Probucol Promotes Macrophages Reverse Cholesterol Transport In Vitro And In Vivo In Mice

BackgroundReverse cholesterol transport (RCT) is defined as the process ofexcess cholesterol transport from the peripheral cells, includingmacrophages, to the liver and bile, followed by excretion in the feces.RCT is the key pathway of cholesterol excretion, and one of theimportant mechanisms of anti-atherosclerosis. Probucol is only alipid-lowering drug, which regresses xanthomas in familyhypercholesterolemic patients. It attenuates atherogenesis, even regressesthe plaque in animals. Although the exact mechanism of probucol againstatherosclerosis is not completely understood, the role of probucol inreverse cholesterol transport may have contributed to theanti-atherosclerosis. A number of studies indicated that probucol mightpromote RCT through modifying cholesterol ester transport protein,lecithin-cholesterol acetyl-transferase, apolipoprotein E and scavengerreceptor class B typeⅠ(SR-BI). However, probucol inhibits ATP bindingcassette (ABC) A1 mediated cholesterol effiux, which is the first step ofRCT. Most of previous studies investigated each single step of RCT,which has been adjusted by probucol in vitro. Recently the critical role ofABCG1, ABCG5/ABCG8 for RCT has been recognized, but it has notbeen reported that they were related or not to the role of probucol on RCT. Foam cells derived from macrophages deposit in the arterial wall formthe primary atherosclerotic plaque. Macrophages RCT is most directlyrelevant to the atherosclerotic progress. The effect of probucol onintegrated macrophage RCT remains unknown. For the absence of themethod to assess RCT in vivo, it has no the direct evidence. In this study,we try to establish one method to directly assess RCT in vivo accordingto the methods used recently by Rader's lab, we observe the effect ofprobucol on macrophage RCT in vitro and in vivo and explore thepossible mechanism by which probucol promotes macrophage RCT.ObjectiveThis study establishes one method to directly assess macrophagesRCT in vivo; investigates the effect of probucol on macrophages RCT invivo, observes the effect of probucol on cholesterol effiux frommacrophages in vitro, and explores the possible mechanisms by whichprobucol promotes macrophage RCT.Methods~3H-cholesterol-labeled and acetyl-low density lipoprotein cholesterol(Ac-LDL)-loaded RAW264.7macrophages were injected intraperitoneallyinto the C57BL/6J mice. The appearance of ~3H-cholesterol in serum, liver,and feces were monitored by liquid scintillation counter.~3H-cholesterol-labeled and Ac-LDL-loaded RAW264.7 macrophages were injected intraperitoneally into the C57BL/6J mice, which weretreated with either vehicle or different dosages of probucol (0.1%, 0.5%,1% W/W) for 4 weeks. The appearance of ~3H-tracer in serum, liver, andfeces were monitored also. Cholesterol effiux from ~3H-cholesterol-labeledRAW264.7 macrophages treated with different concentrations ofprobucol and incubated with serum from mice administrated with 0.5%probucol in vitro was measured; The mRNA and protein expression ofABCA1/ABCG1/SRBI in macrophages, the gene and protein expressionof SR-BI/CYP7a/ABCG5 in liver and ABCG5 in intestine werequantified with RT-PCR and Western-blot respectively.Results1. After ~3H-cholesterol-labeled and acetyl-low density lipoproteincholesterol (Ac-LDL)-loaded RAW264.7macrophages were injectedintraperitoneally, ~3H-cholesterol could be monitored in serum, liverand feces of the mice, the percents to the total injected were2.81±0.06, 0.68±0.03 and 0.69±0.03, respectively.2. Probucol (0.1%, 0.5%, 1%) dose-dependently redused serum totalcholesterol (TC, 1.78±0.22, 1.43±0.20, 1.42±0.15 VS 2.71±0.43) andhigh-density lipoprotein (HDL-C, 0.94±0.16, 0.71±0.14, 0.70±0.10VS 1.60±0.10)(P＜0.01), low-density liprotein (LDL-C, 0.37±0.08,0.32±0.08, 0.31±0.07 VS 0.47±0.06) and triglyceride (TG, 0.91±0.12, 0.82±0.07, 0.84±0.12 VS 1.16±0.21) were lowered also (P＜0.05), butthere were no significant difference between 0.5% and 1% probucolgroups.3. The serum ~3H-cholesterol levels in the mice treated with differentdosage of probucol (0.1%, 0.5%, 1%) were dose-dependently andsignificantly higher at 6hrs (6h: 1.27±0.01, 1.42±0.01, 1.43±0.02 VS1.08±0.02), 24hrs (24h: 1.86±0.03, 2.52±0.02, 2.54±0.02 VS 1.41±0.02), and 48hrs (48h: 1.05±0.01, 1.19±0.01, 1.17±0.01 VS 0.94±0.03) hours than those in control group (P＜0.05), but there were nosignificant difference between 0.5% and 1% probucol groups.4. The liver ~3H-cholesterol levels in the mice treated with differentdosage of probucol (0.1%, 0.5%, 1%) were dose-dependently lowerthan those in control group (1.65±0.05, 1.57±0.03, 1.56±0.02 VS1.86±0.01, P＜0.05); The fecal total 3H-cholesterol levels weredose-dependently and significantly higher than those in control group(2.81±0.04, 3.75±0.03, 3.79±0.07 VS 1.37±0.04, P＜0.01), but therewere no significant difference between 0.5% and 1% probucolgroups.5. The mRNA expression of liver CYP7A1 dose-dependently wasupregulated in mice treated with probucol (0.1%, 0.5%, 1%)compared with those in the control group (0.68±0.01, 0.96±0.02, 0.98±0.04 VS 0.51±0.02, P＜0.05); The mRNA expression (0.64±0.02,0.86±0.04, 0.85±0.03 VS 0.45±0.02, P＜0.05) and the proteinexpression (0.61±0.02, 0.95±0.03, 0.96±0.04 VS 0.45±0.02, P＜0.05)of liver ABCG5 dose-dependently increased also in mice treated withprobucol (0.1%, 0.5%, 1%). No significant difference exists between0.5 % and 1% probucol groups.6. The SR-BI mRNA and protein levels of the liver in mice treated withprobucol did not change significantly compared with control mice(P＞0.05).7. The serum from the mice treated with probucol for 4 weeks mediatedmore cholesterol effiux from the cultured macrophages (38±0.03% VS20±0.03%, P＜0.01).8. Different concentrations of probucol (10, 20, 50, 100μmol/L vs0μmol/L) dose-dependently upregulated the mRNA (0.75±0.03,0.94±0.03, 1.18±0.02, 1.22±0.04 VS 0.52±0.04) and protein(0.68±0.03, 0.85±0.05, 1.17±0.03, 1.19±0.04 VS 0.49±0.04)expression of ABCG1 in macrophages, and promoted cholesteroleffiux (25±0.06%, 29.1±0.04%, 34.1±0.04%, 34.9±0.08% VS20±0.03%), (P＜0.05).9. Different concentrations of probucol (10, 20, 50, 100μmol/L vs0μmol/L) had no effect on the mRNA and protein expression of SR-BI and ABCA1.Conclusions1. The method that mornitering ~3H-tracer in the plasma, liver, and fecesof mice after being injected intraperitoneally with ~3H-cholesterollabled macrophages could be used as a kind of feasible way tomeasure the integrated macrophages RCT in vivo.2. Probucol significantly and dose-dependently reduced serum levels ofTC/HDL-C/LDL-C and TG, but the dose of 0.5%probucol had themost effects.3. Probucol dose-dependently promoted macrophages RCT in vivo inmice.4. Probucol promoted macrophages RCT in vivo possiblely throughupregulating the expression of liver CYP7A1 and ABCG5 in liver andintestine.5. Probucol promoted macrophages RCT in vivo possiblely had norelation to the expression of liver SR-BI in mice.6. The serum from the mice treated with probucol markedly promotedcholesterol efflux from the cultured macrophages.7. Probucol maybe promoted cholesterol efflux from the macrophagesthrough increasing the expression of ABCG1. 8. Probucol promoted cholesterol effiux from the macrophagespossiblely had no relation to the expression of ABCA1 and SR-BI.