脂多糖通过LDLr诱导巨噬细胞泡沫化:炎症应激下巨噬细胞泡沫化的另一条通路

目的研究炎症介质-脂多糖(LPS)诱导的炎症应激是否通过扰乱固醇调节元件结合蛋白裂解激活蛋白-固醇调节元件结合蛋白2(SCAP-SREBP2)复合物介导的低密度脂蛋白受体(LDLr)负反馈调控,增加THP-1巨噬细胞对非修饰低密度脂蛋白胆固醇(LDL)摄取,导致泡沫细胞形成。方法诱导分化成功的THP-1巨噬细胞在无血清培养基中培养4 h后,分为对照组(继续无血清培养),高脂组(加入LDL负荷,终浓度为25 mg·L-1),高脂加炎症刺激组(加入LDL负荷,终浓度为25 mg·L-1,同时加入炎症介质LPS,终浓度200μg·L-1),单纯炎症刺激组(加入炎症介质LPS,终浓度200μg·L-1),以上各组细胞培养24 h后收获。ELISA法检测细胞培养基中TNF-α浓度,酶化学法检测细胞内胆固醇浓度,琼脂糖电泳检测LDL氧化程度,Real time PCR法检测LDLr、SREBP2和SCAP mRNA表达水平,Western blot法检测LDLr、SREBP2和SCAP蛋白表达,激光共聚焦检测SCAP在内质网与高尔基体间的转位情况。结果细胞内胆固醇测定发现,炎症介质LPS通过增加THP-1巨噬细胞对非修饰LDL摄取,导致巨噬细胞转变为泡沫细胞。在无炎症介质LPS时,25 mg·L-1LDL减少LDLr mRNA和蛋白表达(P<0.05)。然而,LPS增加LDLr mRNA和蛋白表达,逆转25 mg·L-1LDL对LDLr的抑制效应,不恰当的增加了巨噬细胞对LDL摄取(P<0.05),LPS刺激也导致了SCAP和SREBP2的mRNA和蛋白过表达(P<0.05),同时促进了SCAP从内质网转移到高尔基体。这些结果提示LPS干扰了胆固醇介导的LDLr负反馈调控,使非修饰LDL在巨噬细胞内堆积,导致泡沫细胞形成。结论本研究提示,在LPS诱导的炎症应激状态条件下,天然LDL可以通过LDLr被巨噬细胞大量摄取入细胞内,导致泡沫细胞形成,这可能是巨噬细胞泡沫化的另一条通路。

Macrophages converted into foam cells via LPS-induced LDLr:another important pathway of foam-cell formation under inflammatory stress

Aim To study if inflammatory agent-LPS increases intracellular accumulation of unmodified low-density lipoprotein(LDL) in THP-1 macrophages by disrupting the sterol regulatory element binding proteins(SREBPs) cleavage-activating protein(SCAP)-SREBP2 mediated feedback regulation of LDL receptor(LDLr),causing foam-cell formation.Methods THP-1 macrophages were incubated in serum free medium in the absence or presence of LDL alone,LDL plus lipopolysaccharide(LPS) and LPS alone,then intracellular cholesterol content,tumor necrosis factor alpha(TNF-α) level in the supernatants,oxidation of LDL,SCAP translocation and mRNA and protein expression of LDLr,SREBP2 and SCAP in the treated cells were assessed by cholesterol enzymatic assay,enzyme-linked immunosorbent assay(ELISA),agarose gel electrophoresis,confocol microscopy,real time quantitative polymerase chain reaction(PCR) and western blotting analysis,respectively.Results LPS enhanced transformation of THP-1 macrophages into foam cells by increased uptake of unmodified LDL as evidenced by direct assay of intracellular cholesterol.In the absence of LPS,25 mg·L-1 LDL decreased LDL receptor mRNA and protein expression(P<0.05).However,LPS enhanced LDL receptor expression,overcoming the suppression of LDL receptor induced by 25 mg·L-1 LDL and inappropriately increasing LDL uptake(P<0.05).Exposure to LPS also caused over-expression of mRNA and protein of SCAP and SREBP2(P<0.05),resulting in an escape of SCAP-SREBP2 complex from the endoplasmic reticulum(ER) to the Golgi in the presence of high concentration of intracellular cholesterol.Conclusions These observations indicate that LPS disrupts cholesterol-mediated LDLr feedback regulation,permitting intracellular accumulation of unmodified LDL and causing foam cell formation.The implication of these findings is that LDLr may be the other important pathway for foam cell formation in THP-1 macrophages,especially under inflammatory stress.