The Study Of Expression Of Wnt And NF-κB Sigal Pathway Post Myocardial Infarction In Old Mice And Interaction Between These Two Pathways

Objective:The research is aimed at mechanisms of higher rate of cardiac rupture,more severe cardiac(?)emodehng in old mice by study of expressions of Wnt and NF-κB signal pathway post infarction,as well as crosstalk between these two pathways.The present study focuses on the following issues:1) To establish the acute myocardial infarction mouse model,study differential expression of dvl-1,glucogen synthetic kinase(GSK) -3β,β-catenin and connexin(cx) 43 in different age groups post-infarction.2) To study differential expression of p65/p50,inter- celluar adhesion molecule(ICAM) - 1 and vascular celluar adhesion molecule(VCAM) - 1 in different age groups post-infarction.3) To study effects of pyrrolidine dithiocarbamate(PDTC,an inhibitor of NF-κB) to cardiac remodeling,rate of cardiac rupture and expression of Wnt signal pathway for advanced age-ralated pathogenesis of cardiac rupture and cardiac remodeling.Methods:Part one:The part is about the expression of Wnt sigal pathway in different age groups post myocardial infarction.1) The acute myocardial infarction model of mouse has been established by ligation of left coronary artery in 160 adult C57BL mice(80 3 month Y,80 18 month O)in which 20 were sham operations group(SH).Compare infarct size,rate of cardiac rupture and cardiac remodeling between different age-group.2)96 mice(48 3 months,48 18 months) were randomly divided into four different groups according to time of infarction:SH,MI 3d,7d and 14d(12 / group,whether old or young).The mice were executed at the time postinfarction according to group division and hearts were kept in liquid nitrogen then moved to -80℃refrigerator.3) Expression of dvl-1,GSK-3β,β-catenin and connexin 43 were detected by western-blot.Dvl-1 mRNA were detected by RT-PCR.Part two:The part is about the expression of NF-κB signal pathway in mice with different age post myocardial infarction.Choose another 96 mice:48 3 months old,other 48 18 months.set up AMI model by ligation of left coronary artery.They were randomly divided mto four different groups according to time of postinfarction:SH,MI 3d,7d and 14d.The mice were sacrified at the time postinfarction according to group division and hearts were kept in liquid nitrogen then moved to -80℃refrigerator or in 4% paraformaldehyde.2)Expression of p65/p50 were detected by immunohistochemistry method and western-blot,while expression of ICAM-1,VACM-1 were detected by western-blot.Part three:1)We used 138 old mice(18 month old) to set up acute myocardial infarction mode 1.69 were given PDTC(5mg/ml) by intraperitoneal injection(PDTC group,P,120 mg/kg/d),others in physiological saline(myocardial infarction group,M).2) Echocardiography were carried on 7 days post-operation.Compare infarct size,rate of cardiac rupture and cardiac remodeling between these two groups.3)Compare activities of matrix metalloproteinases 2,9,expression of TNF-αmRNA,and content of collagen to discuss mechanisms.4)Expression of GSK-3β,β-catenin and connexin 43 were detected by Western blot,while dvl-1 mRNA were detected by RT-PCR to study "crosstalk" between Wnt sigal pathway and NF-κB pathway.Results:Four results have been obtained through the study,1) There was no difference in infarct size between old and young group.Rate of cardiac rupture is higher in old group than it in young(35.3%vs 17.7%,P =0.000).Dilation of left ventricle (LV)and contractile dysfunction were more severe in old group.Expression of dvl-1,β-catenin in left ventricle is higher in MI group compared with sham group(P =0.000 ) which implied that Wnt signal pathway be activitied post infarction.There were statistically magnificent differences of dvl-1 expression in SH and MI 14d group in old mice compared with young mice(P =0.000);there were no differences ofβ- catenin expression between two age groups except in MI 14d(P=0.003) group.Expression of dvl-1 andβ-catenin in infarction region in MI 3d group in old animals is much lower than it in young one(P =0.000).dvl-1 andβ-catenin is associatied with cellular adhension and repair.This results show that function of celluar adhension and repair in old mice be lower than in young.Expression of cx43 is 2.15 fold in young mice than it in old one (P=0.001).Compared with SH group expression of cx43 in MI group decreased greatly (P=0.002,0.000 respectively).Expression of cx43 in infarction region MI 3d is much lower in old mice than in young group(P=0.000),while it is much higher MI 7d in old mice(P=0.000).After that it tends to lower level MI 14d in old mice.cx43 decreased post infarction will be helpful to cardiac protection,while in old group cx43 increased greatly at 7d postinfarction which tell us that cardiac protection attenuated in old mice which maybe one of mechanisms of age-related cardiac remodeling.2) It is observed that p65/p50 positive cells are much higher in old mice than in young through immunohistochemistry methods(p65 400 vs 154,p50 179 vs 83,P＜0.05).Expression of p65 in SH,MI 3d,7d group in old mice is higher than in young mice,which meant higher activities of NF-κB in old animals post infarction.There was no statistics differences in expression of ICAM-1 between old and young.Expression of VCAM-1 is higher in old than in young in SH and MI 14d group(P=0.001,0.034 respectively).It implied that there might not be age-related differences between old and young mice in expression of ICAM-1 and VCAM-1 post infarction.3) Rate.of cardiac rupture decreased greatly in PDTC group(35.3%vs 15.6%, P=0.000).Diameter of LV decrease(P=0.000),ventricular wall thickness(P=0.000), LV mass(P=0.023) and fraction shortening(P=0.001) increased in PDTC group hy echocardiaography.Expression of MMP-9 and MMP-2(P=0.023),TNF-αmRNA (P=0.901) decreased in PDTC group.While CVF also decreased in PDTC group (P=0.000).But there were no charges of expression of ICAM-1 and VCAM-1 in PDTC group.Inhibition of NF-κB leads to lower rate of cardiac rupturc and lower expression of cytokines which identified action of NF-κB signal pathway in cardiac rupture.And age-related change in NF-κB signal pathway maybe associated with age-related CR.But the differences may not be associated with ICAM-1 and VCAM-1.4) Expression of dvl-1 mRNA is lower in PDTC group than it in MI group(P＜0.05 )..While ratio of p-GSK-3β/GSK-3βis higher in PDTC group than it in MI group(P value all 0.000).Expression ofβ-catenin is lower in PTDC group in 3d postinfarction, while it increases in PDTC group 7d postinfarction(P=0.000).Expression of connexin 43 is much lower in PDTC group,and difference has statistic magnificence (P=0.000).Expression of Wnt signal pathway altered when inhibiting activities of NF-κB.That means "crosstalk" between Wnt and NF-κB existed post infarction.Conclusions:The conclusion of the research can be drawn from the following four aspects,1)Rate of cardiac rupture in old mice is higher than it in young.Expression of dvl-1 andβ-catenin in infarct region in old mice is much lower than in young,downregulation of cx 43 which means cardiac protection in old mice lasted for a shorter time than in young,these might be the mechanisms of higher rate of cardiac rupture in old mice 2) NF-κB signal pathway was activitied postinfarction and activities in old group is higher than it in young group.This results identified age-related expression differences of NF-κB signal pathway in old mice postinfarction.But age-related differences were not associated with change of expression of ICAM-1 and VCAM-1.3) Rate of cardiac rupture and cardiac(?)emodeling decreased greatly in old mice when given NF-κB inhibiter(PDTC).It maybe ca(?)sed by decreased collagen is the result of decreased MMP-2,9,TNF-αmRNA which proved that NF-κB signal pathway be associated with age-related cardiac rupture and cardiac remodeling.4) Expression of dvl-1 mRNA,connexin 43 downregulated,p-GSK-3β/GSK-3βupregulated in PDTC group.While expression ofβ- catenin fall down first then rise up in PDTC group compared with infarction group which implied that Wnt pathway might have "crosstalk" with NF-κB pathway post infarction.