Effect Of The Endocannabinoid System In Rabbit Carotid Atherosclerosis By Removal Of Membrana Adventitia

BACKGROUND AND OBJECTIVE :The researches about the mechanisms of atherosclerosis are hotspot in the fleld of cardiovascular disease.A great deal researches indicated that atherosclerosis was resulted by the interaction of multiple factors. The inflammatory cells and inflammatory factors take part in the whole procedure of atherosclerosis.In recent years the influence of adventitia in atherosclerosis was noticed. Many researches proved adventitial imflammation was the triggering factor and characteristic of advanced stage in atherosclerosis. So it is promising that constructing animal atherosclerotic model by interfering in the membrane adventitia.The endocannabinoid is a lipoid substance which has the similar function of THC. It has the function of anti-inflammatory and immunosuppressive effects in vivo and in vitro. The endocannabinoid system is composed of the cannabinoid receptors , endocannabinoids and the enzymes required for their synthesis and degradation. Some studies suggested that THC has the action of inhibiting atherogenesis through activing the cannabinoid recepor and the endocannabinoid can influence the chemotaxis, immigration,proliferation and apoptosis of the cells which are related to atherosclerosis. These studies suggested the endocannabinoid may influce the all stage of atherosclerosis. However, the changes of the endocannabinoid system in atherosclerosis are not clear elucidated.According to the above research background, we constructed atherosclerotic animal model by removing the arteries adventitia. Through it we observed the changes of the endocannabinoid and the cannabinoid receptors to find out the effect of endocannabinoid system in atherosclerosis. we observe the effect of Tongxinluo in atherosclerosis to find out the possible mechanism .PART I: The distribution and expression of the endocannabinoid system in Rabbit Carotid Atherosclerosis by removal of Membrana AdventitiMETHODS: 24 male New Zealand White rabbits were divied into two groups randomly,one group were fed with normal diet and the other group were fed with high-cholesterol diet. Digestion and blunt dissection injury in the left carotid artery adventitia was performed, while sham operation was performed in the right artery. After that, they were fed for 4 weeks or 8 weeks until the end of the study. The carotid arteries were collected for histological analysis of paraffin sections stained with Hematoxylin and Eosin, the levels of IL-6 and TNF-αdetermined by ELISA, the CB2 receptor and the CD68 by immunohistochemical staining, and the endocannabinoid by LC-MSRESULTS: Combination of digestion with collagenase and mechanical dissection could injury vascular adventitia efficiently.The level of serum lipids were elevated after high-cholesterol diet;We did not find the intimal hyperplasia in the normal diet group, Intimal hyperplasia lesion could be observed in adventitial injured carotid arteries of high-cholesterol diet group compared with control side and normal diet group, and classical atherosclerotic plaque with macrophages infiltrated at 8 weeks.There is no different of inflammatory factor in the vessel between normal diet group.But the level of IL-6 and TNF-αwere elevated in high-cholesterol diet compared with the normal dietgroup.The CB2 was detected in the in the thickered intima.The expression of CB2 and 2-AG were elevated with the prolongation of the high-cholesterol diet ,and the level of 2-AG was significantly associated with the level of IL-6 and TNF-α,but the level of AEA was not changed.PART II: The effect of endocannabinoid system in rabbit carotid atherosclerosis by removal of membrana adventitiaMETHODS:36 male New Zealand White rabbits were divied into three groups randomly:the AS model group,the URB602 gel group(administered with URB602 dissolved in F-127 gel solution around carotid) and the Tongxinluo group(administered with tongxinluo 0.4g·kg-1·d-1). Digestion and blunt dissection injury was performed in the left carotid artery adventitia, and sham operations were performed in the right artery before they were fed high-cholesterol diet.After 4 weeks or 8 weeks,the animals were executed to collect the carotid arteries to analysis of paraffin sections by stained with Hematoxylin and Eosin,the CB2 receptor and CD68 by immunhistochemical staining,the IL-6 and TNF-αby ELISA,the endocannadinoid by LC-MS.RESULTS:The level of serum lipids were higher in 8 weeks model group than 4 weeks model group.There is no different between the gel group and the model group.The level of serum lipids of the Tongxinluo groups were lower than the the corresponding time model group.Compared with 4 weeks model group, the IMR of 4 weeks gel group was significantly lower,but there is no significantly different between the 8 weeks gel group and the 8 weeks model group;The IMR of tongxinluo group was significantly lower than the model group. The inflammatory factors of the 8 weeks were higher than the 4 weeks in every group.In the gel group and the tongxinluo group ,the inflammatory factors were significantly lower than the model group,the level of inflammatory factors in the 8 weeks Tongxinluo group was the lowest in all groupsThe CB2 receptor was dectected in the thickered intima and the level of the expression of CB2 receptor was higher with the thicker of the intima.The expression of CB2 receptor in the early gel groups and the all tongxinluo groups were lower than the model group following the decrease of the intima thickness.The level of 2-AG of the 8 weeks model group was higher than the 4 weeks,and the 4 weeks URB602 gel group was higher than the model groups,there is no different between the 8 weeks gel group and the 8 weeks model group;the tongxinluo group was significant higher than the model groups in 4 weeks and in 8 weeks and the level of the 2-AG of the 8 weeks tongxinluo group was the highest in the all groups.CONCLUSION:1,We can successfully construct rabbit carotid atherosclerotic model by high-cholesterol diet and removal of membrana adventitia.2,With the progress of the atherosclerosis,the level of the inflammatory factors in the artery were elevated,and the endocannabinoid system was up-regulation,the expressiong of CB2 receptor and the level of 2-AG were elevated.3.It can delay the progress of the atherosclerosis and the level of the inflammatory factors of the lesion with the means of evevating the level of the 2-AG through locally applied the specificity MAGL inhibitor URB602 in the lesion .4,Tongxinluo could inhibit atherogenesis in part by further elevating the level of 2-AG and inhibit the inflammatory level of the artery.It may be the new mechanism of inhibiting atherogenesis.