TLR4通过下调ABCA1表达促进平滑肌源性泡沫细胞形成

TLR4 promotes VSMCs-derived foam cells formation via down-regulating ABCA1 expression

目的探讨Toll样受体4(Toll-like receptor 4,TLR4)促进平滑肌源性泡沫细胞的形成是否与三磷酸腺苷-结合盒转运子A1(ATP-binding cassette transporter,ABCA1)表达异常相关。方法组织贴块法培养原代血管平滑肌细胞(vascular smooth muscle cells,VSMCs);细胞分为野生型(Wild type)空白对照组、Wild type氧化低密度脂蛋白(oxLDL)组、TLR4敲除(TLR4-/-)空白对照组和TLR4-/-oxLDL组;油红O染色观察VSMCs内脂质聚集,并用异丙醇萃取油红O测定光密度值;RT-qPCR、Western blot检测VSMCs中TLR4、ABCA1、IL-1β及TNF-αmRNA和蛋白表达水平;细胞免疫荧光染色鉴定原代VSMCs及观察核转录因子-kappa B(NF-κB)核转位。结果①Wild type oxLDL组中见细胞内大量脂滴形成,油红O光密度值为Wild type空白对照组的2.3倍(P<0.01);TLR4-/-oxLDL组中只见少许脂质沉积于细胞内,油红O光密度值与TLR4-/-空白对照组相比无明显变化(P>0.05)。②Wild type oxLDL组ABCA1 mRNA及蛋白表达水平较Wild type空白对照组分别下降61%(P<0.05),44%(P<0.01);TLR4-/-oxLDL组ABCA1 mRNA及蛋白表达水平分别为TLR4-/-空白对照组的2.3倍(P<0.05),1.7倍(P<0.05)。③Wild type oxLDL组VSMCs中NF-κB核转位明显增多,IL-1β和TNF-αmRNA表达较Wild type空白对照组分别升高3.9倍(P<0.05),1.7倍(P<0.05);TLR4-/-oxLDL组VSMCs中无明显NF-κB核转位,仅IL-1βmRNA表达较TLR4-/-空白对照组升高1.2倍(P>0.05)。结论 TLR4及其调节的炎性反应参与平滑肌源性泡沫细胞形成过程中ABCA1表达异常。

Objective To determine whether Toll-like receptor 4 （TLR4） promoting VSMCs-derived foam cells formation is related with the abnormal expression of ATP-binding cassette transporter 1 （ ABCA1 ）. Methods Vascular smooth muscle cells （VSblCs） were isolated from male C57BL/6J （TLR4-/-） and C57BL/lOScNj （ TLR4 -/- ） mice respectively, and primary cultured and identified. The obtained VSblCs were divided intowild type blank control cells, oxLDL-treated wild type cells （50 μg/mL oxLDL）, TLR4-/- blank control cells and oxLDL-treated TLR4 -/- cells. Neutral lipid inclusions in the VSblCs induced by oxLDL were assessed by Oil Red O staining. Lipid accumulation was quantitated by isopropanol extraction of Oil Red O from stained cells and optical density determinations at 518 nm. The mRNA and/or protein levels of TLR4, ABCA1, IL-1 β and TNF-α were determined by Western blotting and RT-qPCR. The NF-KB translocation was identified by immunofluorescence assay. Results Oil red O staining indicated that a large amount of lipid was accumu-- lated in oxLDL-treated wild type VSMCs, with the OD value increased by 2.3 folds compared with that of blank control （ P 〈 0.01 ）. Only few lipids were found in oxLDL-treated TLR4 -/- VSMCs, with no significant difference with the untreated cells （ P 〉 0.05 ）. The ABCA1 expression at mRNA and protein levels wasreduced by 61% （P 〈 0.05 ） and 44% （ P 〈 0.01 ）, respectively, in oxLDL-treated wild type VSMCs than in untreated cells. However, in the TLR4-/- VSMCs stimulated by oxLDL, the ABCA1 mRNA and protein expression were 2.3-fold （ P 〈 0.05 ） and 1.7-fold higher （ P 〈 0.05 ） than the TLR4 -/- blank control, respec- tively. The translocation of NF-KB was activated by TLR4 in oxLDL-treated wild type VSMCs, and the IL-1β and TNF-α mRNA expression were 3.9 folds （P 〈 0.05 ） and 1.7 folds （ P 〈 0.05 ） respectively of wild type blank controls. However, in oxLDL-treated TLR4 knockout VSMCs, NF-KB translocation and the expression of TNF-c~ had no significant change, but the expression of IL-113 was enhanced by 1.2 times （ P 〉 0.05 ） when compared with untreated TLR4 -/- VSMCs. Conclusion TLR4 and its mediating inflammation are involved in the abnormal expression of ABCA1 during VSMCs-derived foam cells formation.