绞股蓝总皂苷调控CLIC1抑制巨噬细胞脂质累积的作用研究  

Effect of Gypenosides Inhibiting the Accumulation of Lipid in Macrophage by Regulating CLIC1

作　　者：宗磊[1] 任广岩 胡潇[1] 郝正亮 王萃[1] 祝骥[1] 杨贞[1] 卢德赵[1] ZONG Lei REN Guangyan HU Xiao et al(Col lege of Life Science, Zhejiang Chinese Medical University, Hangzhou（310053)

机构地区：[1]浙江中医药大学生命科学学院,杭州310053

出　　处：《浙江中医药大学学报》2017年第10期777-783,789,共8页Journal of Zhejiang Chinese Medical University

基　　金：国家自然科学基金(81403133);浙江省自然科学基金(LQ14H280004;LY15H280005)~~

摘　　要：[目的]研究绞股蓝总皂苷(gypenosides,GPs)对RAW264.7细胞脂质累积及细胞内氯离子通道蛋白1(chloride intracellular channel 1,CLIC1)表达的影响,探讨GPs抗动脉粥样硬化的分子机制。[方法]将RAW264.7细胞接种于六孔板中,分为NC组、氧化型低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)组、GPs 10组、GPs 50组、GPs 100组及CLIC1 si RNA组。各组细胞处理24h后,油红O染色检测各组细胞内脂质的累积,化学法检测细胞内总胆固醇(total cholesterol,TC)及游离胆固醇(free cholesterol,FC)的含量,荧光定量PCR和免疫印迹法检测CLIC1、CD36m RNA和蛋白质的表达,免疫荧光技术观察CLIC1的细胞定位,并用MQAE法检测细胞内氯离子浓度的变化。[结果]与NC组相比,ox-LDL组CLIC1、CD36 m RNA(P<0.01,P<0.001)及蛋白(P<0.01,P<0.001)表达水平显著升高,细胞内TC(P<0.001)、FC(P<0.01)显著升高,细胞膜上CLIC1表达显著升高,细胞内氯离子浓度显著增加(P<0.01);经CLIC1 si RNA或GPs处理后,细胞内TC(P<0.001,P<0.001)、FC(P<0.01,P<0.01)含量降低,脂质累积减少,CLIC1、CD36 m RNA及蛋白表达水平显著下调(P<0.01,P<0.001)。[结论]CLIC1在巨噬细胞脂质累积中发挥着重要的作用,GPs可以通过调控CLIC1的表达抑制巨噬细胞脂质累积。[Objective] To study the effect of gypenosides（GPs） on the accumulation of lipid in RAW264.7 cells and the expression of intracellular chloride channel 1（CLIC1）, and investigate the molecular mechanism of anti-atherosclerosis of GPs. [Methods] RAW264.7 cells were inoculated into 6-well plates and divided into NC group, ox-LDL group, GPs 10 group, GPs 50 group, GPs 100 group and CLIC1 si RNA group. After each group had been processed,the accumulation of lipid was detected by oil red O, the total cholesterol（TC） and free cholesterol（FC） in cells were measured by chemical method, the expression of CLIC1 and CD36 were measured with the technologies of q PCR and immunoblotting. Furthermore, immunofluorescence technique was used to observe the localization of CLIC1 and the method of MQAE was used to detect the change of chloride ion concentration. [Results] Compared with the NC group, CLIC1 and CD36 were significantly increased at the m RNA level（ P〈0.01, P〈 0.001） and protein level（P〈0.01, P 〈0.001）, the intracellular TC（P〈 0.001） and FC（P〈0.01） were significantly increased, the CLIC1 expression on the cell membrane and the concentration of chloride ion were significantly increased（P〈0.01） in the ox-LDL group; however, after treated with CLIC1 si RNA and different concentrations of GPs, intracellular TC（ P〈 0.001, P〈0.001）, FC（P〈0.01, P〈0.01） content and lipid accumulation were decreased, CLIC1 and CD36 expression at the m RNA level and protein level were significantly reduced（P〈0.01,P 〈0.001）. [Conclusion] CLIC1 plays an important role in lipid accumulation of macrophage. GPs can inhibit accumulation of lipid by regulating CLIC1, and retard the progress of atherosclerosis.

关 键 词：GPs CLIC1 巨噬细胞 总胆固醇 游离胆固醇 CD36 mRNA 脂质累积 动脉粥样硬化 

分 类 号：R282[医药卫生—中药学]