| Objective: Atherosclerosis is the most important pathophysiological basis for cardiovascular disease.Current studies have shown that reverse cholesterol transport is an effective mechanism for preventing or even reversing atherosclerotic plaques.Cholesterol efflux from macrophages in plaques is the initial step of cholesterol reverse transport.This study focuses on the protective mechanism of estrogen on atherosclerosis,and uses macrophage cholesterol efflux as the research content.Through the analysis of cholesterol efflux related indicators in young women with coronary heart disease,the clinical characteristics of young women with coronary heart disease are explored;in vitro studies are conducted to explore the target of estradiol on cholesterol efflux from macrophages and validated in mice.Provide a new theoretical basis for estrogen in cardiovascular protection.Methods: 1)The young women who had confirmed coronary artery disease by coronary angiography were used as the case group,and the women with negative coronary angiography in the same age group were used as the control group.The clinical data of the two groups were compared,as well as estrogen levels,plasma cholesterol efflux capacity,and the expression of ABCA1,ABCG1,ESR1 m RNA in peripheral blood is different.2)Divide J774 A.1 cells into three groups: control group,estradiol group,fulvestrant +estradiol group,and observe the phenotypic differences between the three groups through cholesterol efflux experiment and foam cell induction experiment.Detection of macrophage cholesterol efflux gene expression is affected by estradiol.Construct the small interfering RNA of the target gene according to the actual situation to further verify the regulatory relationship.3)Take the C57BL/6 female mice with apo E knockout as the research object,they are divided into sham operation group(sham),ovariectomized group(OVX),ovariectomized group + estradiol group(OVX+E2),The atherosclerosis model was constructed with high-fat diet for 8 weeks.The differences in the formation of aortic root plaque and the expression of ABCA1 in coronary arteries were detected between the groups;the peritoneal macrophages were isolated and the expressions of ABCA1,ABCG1 and ERαwere detected.Results: 1)Taking 55 years as the demarcation point,the baseline data between the coronary heart disease group and the control group of different age groups show that the BMI of the coronary heart disease group of women ≤55 years old is higher than the other three groups,and the HDL-c is lower than the other three groups,the differences were statistically significant;the TG of the control group ≤55 years old was lower than that of the coronary heart disease group;the proportion of hypertension and diabetes in the coronary heart disease group was higher than that of the control group of the same age.Women subjects ≤55 years of age were divided into groups according to whether they were menopausal or not.The baseline data of the coronary heart disease group and the control group showed that regardless of menopause,the BMI,proportions of hypertension and diabetes,TC in the coronary heart disease group were greater than those of the control group;The levels of HDL-c and apo A-1 in the coronary heart disease group are lower than those of the control group in the same menopausal state.The total RNA in the peripheral blood of female subjects ≤55 years of age was extracted,and the m RNA expression level of the target gene was detected by real-time fluorescent quantitative PCR.The results showed that the expression levels of ABCA1 and ESR1 m RNA in the coronary heart disease group were lower than those in the control group.Through the analysis of the GEPIA online database,it is found that the m RNAs of ESR1 and ABCA1 in the peripheral blood of healthy people have a strong correlation,with the correlation coefficient R=0.75 and P=0.The Logistic multivariate analysis of coronary heart disease in women aged ≤55 years showed that without ABCA1 m RNA model,the risk of coronary heart disease in young women with hypertension is 2.11 times that of non-hypertension.Young women with diabetes suffer from coronary heart disease risk of coronary heart disease is 3.91 times that of non-diabetic patients;for every 1 unit increase in TC,the risk of coronary heart disease in young women increases by 61%.Included in ABCA1 m RNA model,young women increase their risk of coronary heart disease by27%for every 1 year old;and the risk of coronary heart disease increases by 40%for every1 unit increase in BMI;and for every 1 unit increase in TC,the risk of coronary heart disease increases by 383%.2)In in vitro studies,mouse mononuclear macrophages J774 A.1 were incubated with estradiol,estradiol + fulvestrant,and NBD fluorescently labeled cholesterol was used to detect the cholesterol outflow rate,whether it was HDL or special treatment standard serum was used as cholesterol efflux induction solution,the cholesterol efflux rate of J774 A.1 cells in the estradiol incubation group was higher than that of the control group.The estradiol + fulvestrant incubation group could reverse the effect of estradiol in promoting cholesterol efflux,and Lower than the cholesterol outflow rate of the control group.In the foam cells induced by ox-LDL in J774 A.1,the lipid droplet content in the estradiol incubation group was lower than that in the control group,and the lipid droplet content in the estradiol + fulvestrant incubation group was higher than that in the control group and estradiol group.In estradiol-incubated cells,ABCA1 m RNA was significantly different in the upregulation folds of the 100 n M and 1000 n M estradiol treatment groups compared to the control group,and ABCA1 m RNA levels were reduced in the estradiol+fulvestrant group.At the protein level,the total ABCA1 protein and membrane protein levels of the estradiol treatment group were higher than those of the control group,while the ABCA1 protein of the estradiol + fulvestrant co-incubation group was lower than that of the estradiol treatment group.After knocking down ESR1 with si RNA,the expression of ABCA1 m RNA and protein decreased,the expression of ABCA1 did not increase after adding estradiol;after knocking down si-LXRα with si RNA,the expression of ABCA1 m RNA and protein decreased.After adding estradiol on the basis of LXRα,the expression of ABCA1 increased relative to the LXRα knockdown group alone.3)In animal experiments,the weight of mice in the OVX group was higher than that of the sham group,and the weight of the mice in the OVX+E2 group was also higher than that of the sham group.There was no difference between the OVX group and the OVX+E2 group;the aortic plaque area and plaque in the OVX group The lipid content in the block was greater than that in the sham group and the OVX+E2 group.The aortic plaque area and the lipid content of the plaque in the OVX+E2 group were greater than that in the sham group.The Western blot results of peritoneal macrophages showed that ABCA1,ABCG1 and ERα protein were the lowest in the OVX group,and were statistically different from the sham group and the OVX+E2 group.The ABCA1,ABCG1,ERα protein expression in the OVX+E2 group was high In the OVX group,it is also lower than the sham group.Coronary artery immunohistochemistry showed that the coronary arteries had ABCA1 expression.The expression of ABCA1 in the OVX group was lower than that of the sham group and OVX+E2 group,and the expression of ABCA1 in the OVX+E2 group was lower than that of the sham group.Conclusion: 1)Young women with coronary heart disease show high BMI,high blood pressure and diabetes combined rate,as well as high TG,low HDL-c,which is more obvious in young women who are not menopausal.ESR1 and ABCA1 m RNA have a good correlation,and their expressions in young women with coronary heart disease are lower than those in the control group.In without ABCA1 m RNA model,hypertension,diabetes,and TC are independent risk factors for CHD in young women;in contained ABCA1 m RNA model,age,BMI,and TC are independent risk factors for CHD in young women.2)Estradiol can promote cholesterol efflux from mouse mononuclear macrophages J774 A.1 and reduce the formation of lipid droplets in macrophage-derived foam cells;this is achieved through the positive regulation of ABCA1 after estradiol and ERα are combined It is independent of the positive regulation of LXRα on ABCA1.3)The lack of endogenous estrogen can cause female mice to gain weight,increase the area of aortic atherosclerotic plaques,and down-regulate ABCA1 in the coronary arteries;supplementation of estradiol can delay mice due to endogenous estrogen to a certain extent Atherosclerosis caused by deletion is partly caused by the up-regulation of ABCA1;the expression of ABCA1,ABCG1 and ERα in the peritoneal macrophages of female mice lacking endogenous estrogen decreases,and supplementation of estradiol can improve this effect,But it cannot be completely corrected. |
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