COX2)protein in macrophage cytoplasm.The inflammatory cytokines were produced by LPS-stimulated RAW264.7 macrophages as stimulators(conditioned medium,CM),thereby establish a H9C2 myocardial inflammation injury model;cell viability were detected by CCK-8 assay,NO and LDH levels of cell supernatants were also detected.Then using this model to explore the mechanism of QSKL on anti-apoptosis and inhibiting inflammation injury;using cell viability assay binding cell supernatant NO,LDH and PGE2 levels to evaluate the efficacy of QSKL.WB assay were used to detect the expression of COX2 in myocardial cells and the expression of P-NF-κB in myocardial cells were detected by immunofluorescence assay.Fully discussing the mechanism of QSKL on multi-target regulation of AA metabolism thereby inhibiting the inflammatory response in AMI model combines the molecular biology techniques.The circulation TXB2,6-keto-PGFla content were detected by using radioimmunoassay method.WB assay were used to detect AA metabolic pathways associated proteins(such as sPLA2,COX1,COX2,5LOX,15LOX)expression levels.3.Utilizing myocardial cell inflammation injury model and AMI animal model,revealing the pathway and mechanism of QSKL on inflammatory injury and myocardial apoptosis inhibition from in vivo and in vitro levels.Determination of H9C2 myocardial apoptosis were detected by Hoechst 33258 staining and mitochondrial membrane potential analysis;WB assay to detect Bax and Bcl-2 protein expressions in each treatment group.In addition,with AMI animal models,using WB method to detect the protein expression of apoptosis pathway-related proteins such as P53,Bax,Bcl-2,FasL,Caspase3/8/9.Results:1.The AMI model was produced to evaluate the efficacy of QSKLUltrasonic testing showed that ejection fraction(EF)(P<0.001,vs.Sham)and fractional shortening(FS)(P<0.001,vs.Sham)showed a sharp decline,which indicates successful model.QSKL can significantly improve cardiac function in the acute and repair period of myocardial infarction,manifested in EF,FS compared with the model group(P<0.05)were significantly improved.In opposition to the acute phase of expansion of Left Ventricular Internal Diameter(LVID),the QSKL and Celecoxib had no significant improvement.The results of myocardial enzyme showed that QSKL can be effectively reduce the content of CK-MB and LDH(vs.Model,P<0.05)which were released after AMI.In addition,QSKL significantly inhibited AMI induced proinflammatory cytokines(TNF-α,PGE2)(P<0.05)and ET/NO showed a downward trend,but had no significant difference;but WB showed QSKL can effectively inhibit the expression of iNOS which was associatee with inflammatory mechanisms rather than eNOS;HE staining showed that QSKL significantly improved the morphology of myocardial cells,and there are rare inflammation cell infiltration.TUNEL staining showed QSKL could inhibit the number of cardiomyocyte apoptosis in AMI mice.2.The LPS-induced RAW264.7 macrophages and H9C2 myocardial cell inflammation model as well as AMI animal models were used to explore the mechanism of QSKL on regulating AA metabolism and inflammation injury inhibition.Griess assay suggested that different concentrations of QSKL can inhibit NO produce by LPS-stimulated RAW264.7 macrophages;ELISA results showed QSKL and Celecoxib could inhibit the expression of related inflammatory cytokines,TNF-a,IL-6,MCP-1 in cell supernatants and showed a concentration-dependent manner;WB test results show that QSKL and Celecoxib could inhibit macrophage NF-κB/COX2 expression.Using 1 μg/ml LPS to stimulate RAW264.7 macrophages produce inflammatory cytokines as a stimulant(conditioned medium,CM),thereby intervention H9C2 myocardial cell,to establish a myocardial inflammation injury model,the results showed that the cell death induced by the CM myocardial inflammatory injury model up to 40-50%,significantlyhigher than LPS or single-inflammatory cytokines induced mortality,this model could be a tool to further investigate the anti-inflammatory effects of QSKL.CCK-8 assay showed different concentrations of QSKL(400,600,800,1000μg/ml)significantly inhibited CM-induced myocardial cell injury;the NO and LDH released by CM-stimulated H9C2 myocardial cells were significantly inhibited in a dose-dependent manner;this shows that the QSKL have a good myocardial inflammation injury protection.ELISA and immunofluorescence results suggest QSKL can effectively inhibit the release of PGE2 and NF-κB in CM-stimulated H9C2 myocardial cells.WB assay showed that different concentrations of QSKL could decrease the expression of COX2 in cardiac myocytes.With the AMI animal model given QSKL intervention,RIA results showed that the model group mouse plasma TXB2 was significantly higher(vs.sham,P<0.01),6-keto-PGF1αwas significantly lower(vs.sham,P<0.01),and TXB2/6-keto-PGF1α ratio was significantly higher(vs.sham,P<0.01);QSKL can effectively reduce the levels of circulation TXB2 and TXB2/6-keto-PGF1α,and Celecoxib group was only inhibits the 6-keto-PGF1α.WB test results shows that QSKL and Celecoxib are able to effectively inhibit the expression of COX2 in the myocardial infarct border zone;However,compared with Celecoxib,QSKL may also significantly inhibited the expression of other key enzyme of AA metabolic pathway as COX1 and lipoxygenase(5LOX,15LOX);In addition,the inflammatory signal transduction pathway JAK2/STAT3 also have a good significant inhibition.3.The myocardial cell inflammation model as well as AMI animal models were used toexplore the mechanism of QSKL on inhibiting myocardial apoptosis meditated by inflammation in AMI.In the CM-induced H9C2 myocardial cell inflammation injury model,Hoechst 33258 staining showed that CM can induce H9C2 myocardial apoptosis;different concentrations of QSKL(400,600,800,1000μ.g/ml)intervention can effectively suppress the number of apoptotic cells induced by myocardial inflammation injury.In addition,combining with the Rhodamine 123 staining,CM enables myocardial mitochondrial membrane potential loss,triggering endogenous myocardial apopt6sis;the different concentrations of QSKL intervention can significantly stabilize mitochondrial membrane potential,and reduce the number of myocardial cells caused by the endogenous mitochondrial pathway of apoptosis.WB showed QSKL can effectively downregulate the expression of Bax,and increase the expression of Bcl-2 protein.With the AMI animal model given QSKL and Celecoxib intervention inhibited the number of myocardial apoptotic cells in the infarct border zone.WB showed QSKLand Celecoxib can inhibit the expression of P53,FasL,Caspase3 and Caspase8(vs.Model,P<0.05)in myocardial tissue;In addition,QSKL can also inhibit the expression of COX2,Bax,Caspase9(vs.Model,P<0.05),and up-regulated the expression of Bcl-2(vs.Model,P<0.05)in myocardial tissue.Conclusions:1.Early efficacy studies suggest that QSKL can effectively improve the sharp decline in AMI acute phase and restoration phase in left ventricular function;for the acute phase of AMI,QSKL can reduce the infiltration of inflammatory cells in myocardial tissue and inhibit myocardial apoptosis as well as cardiac local inflammatory cytokines release,suggesting that anti-inflammatory and anti-apoptosis mechanism of QSKL in improving cardiac function in the AMI acute phase.2.Anti-inflammatory mechanism of QSKL is associated with inhibition of COX2 in macrophages and myocardial cells in the myocardial tissue,in addition,it can be reduced the release of inflammatory cytokines such as TNF-α,IL-6,MCP-1 in macrophages,which related to the regulation of NF-κB,JAK2/STAT3 pathway protein;compared with Celecoxib,an anti-inflammatory characteristics of QSKL is within the multi-target regulation of myocardial cells AA metabolism,especially the suppression sPLA2 expression caused by AMI within the myocardial cells,and it can be regulated the AA metabolism caused by inflammation from different subtypes of cyclooxygenase pathway,lipoxygenase pathways.3.QSKL inhibits the inflammatory lesion thereby regulate myocardial apoptosis,which is twofold:First,QSKL and Celecoxib significantly inhibit the intrinsic apoptosis pathway of TNF-a and FasL-mediated with specific performance of regulatory role in TNF-α-NF-κB-P53 and FasL-Caspase8-Caspase3 pathway,and the regulation is associated with inhibit the myocardial tissue macrophages COX2 activity thereby reducing the release of inflammatory factors;in addition,QSKL can also by inhibiting myocardial cells COX2 protein,thereby regulating the expression of Bax/Bcl-2 in mitochondrial membrane,inhibiting endogenous apoptotic pathway induced by Caspase9,Celecoxib does not have this role.4.This study elucidate the mechanism of QSKL intervention of AMI myocardial apoptosis mediated by inflammatory damage,that for the treatment of coronary heart disease AMI provides new ways and therapeutic intervention ideas;providing experimental basis to fully reveal the role of the characteristics and pharmacology mechanism of traditional Chinese medicine,but also for providing ideas and approaches for the pharmacological mechanisms research and new drugs treating the coronary heart disease,heart failure which based on myocardial ischemia.