The Impact Of TCDD On VSMC And Its Role In The Repairing Of Artery Intimal Injury

Today our country has entered aging society period.With the increase of the aged population,the incidence of cerebrovascular disease is also increasing.Many studies have shown that cerebrovascular diseases have become the leading cause of death and disability in China,so it is of great significance to study the occurrence and development mechanisms and influencing factors of cerebrovascular disease.Researches have shown that the occurrence and development of cerebrovascular diseases are related to the imbalance between vascular damage factors and vascular repair factors.Inflammatory factors and hemodynamics are considered to play an important role in them..In recent years,more and more attention has been paid to the relationship between environmental factors and diseases.As a harmful environmental pollutant,TCDD is widely distributed and has many sources.It has been detected in the atmosphere and many organisms.Many countries,including China,have placed the control of TCDD emissions in an extremely important position.TCDD is relatively stable,not easy to degrade,and will accumulate in organisms with the food chain.It has a strong affinity for AhR in vivo and plays an important role in immunity,inflammation,toxicology and cancer by activating AhR.TCDD has been found to be related to the occurrence of many diseases.However,there are few studies on the relationship between TCDD and cerebrovascular diseases.Previous studies have shown that smoking can induce inflammation through AhR and play a role in diseases such as aneurysms and atherosclerosis.Previous studies have also shown that environmental pollutants such as TCDD can affect the production of IL-1? in many cells,and inflammatory cytokines such as IL-1? are thought to be associated with the occurrence of cardiovascular and cerebrovascular diseases such as atherosclerosis.Therefore,we speculate that TCDD can affect the balance of vascular wall repair by activating AhR to produce inflammation on vascular wall,thus affecting the occurrence and development of cerebrovascular diseases.The main purposes of this study are as follows: 1.To study the impact of TCDD on vascular endothelial repair in two different mice models of carotid artery injury;2.To study the impact of TCDD on vascular smooth muscle cells and the related mechanisms.The main technical methods in this study include: two different models of carotid artery injury in mice,fixed embedding sections of specimens,HE and immunohistochemical staining,immunofluorescence staining,real-time quantitative PCR,cell culture and passage,establishment of phenotypic transformation model of vascular smooth muscle cells,CCK8 cell viability test,cell migration ability test,enzyme-linked immunosorbent assay(ELISA),Western blotting,medical statistics,etc.Part ? The impact of TCDD on the repair of endothelial injury in carotid artery guide wire injury model in miceObjectives: 1.To investigate the impact of TCDD on the repair of endothelial injury in carotid artery guide wire injury model in mice.2.To detect the main cell types and the expression of IL-1? in neointima.Methods: 1.Acute administration of TCDD: TCDD group,corn oil group,control group and sham operation group were randomly divided into three groups.Different doses of TCDD were injected into abdominal cavity in advance,and then the model of carotid artery guide wire injury was established in mice.2.Chronic administration of TCDD was divided into one month group and two months group.Each group was further randomly divided into TCDD group,corn oil group,control group and sham operation group.TCDD group was divided into three groups: 0.1 ug/kg,1 ug/kg and 10 ug/kg.Establishment of carotid artery guide wire injury model in mice after long-term administration;3.Detection of the antagonistic effect of CH-223191 on TCDD: corn oil group,TCDD group and CH-223191 + TCDD group.4.After 2 weeks,carotid artery specimens were taken for HE staining to detect intimal hyperplasia,and Image-Pro Plus software was used to analyze and calculate intimal/medial area.Immunohistochemistry and immunofluorescence were used to detect ?-SMA,CD31,IL-1? and other indicators.5.Differential expression of IL-1? and other genes in carotid artery specimens was detected by QPCR.Results: 1.The effect of carotid artery endothelial exfoliation caused by wire injury in this model is obvious and the model is successful.2.Acute and chronic administration of TCDD can promote intimal hyperplasia induced by carotid artery guide wire injury model and there is a dose-effect relationship between chronic administration of TCDD and intimal hyperplasia.3.TCDD promotes the expression of IL-1? in neointima induced by carotid artery guide wire injury model.4.TCDD antagonist CH-223191 attenuates intimal hyperplasia in carotid artery guidewire injury model induced by TCDD.Conclusion: 1.TCDD promotes intimal hyperplasia induced by carotid artery guide wire injury model,and the intimal hyperplasia is proportional to the dose-effect of TCDD administration;2.Smooth muscle cells are the main components of intimal hyperplasia;3.TCDD promotes the expression of inflammatory factor IL-1? in intimal hyperplasia;IL-1? may be associated with intimal hyperplasia;4.The role of TCDD in promoting intimal hyperplasia is AhR dependent.Part ? The impact of TCDD on the repair of endothelial injury in carotid artery ligation model in miceObjectives: 1.To investigate the effects of TCDD on intimal hyperplasia in carotid artery ligation model of mice and the cell types of intimal hyperplasia.2.To detect the expression of IL-1? in proliferative intima.Methods: 1.Establishment of acute carotid artery ligation model in mice after intraperitoneal injection in advance;2.2 weeks later,carotid artery specimens were taken for HE staining to detect intimal hyperplasia,and Image-Pro Plus software was used to analyze and calculate intimal/medial area;immunohistochemistry and immunofluore-scence were used to detect ?-SMA,CD31,IL-1? and other indicators.3.Differential expression of IL-1? and other genes in carotid artery specimens was detected by QPCR.Results: 1.TCDD promotes intimal hyperplasia induced by carotid artery ligation model;2.TCDD promotes the expression of inflammatory factor IL-1? in proliferative intima;IL-1? may be associated with intimal hyperplasia;3.CH-223191 attenuates the intimal hyperplasia induced by TCDD in carotid artery ligation model.Conclusion: TCDD promotes intimal hyperplasia induced by carotid artery ligation,and the main component of intimal hyperplasia in this model is vascular smooth muscle cells.TCDD promotes the expression of inflammatory factor IL-1? in intimal hyperplasia.The effect of TCDD on intimal hyperplasia is AhR-dependent.Part ? Effect and mechanism of TCDD on human cerebral vascular smooth muscle cellsObjectives: 1.To study the effects of four common phenotypic transformation stimulators on phenotypic transformation of human cerebrovascular smooth muscle cells;2.To study the effects of TCDD and IL-1? on phenotypic transformation of human cerebrovascular smooth muscle cells and the changes of proliferation and migration after phenotypic transformation;3.To study the effects of TCDD on the expression of IL-1? in human cerebrovascular smooth muscle cells;4.To study the effects of TCDD on IL-1? expression in human cerebrovascular smooth muscle cells Mechanisms for reaching impact.Methods: 1.Human cerebrovascular smooth muscle cells were stimulated with four commonly used phenotype transformation stimulators: PDGF-BB(20ng/ml),IL-1?(40ng/ml),TNF-alpha(20ng/ml)and TGF-?(10ng/ml),respectively,and the changes of alpha-SMA and calponin levels were detected.2.Human cerebrovascular smooth muscle cells were stimulated with 0.1nM,1nM and 10 nM TCDD,respectively.Changes of onin and IL-1? levels;3.Select 1nM TCDD and 40ng/ml IL-1? to stimulate human cerebrovascular smooth muscle cells individually and jointly to detect the changes of proliferation and migration of vascular smooth muscle cells;4.Detect the antagonistic effect of CH-223191 on TCDD;5.Detect the effect of TCDD on the secretion of IL-1? by human cerebrovascular smooth muscle cells;6.Detect the inflammation of human cerebrovascular smooth muscle cells NLRP3 by TCDD.The changes of IL-1? after TCDD stimulation were detected by using NF-?B inhibitor.Results: 1.IL-1? could induce phenotypic transformation of human cerebrovascular smooth muscle cells and enhance their proliferation and migration after phenotypic transformation.2.TCDD could promote phenotypic transformation of human cerebrovascular smooth muscle cells,and also increase the expression and secretion of IL-1? in vascular smooth muscle cells.The level of NLRP3 did not change with IL-1?.NF-?B inhibitor can inhibit the increase of IL-1? expression induced by TCDD.Conclusion: IL-1? mediates human cerebrovascular smooth muscle cells phenotypic switching.After phenotype switching,the proliferation and migration ability of human cerebrovascular smooth muscle cells increases.TCDD can promote the increase of IL-1? expression in human cerebrovascular smooth muscle cells.The mechanism is mediated by AhR and associated with NF-?B signaling pathway.But the NLRP3 inflammasome is not activated.Summary: 1.TCDD can promote the increase of intimal hyperplasia and the expression of IL-1? in carotid artery guide wire injury model and carotid artery ligation model in mice;2.TCDD can promote the phenotypic switching of vascular smooth muscle cells and the expression of IL-1?;IL-1? can promote the phenotypic switching and proliferation and migration of vascular smooth muscle cells,which may be the origin of TCDD promoting intimal hyperplasia.3.The increased expression of IL-1? induced by TCDD may be mediated by NF-?B signaling pathway,but the NLRP3 inflammasome is not activated.