| BackgroudThe adventitia is an "active participant" of vascular remodeling diseases such as atherosclerosis(AS)and restenosis after percutaneous coronary intervention(PCI).Hyperhomocysteinemia is an independent risk factor for atherosclerosis and intervention.Previous studies have focused on the effects of homocysteine on vascular endothelial cells and smooth muscle cells,but less on adventitial fibroblasts.Based on the theory of "fussing evils",Huotan Jiedu Tongluo decoction which is adept at tonifying qi,eliminating phlegm and dredging collaterals,has a good effect on clinical treatment of atherosclerotic diseases.This study use a method of hyperhomocysteinemia combined with balloon injury in rabbit common carotid artery and homocysteine-induced injury of adventitial fibroblasts to observe the vascular remodeling,the changes of adventitia's NO/NOS system and the effects of proliferation of adventitial fibroblasts.We aim to explore the intervention effect and mechanism of Huotan Jiedu Tongluo decoction,and to find a new target for prevention and treatment of atherosclerosis and restenosis after percutaneous coronary intervention.Objectives1.To establish a rabbit model caused by hyperhomocysteinemia combined with balloon injury and observe the vascular remodeling,the curative effect of Huotan Jiedu Tongluo decoction on the prevention of vascular remodeling.2.To observe the changes of NO/NOS system in the adventitia of rabbit model and the regulation of Huotan Jiedu Tongluo decoction on it.3.To observe the changes of proliferation of adventitial fibroblasts induced by homocysteine and to elucidate the effects of Huotan Jiedu Tongluo decoction.Methods1.Japanese purebred large-ear white rabbits were randomly divided into sham operation group,model group,Huotan Jiedu Tongluo decoction medium dose group,Huotan Jiedu Tongluo decoction high dose group,atorvastatin group.Model group and drug groups are all fed with methionine and was treated with parallel balloon to injury the left common carotid artery.The sham operation group was fed with normal feed without balloon injury but cut the neck.Vascular remodeling,blood lipids levels and homocysteine levels in animal models was evaluated by HE staining,Masson staining,biochemical method and Elisa.2.We test the hanges of nitric oxide,endothelial nitric oxide synthase,inducible nitric oxide synthase and reactive oxygen species of serum in each group by nitrate reductase method and Elisa.Semi-quantitative analysis of eNOS and iNOS protein expression in intima and adventitia of carotid artery was performed by immunohistochemistry.3.The rabbit adventitial fibroblasts were extracted by tissue block adherence method and identified by immunocytochemistry.Cell proliferation was detected by CCK-8 method.The effects of Huotan Jiedu Tongluo decoction on proliferation of adventitial fibroblasts induced by homocysteine(Hcy)were observed.Results1.HE staining showed that the intima thickness,media thickness,adventitial thickness,adventitial area and stenosis rate were significantly increased in the model group compared with the sham operation group(p<0.05).Medium dose of Huotan Jiedu Tongluo decoction could reduce outer membrane thickness and outer membrane area(p<0.05)and has a tendency to reduce the thickness of the media(p=0.057).High dose of Huotan Jiedu Tongluo decoction could reduce the media thickness of the model group(p<0.05).The serum cholesterol(TC),high-density lipoprotein(HDL)and low-density lipoprotein(LDL)in the model group were significantly higher than those in the sham operation group(p<0.05),and the drug group could lower the level of high-density lipoprotein in the model group(p<0.01).Compared with the model group,the mean value of triglyceride(TG),TC in drug groups were lower than that of the model group,but there was no statistical difference.The mean value of Hcy in the model group increased,but there was no statistical difference compared with the sham operation group.Compared with the model group,the mean value of Hcy in drug groups were lower than that of the model group,but there was no statistical difference.2.Masson staining showed that the ratio of collagen in the inner,middle and outer membranes was significantly increased in the model group compared with the sham operation group(p<0.01).The medium dose of Huotan Jiedu Tongluo decoction could reduce the ratio of collagen in the inner,middle and outer membranes of the model group(p<0.05),the high dose of Huotan Jiedu Tongluo decoction and atorvastatin calcium could reduce the ratio of adventitial collagen in the model group(p<0.01).3.The results of nitrate reductase method showed that there was no significant difference in serum NO content between the groups.Elisa assay showed that the ratio of eNOS and iNOS in the model group was significantly lower than that in the sham operation group(p<0.01).Compared with the model group,the medium dose of Huotan Jiedu Tongluo decoction,high dose of Huotan Jiedu Tongluo decoction and calcium atorvastatin increased the ratio of eNOS and iNOS(p<0.01).However,there were no significant changes in serum levels of eNOS,iNOS and ROS in the drug groups.4.According to the results of immunohistochemistry,we found that there was no statistically significant difference in the expression of eNOS and iNOS protein in carotid artery and adventitia between the groups.The mean optical density of eNOS protein in adventitia of carotid artery of rabbits in the medium dose of Huotan Jiedu Tongluo decoction and the high dose of Huotan Jiedu Tongluo decoction and the atorvastatin calcium group increased relatively,and the mean average optical density of the iNOS protein in intima and adventitia reduced.Immunofluorescence showed eNOS expression in the adventitia.5.When the concentration of homocysteine was 100?mol/L,it could induce the proliferation of fibroblasts at 6 hours.The concentration of Hcy was 100 ?pmol/L,and the time was 6h for the experimental stimulation.After using lyophilized powder of Huotan Jiedu Tongluo decoction for 24 hours,it was found to inhibit the proliferation of adventitial fibroblasts induced by homocysteine.The strongest inhibitory concentration of lyophilized powder was 300?g/ml(p<0.01).Conclusions1.The thickness and collagen of the inner,middle and outer membranes are significantly increased and the ration of eNOS and iNOS of serum is abnormal in rabbit model caused by hyperhomocysteinemia combined with balloon injury,and the dyslipidemia also occurs in the model.We also find that the eNOS express in the adventitia.Above results suggest that vascular remodeling is associated with eNOS/iNOS abnormalities in this model.2.Huotan Jiedu Tongluo decoction can regulate blood lipids,reduce the vascular proliferation and collagen content in rabbit model,inhibit vascular remodeling,raise the ratio of eNOS and iNOS,increase the eNOS protein of carotid artery and decrease the expression of iNOS protein in inner and outer membranes.Simultaneously,it can significantly reduce homocysteine-induced proliferation of adventitial fibroblasts,suggesting that Huotan Jiedu Tongluo decoction can improve vascular remodeling especially the remodeling of adventitia,by regulating eNOS/iNOS system probably.And the more in-depth mechanism needs further study. |
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