Bezafibrate Promots Reverse Cholesterol Transport From Macrophages In Vivo And In Vitro In Mice

BackgroundIt has been found from numerous clinical epidemiological studies that the risk of developing coronary heart disease(CHD) is inversely related to high-density lipoprotein-cholesterol(HDL-C),which implies that HDL plays a role on the anti-atherosclerosis.Reverse cholesterol transport(RCT) is the only pathway from human body for excess cholesterol and one of the key mechanisms of HDL anti-atherosclerosis. RCT is a process that free cholesterol in peripheral cells,including macrophages,is transported to the liver in the form of HDL and excreted from liver.Macrophages are major cellular components of atherosclerotic lesions.Continuous accumulation of excess cholesterol in macrophages eventually leads to the formation of foam cells,which deposit on the arterial wall to form the characteristics of early atherosclerotic plaque. Therefore,Macrophages reverse cholesterol transport is the most crucial factor to atherosclerotic progress.Many proteins,receptors and transporters,such as ATP binding cassette transporter family(ABCA1, ABCG1,ABCG5 and ABCG8 etc.),scavenger receptor BI(SR-BI) and cholesterol 7alpha-hydroxylase(CYP7A1),all participate in RCT.And they are regulated by liver X receptorα(LXRα).Bezafibrate is a commonly used medicine that can efficiently lower triglyceride and increase HDL-C.It has proven from numerous clinical studies that bezafibrate significantly retards atherosclerosis progression and reduces the CHD death.Besides the effect of regulating the blood lipid,such as lowering triglyceride and increasing HDL-C,Bezafibrate has other effects, e.g.anti-inflammatory and anti-coagulation.A few studies indicate that the effect of bezafibrate on RCT is limited to affecting ABCA1 and ABCG5.However,no effective approach to assess RCT in vivo has been reported.In this work,an alternative approach has been developed on the basis of methods employed by Rader laboratory.We have evaluated the effect of bezafibrate on macrophages RCT in vivo and in vitro and discussed its possible mechanism.ObjectivesThe objectives of this thesis are to investigate the effect of bezafibrate on macrophage RCT in mice in vivo and the effects of bezafibrate and mice serum on cholesterol efflux from macrophage in vivo,by determining the changes of lipid concentration and the ratio of 3H-cholesterol in serum,liver and feces to the total dose of 3H-cholesterol intraperitoneally injected after treating mice with bezafibrate,and to discuss the possible mechanisms of bezafibrate promotes macrophage reverse cholesterol transport.MethodsAfter being treated with bezafibrate(0.1%,0.25%,0.5%W/W) for 4 weeks,C57BL/6 mice were injected intraperitoneally with ~3H-cholesterol-labeled and cholesterol-loaded(Ac-LDL) macrophages. The concentration of ~3H-tracer in samples of serum,liver and feces were determined by using liquid scintillation counter.Cholesterol efflux from 3H-cholesterol prelabled RAW264.7 macrophages incubated from mice serum with 0.25%bezafibrate in vitro was measured after being treated with different concentrations of bezafibrate.Reverse transcription polymerase chain reaction(RT-PCR) was used to evaluate mRNA expression of CYP7A1 and SR-BI of liver,ABCG5 and LXRαof liver and intestine as well as SR-BI,ABCA1,ABCG1 and LXRαof macrophage.And protein expression of ABCG5 and LXRαof liver as well as ABCG1 and LXRαof macrophage was evaluated with western-blot method.Results1.Being treated by bezafibrate(0.1%,0.25%,0.5%) for 4 weeks,the level of triglyceride(TG) and HDL-C was correlated to the bezafibrate dose.Compared to the control,HDL-C level increased (P＜0.01) by 26.4%,while TG level decreased(P＜0.01) by 34%in 0.5%bezafibrate group.2.Compared to those in the control group,the ~3H-cholesterol levels in serum of the mice of treated group(0.1%,0.25%,0.5%) were significantly higher by 100%,131%and 110%,respectively. Similarly,the ~3H-cholesterol levels in liver and feces were significantly higher by 86.4%,52.3%and 51.6%,by 110%,140% and 160%,respectively.3.Being treated with bezafibrate,Cholesterol efflux from macrophages mediated by the mice serum increased significantly,compared to those from the control group(P＜0.05).4.Bezafibrate(0,50,100,200μM) dose-dependently promoted cholesterol efflux from macrophages mediated by the mice serum.5.The experiements in vitro showed that the Bezafibrate(0,50,100, 200μM) with different concentrations dose-dependently increased the mRNA expression of ABCA1,ABCG1 and SR-BI as well as the protein expression of ABCG1,the mRNA and protein expression of LXRαof macrophages.6.The SR-BImRNA expression of liver was dose-dependently upregulated in mice treated with different dosage of bezafibrate (0.1%,0.25%,0.5%),compared to those in control group(P＜0.05).7.For treated group(0.1%,0.25%,0.5%),mRNA and protein expression of ABCG5 and LXRαof liver and intestine increased significant(P＜0.05),8.For treated group(0.1%,0.25%,0.5%,the CYP7A1mRNA expression of mice liver were dose-dependently lower than those of control group(P＜0.05).Conclusions1.Bezafibrate has following effects:1) Reduced the triglyceride(TG) level and increased the HDL-C level.2) Promoted macrophages RCT in vivo in mice.3) Promoted the cholesterol efflux from the cultured macrophages.2.The key mechanisms of Bezafibrate promoting macrophages reverse cholesterol transport:1) Bezafibrate increased the expression of ABCA1,ABCG1 and SR-BI to promote cholesterol efflux,which might be due to the up-regulation of LXRα.2) Bezafibrate promoted macrophages reverse cholesterol transport in vivo possibly by upregulating the expression of SR-BImRNA to mediate the selective uptake of cholesteryl esters from HDL in the liver and through upregulating expression of ABCG5 of liver and intestine to promote cholesterol efflux via bile and to reduce uptake of cholesterol in intestine,which might be due to the up-regulation of LXRα.3) Bezafibrate promoting macrophages reverse cholesterol transport in vivo had no relation to the expression of liver CYP7A1mRNA in mice.