熊果酸对RAW264.7巨噬细胞源性泡沫细胞胆固醇逆转运及对PPAR-γ的基因及蛋白表达的影响

Effect of Ursolic Acid on Cholesterol Efflux and PPAR-γ Gene and Protein Expression in RAW264.7 Macrophage-derived Foam Cells

目的:观察不同质量浓度熊果酸(UA)对RAW264.7巨噬细胞源性泡沫细胞促进胆固醇流出的影响及过氧化物酶体增殖物激活受体-γ(PPAR-γ)转运蛋白的表达,探讨可能作用机制。方法:体外培养RAW264.7巨噬细胞,用20 mg·L^(-1)的氧化型低密度脂蛋白(ox-LDL)孵育48 h诱导成泡沫细胞,采用油红O染色,光镜下鉴定泡沫细胞形态变化,液体闪烁计数仪检测胆固醇的流出率,并用实时荧光定量聚合酶链式反应(Real-time PCR)及酶联免疫吸附(ELISA)测定PPAR-γ的基因及蛋白表达。结果:巨噬细胞经ox-LDL诱48 h后转化为泡沫细胞,与空白组比较,10,15,20,25 mg·L^(-1)质量浓度UA干预组泡沫细胞的胆固醇流出率上升(P<0.01);10,15,20,25 mg·L^(-1)质量浓度UA干预组泡沫细胞内PPAR-γ的基因表达上调(P<0.01),在一定质量浓度范围内呈剂量依赖性;10,15,20,25 mg·L^(-1)质量浓度UA干预组泡沫细胞内PPAR-γ的蛋白表达增加(P<0.01),差异有统计学意义。结论:经20 mg·L^(-1)ox-LDL的诱导后,巨噬细胞分化为泡沫细胞,细胞内脂质大量增加,UA促进巨噬细胞内胆固醇流出,可能与上调细胞内PPAR-γ的表达有关。

Objective： To observe the effects of different concentrations of ursolic acid（UA） on cholesterol efflux and peroxisome proliferatros activated receptor-γ（PPAR-γ） transporter expression in RAW264.7 macrophage-derived foam cells,and its possible mechanism. Method： RAW264.7 macrophages were cultured in vitro and incubated with 20 mg·L-1oxidized low density lipoprotein（ox-LDL） for 48 hours to induce foam cells.Oil red O staining was used to identify morphological changes of foam cells. The mRNA and protein expressions of PPAR-γ were detected by Real-time polyerse chinrection（PCR） and enzyme-linked immunosorbent assay（ELISA）. Result： Macrophages were transformed into foam cells by being induced with ox-LDL for 48 h.Compared with the blank group,the cholesterol efflux rate of foamed cells in the UA intervention group increased at concentrations of 10,15,20,25 mg·L-1（P〈0.01）,and were dose-dependently within a certain concentration range. At concentrations of 10,15,20,25 mg·L-1,the protein expression of PPAR-γ mRNA in foam cells in the UA intervention group was up-regulated（P〈0.01）,and wasdose-dependently within a certain concentration range. The protein expression of PPAR-γ in foam cells of UA treated at concentrations of 10,15,20,25 mg·L-1increased（P〈0.01）,and the difference was statistically significant. Conclusion： After 20 mg·L-1ox-LDL induced macrophages are differentiated into foam cells,a large number of intracellular lipid increases,and UA promotes macrophage cholesterol efflux,which may be related to increase in intracellular expression of PPAR-γ.