激活视黄醇类X受体抑制轻度氧化低密度脂蛋白诱导的小鼠巨噬细胞系RAW264.7增殖

目的 探讨激活视黄醇类X受体(RXR)对轻度氧化低密度脂蛋白(LoxLDL)诱导巨噬细胞增殖的影响及其作用机制.方法 饥饿处理后的小鼠巨噬细胞系RAW264.7经LoxLDL(20μg/ml)刺激24 h诱导细胞增殖,干预组同时给予不同浓度的RXR特异性激动剂顺式维甲酸9(9-cisRA),四甲基偶氮唑蓝方法检测细胞活力,脱氧尿嘧啶核苷免疫组化方法检测细胞DNA合成,流式细胞仪PI单染法检测细胞凋亡,Western印迹方法检测Nur77和RARB蛋白表达.结果 LoxLDL诱导24 h后,细胞活力和DNA合成(A值)分别升高0.79±0.12和1.81±0.31,RXR的特异性配体9-cisRA能够显著抑制LoxLDL的作用,在10-8mol/L和10-7mol/L浓度分别使细胞活力下降到0.43±0.10和0.26±0.07(P＜0.05),DNA合成升高为1.14±0.43和0.72±0.06(P＜0.05),且对细胞凋亡没有影响.Western印迹检测表明,孤儿核受体Nur77蛋白表达在LoxLDL刺激后显著升高5倍以上,9-cisRA显著下调Nur77表达,同时上调下游靶基因RARB蛋白表达.结论 激活核受体RXR能够显著抑制LoxLDL刺激引起的巨噬细胞增殖,其机制可能与调控RXR/Nur77异源二聚体及其下游靶基因RARβ有关.

Effects of retinoid X receptor activation on lightly oxidized low-density lipoprotein induced cell proliferation of macrophages: experiment with murine RAW264.7 cells

Objective To investigate the effects and mechanism of retinoid X receptor (RXR) activation on macrophages proliferation induced by lightly oxidized low-density lipoprotein (LoxLDL).Methods Serum-starved mouse macrophages of the line RAW264. 7 were stimulated with 20 μg/mLLoxLDL for 24 h to induce proliferation in the absence or presence of varying doses of RXR special agonist 9-cis retinoid acid (RA). The cell viability was assayed by MTT method. The DNA synthesis was assayed by BrdU ELISA. The apoptotic percentage of cells was measured by flow cytometry using propidium iodide (PI) staining. Nur77 and RARβ protein expressions were detected by Western blotting. Results After treated by LoxLDL for 24 h, the cell viability and DNA synthesis ( A value) of the RAW264.7 ceils were increased by 0.79±0.12 and 1.81±0.31 respectively. Co-incubated with LoxLDL and 10-s mol/L and 10-7 mol/L 9-cisRA the ceil viability decreased to 0.43±0.10 and 0.26±0.07 respectively( both P＜0. 05 ), and the DNA synthesis decreased to 1.14±0.43 and 0.72±0.06 respectively(both P＜0.05 ) The apoptotic rate of the ceils treated with LoxLDL was (3.08±0.30) %, not significantly different from those of the cells cocultured with LoxLDL and 10-9-10-7 mol/L 9-cisRA (2.74±0.13 ) % , (2.94±0.24) % , and (2.48±0.42) % respectively, all P＞0.05. The orphan nuclear receptor Nur77 protein expression was up-regulated by more than 5 times after LoxLDL treatment. 9-cisRA down-regulates the Nut77 expression and increased the expression of the downstream gene RARβ. Condnsion RXR activation significantly inhibits the macrophage proliferation induced by LoxLDL. The mechanism may be related to the regulation of RXR/Nur77 heterodimer and its downstream gene RARβ.