沉默CXCL12抑制ox-LDL诱导小鼠巨噬细胞系RAW264.7泡沫化和凋亡

Silenced CXCL12 inhibits ox-LDL-induced foaming and apoptosis in mouse macrophage cell line RAW264.7

目的探讨CXC趋化因子配体12(CXCL12)对氧化低密度脂蛋白(ox-LDL)诱导小鼠腹腔巨噬细胞系RAW264.7泡沫化和凋亡的作用及其机制。方法用60 mg/L的ox-LDL诱导RAW264.7泡沫化,记为ox-LDL组,不添加ox-LDL的作为对照(NC)组;将si-con和si-CXCL12转染至RAW264.7细胞中再用60 mg/L的ox-LDL处理,分别记为ox-LDL+si-con组、ox-LDL+si-CXCL12组。油红O染色法检测细胞泡沫化;Western blot检测ABCA-1、SRB-1、cleaved caspase-3、cleaved caspase-9、CXCL12、p38MAPK和p-p38MAPK蛋白表达;流式细胞计量术检测细胞凋亡;RT-qPCR检测CXCL12 mRNA表达。结果ox-LDL处理的RAW264.7细胞体积增大,形状变为圆形,胞质中可明显见到大量红色脂滴;ABCA-1和SRB-1表达水平显著升高(P<0.05),cleaved caspase-3和cleaved caspase-9表达水平显著升高(P<0.05),细胞凋亡率显著升高(P<0.05),CXCL12表达水平显著升高(P<0.05),p-p38MAPK表达水平显著升高(P<0.05)。沉默CXCL12,大部分细胞内未见红染脂滴,仅少数细胞内有部分脂滴,ABCA-1和SRB-1表达水平显著降低(P<0.05),cleaved caspase-3和cleaved caspase-9表达水平显著降低(P<0.05),细胞凋亡率显著降低(P<0.05),p-p38MAPK表达水平显著降低(P<0.05)。结论沉默CXCL12抑制ox-LDL诱导巨噬细胞RAW264.7泡沫化和细胞凋亡,其可能与p38MAPK信号通路有关。

Objective To investigate the effect and mechanism of CXC chemokine ligand 12(CXCL12)on oxidized low density lipoprotein(ox-LDL)-induced foaming and apoptosis of macrophages.Methods RAW264.7 was incubated with 60 mg/L of ox-LDL to induce foaming of macrophages,which was recorded as ox-LDL group,and to compare with the control without ox-LDL;si-con and si-CXCL12 were transfected to RAW264.7 cells and thenincubated with ox-LDL at 60 mg/L as ox-LDL+si-con group and ox-LDL+si-CXCL12 group,respectively.Oil Red O staining was used to detect cellular foaming;Western blot was used to detect ABCA-1,SRB-1,cleaved caspase-3,cleaved caspase-9,CXCL12,p38 mitogen-activated protein kinase(p38MAPK),and phosphorylated p38MAPK(p-p38MAPK)protein expression;flow cytometry is used to detect apoptosis;CXCL12 mRNA expression was measured by real-time fluorescence quantitative PCR(RT-qPCR).Results The size of ox-LDL-treated macrophages increased,the shape became round,and a large number of red lipid droplets were found in the cytoplasm;the expression of ABCA-1 and SRB-1 and cleaved caspase-3 as well as cleaved caspase-9 were all significantly increased;the apoptosis rate and the expression of CXCL12 and p-p38MAPK were significantly increased(P<0.05).Silencing CXCL12,no red-stained lipid droplets were found in most cells,only a few cells had partial lipid droplets,the expressions of ABCA-1 and SRB-1 were significantly reduced,and the expression of cleaved caspase-3 and cleaved caspase-9,apoptosis rate and the expression of p-p38MAPK significantly reduced(P<0.05).Conclusions Silencing CXCL12 inhibits ox-LDL-induced foaming and apoptosis of mouse macrophage cell line RAW264.7,which may be related to the p38MAPK signaling pathway.