A Comparison Of Intimal And Adventitia Injury Induced Atherosclerosis Formation And MAPK/NF-κB Signal Transduction Pathway

Endothelial injury has ever been regarded as the initiator of atherosclerosis.But recent research revealed that adventitia is not a bystander.What ever the adventitial and intimal injury induced atherosclerosis,Inflammation and oxidative stress and immune disorders might be the common mechanism.Inflammation,oxidative stress and immune conditioning messages pass through all kinds of pathway.Adventitia and endothelial injury induced signal transduction may transduct through the same pathway and different way.So we decided to establish a new model which exert adventitia injury and endothelial injury in two side carotid artery in one animal,which we can compare the different atherosclerosis at the same time.PartⅠ:Establishment of the animal model of adventitia injury and endothelia injury induced atherosclerosis.Object:The aim of this study was to establish rabbit model with carotid artery adventitial and intimal injury.Method:The left carotid artery adventitia of rabbit was injured with collagenase digestion,meanwhile the contralateral one was injected with collagenase.Arteries were collected for paraffin sections and cryosection at 1,2,4,8 weeks after operation.Paraffin sections were stained with Hematoxylin and eosin to assess vascular morphology.Cryotomy sections were used for Oil Red O staining to identify fat deposits in the arteries.Immunohistochemical staining was performed to identify cellular components in intimal lesions.Results:The results showed that intimal hyperplasia lesions occured 1 weeks after adventitial and endothelial injury in carotid arteries of rabbits.IMR(neointima area/media area×100%) of adventitia injurywithout adventitial injury were higher than endothelial injury side.(0.29±0.02 vs 0.11±0.01,p＜0.05).8 weeks later,the IMR of endothelial injury side surpass the adventitia injury side(0.63±0.05 vs 0.52±0.03).Rabbits fed with high fat diet developed classical atherosclerotic plaques were seen in both injured carotid arteries.As the experiment went on,the lesion represent a more lipid deposition and less smoothe muscle character.PartⅡ:Evaluation of inhibitory effects on MAPK and NF-κB signal pathway by delivery through adventitiaObject:We prepared the p38,JNK and NF-κB inhibitor slow release delivery vehicle by Pluronic-F127(PF127),and delivered to the artery adventitia. Then we evaluate the effects of this system.Methods:After PF127 gel was prepared,p38,JNK and NF-κB inhibitor were completely resolve in the gel.After we put the gel around the artery,24h,72h,1w,2w,4w later respectively,we collect the artery and examine the expression of p38,JNK,NF-κB by Immunochemistry and Western blot.Results:(1) p38 inhibitor group:p-p38 expression didn't show significant after drug delivery of 24h to 72h.After 1 week,p-p38 expression reduced,which is consistant with Immunochemistry results.(2) JNK inhibitor group:1 to 4 weeks after delivery of JNK inhibitor,JNK expression obviously reduced in artery.After four weeks,there was no positive expression in artery.(3) NF-κB inhibitor group: Immunochemistry examination found that NF-κB p65 represents no difference in the artery.During 1 to 4 weeks,NF-κB p65 expression was negative in the artery. Western blot proved the same results,there was no difference in 1 week,but reduce during 1 to 4 weeks.Conclusion:Delivery of p38,JNK,NF-κB inhibitor through adventitia may effectively inhibit the expression of p38,JNK,NF-κB. PartⅢ:Role of MAPK,NF-κB in adventitia injury and endothelial induce atherosclerosisObjects:After compared MAPK,NF-κB pathway in adventitia injury and endothelial induced atherosclerosis,we try to find out the role of MAPK,NF-κB in the pathogenesis of adventitia injury and endothelial injury induce atherosclerosis by adventitia delivery of specific MAPK,NF-κB inhibitor,Methods:On the basis of animal in partⅠ,we examined p38,JNK,ERK,NF-κB expression by western blot after the animal model was established for 24h,72h,1w,2w,4w,8w.Then after deliverd of p38,JNK,NF-κB via adventitia,we examined the hyperplasia of intimal and the expression of p38,JNK,NF-κB.Results:Both adventitia and endothelial can up regulate the expression of p38,JNK,NF-κB.Adventitia injury can't up regulate ERK,while endothelial can up regulation ERK1.Adventitia delivery of p-p38,p-JNK and NF-κB inhibitor can effectively inhibit the expression of p38,JNK,NF-κB and slow the progress of atherosclerosis.PartⅣ:Effects and the mechanism of tongxinluo(TXL) on adventitial and endothelial injury induced atherosclerosisObjectives:We hope to find the effects of TXL on adventitial and endothelial injury induced atherosclerosis,and suppression effects on APK,NF-κB signal pathoday.Methods:On the basis of animal model established in partⅠ,TXL was administrator to the animals.After 8 weeks intervention,arteries were collected and examined by HE,immunochemistry and westem blot examination.The degree of intimal hyperplasia and expression of p38,JNK,ERK,NF-κB were examined.Results:TXL may effectively inhibitor adventitia and endothelial injury induce AS, and present a anti-atherosclerosis character.By analysis of MAPK,NF-κB signal pathway via IHC and western blot,we found that TXL may inhibit the p-p38,p-JNK expression of both side,And TXL can just inhibit NF-κB expression in endothelial injury atherosclerosis.