转化生长因子β相关激酶1抑制剂对人肝癌细胞系HepG2脂滴积累的影响及其机制

Effect of transforming growth factor-β-related kinase 1 inhibitor on lipid droplet accumulation in human liver cancer cell line HepG2 and its mechanism

目的观察转化生长因子β相关激酶1(TAK1)抑制剂(NG25)对人肝癌细胞系HepG2脂滴积累的影响,并探讨其作用机制。方法取HepG2细胞并分为两组,处理组细胞加入2μmol/L NG25处理24 h,对照组加入等体积DMSO处理24 h,两组均同时用终浓度为250μmol/L脂肪酸(OA、PA各125μmol/L)共同孵育,采用油红O染色观察细胞脂滴积累情况(油红O染色红光强度),荧光定量PCR法和蛋白免疫印迹法检测细胞过氧化物酶体增殖物激活受体γ(PPARγ)、诱导细胞凋亡DNA分裂因子样效应因子C(CIDEC)mRNA、蛋白相对表达量,双荧光素酶报告基因检测系统检测细胞荧光素酶活性。另取HepG2细胞并分为3组,恢复组细胞预先转染1μg质粒PPARγ后再加入2μmol/L NG25处理24 h,处理组细胞加入2μmol/L NG25处理24 h,对照组细胞加入等体积DMSO处理24 h,3组同时用终浓度250μmol/L脂肪酸(OA、PA各125μmol/L)共同孵育,荧光定量PCR法和蛋白免疫印迹法检测细胞CIDEC mRNA、蛋白。结果与对照组比较,处理组油红O染色红光强度弱(P<0.05)。与对照组比较,处理组PPARγ及CIDEC mRNA、蛋白相对表达量和PPARγ启动子荧光素酶活性降低(P均<0.05);与对照组比较,恢复组和处理组CIDEC mRNA和蛋白相对表达量降低(P均<0.05)。结论NG25对HepG2细胞脂滴积累有抑制作用,机制可能是抑制PPARγ的转录,进而下调CIDEC表达,即NG25通过PPARγ-CIDEC信号途径最终抑制HepG2细胞脂滴积累。

Objective To observe the effect of transforming growth factor-β-activated kinase 1(TAK1)inhibitor(NG25)on the accumulation of lipid droplets in the human liver cancer cell line HepG2 and probe into its mechanism.Methods HepG2 cells were divided into two groups;2μmol/L NG25 was added to the treatment group for 24 h,the same volume of DMSO was added to the control group for 24 h,and cells in both groups were incubated with 250μmol/L free fatty acids(OA,PA,125μmol/L each)simultaneously.The accumulation of lipid droplets in cells was observed by oil red O staining(red intensity of oil red O staining).Quantitative real-time PCR and Western blotting were used to detect the mRNA and protein expression levels of peroxisome proliferator-activated receptorγ(PPARγ)and cell death-inducing DFFA(DNA fragmentation factor-α)-like effector C(CIDEC),respectively.Luciferase activity was detected by the dual luciferase reporter gene assay system.Another part of HepG2 cells was taken and then we divided them into 3 groups;the cells in the recovery group were treated with 2μmol/L NG25 for 24 h after being pretransfected with 1 g plasmid PPARγ,the treatment group with 2μmol/L NG25 for 24 h and the control group with the same volume of DMSO for 24 h,and all three groups were incubated with 250μmol/L free fatty acids(OA,PA,125μmol/L each)simultaneously.Quantitative real-time PCR and Western blotting was used to detect the mRNA and protein expression levels of CIDEC.Results Compared with the control group,the red intensity of oil red O staining in the treatment group was weaker(P<0.05).Compared with the control group,the relative expression of PPARγand CIDEC mRNA and protein and the PPARγpromoter luciferase activity decreased in the treatment group(all P<0.05);compared with the control group,the relative expression levels of CIDEC mRNA and protein in the recovery group and the treatment group decreased(both P<0.05).Conclusion NG25 suppresses the accumulation of lipid droplets in HepG2 cells,which may be caused by the down-regulation of CIDEC expression through the inhibition of PPARγtranscription,that is,NG25 ultimately inhibits lipid droplet accumulation in HepG2 cells through the PPARγ-CIDEC signaling pathway.