The Effect Of Cholinergic Anti-inflammatory Pathway On The Gene Expression Of Injured Myocardium From Ischemia/reperfusion Rat Model

Objectives:to study the effect of cholinergic anti-inflammatory pathway on the gene expression of injured myocardium from ischemia/reperfusion rat model and investigate the involved molecular mechanism.Methods:(1) 20 Sprague Dawley male rats were randomly selected and divided into 2 groups evenly,the ischemia/reperfusion group(I/R group) and the vagus nerve stimulation group(STM group).The left cervical vagus nerve was isolated and the chest was exposed.The left anterior descending coronary artery(LAD) was ligated at the position between arterial cone and the root of the left atria appendage.Each group was subjected to 30 min of myocardial ischemia followed by 2h of reperfusion.In addition,10 min before reperfusion,left cervical vagus nerve of STM group was subjected electronic stimulation at 5V,2ms and 1HZ for 20min.(2) 0.5 ml blood was taken from tail vein before ligation and from right atrium after 120 min reperfusion for plasma troponin cTn-I analysis. After 120 min of reperfusion and every group was randomly divided into two parts.One part that myocardium was collected from left ventricle was applied to determine the area of myocardial infarction by Evan's blue dye perfusion and triphenyl tetrazolium chloride(TTC) staining.The RNA isolated from another part of the ischemic myocardium collected from left ventricle was hybridized to Affymetrix GeneChip Rat Genome U230 2.0 and the quality of hybridized RNA from both I/R and STM group was assessed and analyzed.(3)GeneSpring software was applied to screen out the genes,which show significant difference between I/R group and STM group.Data was assessed by hierarchical clustering analysis and gene ontology(GO) analysis.The involved signaling pathway was investigated as well.Based on the results and relative reported papers,the molecular mechanism of the effect of cholinergic anti-inflammatory pathway on gene expression in ischemia/ reperfusion myocardium was investigated.Real-time PCR was applied to analyze the expression of following important genes:Bdkrb2, Ntrk1,Cd401g,ATP2a2,and Ctnnb1.Results:(1) There is no significant difference of cTn-1 level between I/R and STM group before the ligation.(I/R group:4.52±0.56 ng/ml,STM group:4.50±0.57 ng/ml,p＞0.05) After 120 min of reperfusion,the level of cTn-1 in STM group(32.33±6.25 ng/ml) was significant lower than I/R group(64.35±5.89ng/ml)(P＜0.05).(2)There was no significant difference of the area of myocardial ischemia between two groups(I/R:48.2±4.5%,STM:47.5±4.6%,p＞0.05). However,the area of myocardial infarction of STM(25.5±3.9%) was significant reduced compared with I/R group(45.5±4.8%)(p＜0.05).(3) The expression level of 186 genes were changed significantly,in which 133 genes were upregulated and 53 genes were downregulated. Analyzed by GO software,there were 3 kinds of genes were affected.The first kind is membrane receptors,such as Bdkrb2,Ntrk1 and Cd401g.The second is ATPase,ATP binding protein and calcium binding protein,for example ATP2a2.The last is transcriptional factors,such as Ctnnb1.The upregulated genes,which are Bdkrb2,ATP2a2,Ntrk1 and Ctnnb1,were reported to show protective effect on myocardium.The downregulated gene,Cd401g,was relative to inflammation.These genes involved in 40 signaling pathways,including Wnt pathway.(4) The result of real-time PCR confirmed the genechip assay,which showed the level of Bdkrb2,ATP2a2,Ntrk1 and Ctnnb1 were upregulated and Cd401g was downregulated.Conclusion:The activation of cholinergic anti-inflammatory pathway shows the protective effect on the ischemia/ reperfusion myocardia.Significant change of the gene expression was detected between I/R and STM group,such as membrane receptors,ATPase,ATP binding protein and calcium binding protein and transcriptional factors. The results suggest that activation of cholinergic anti-inflammatory pathway upregulated the Bdkrb2,Ntrk1 and ATP2a2,downregulated Cd401g and activated Wnt/β-catenin(Ctnnb 1) pathway.The sum of the evidences suggests that activation of cholinergic anti-inflammatory pathway relieve and prevent the myocardia from ischemia/ reperfusion injury.