PI3K抑制剂LY294002对鼠前脂肪细胞分化和C/EBPα及PPARγ表达的影响

Effects of PI3K inhibitor LY294002 on the differentiation of mouse preadipocytes and the expression of C/EBPα and PPARγ

目的研究磷脂酰肌醇激酶(PI3K)抑制剂LY294002对小鼠前脂肪细胞分化和CCAAT增强子结合蛋白α(C/EBPα)及过氧化物酶体增殖物激活受体γ(PPARγ)表达的影响,探讨胰岛素受体底物(IRSs)/PI3K信号通路在前脂肪细胞分化中的作用。方法小鼠3T3-L1细胞分为实验组和对照组,实验组加入LY294002(25μmol/L),对照组加入同体积DMSO。应用0.5 mmol/L 3-异丁基-1-甲基黄嘌呤(IBMX)、10-6mol/L地塞米松和5μg/mL胰岛素诱导两组前脂肪细胞分化,在培养的0 d、2 d、4 d、8 d分别收集细胞,实时PCR法及Western blot法检测细胞中C/EBPα和PPARγ表达水平。培养8 d油红O染色,观察细胞分化情况。结果小鼠3T3-L1细胞基础状态下两组C/EBPα及PPARγ表达差异无统计学意义(P>0.05);诱导分化时实验组C/EBPα及PPARγ表达低于对照组(分别P<0.05,P<0.01)。油红O染色示实验组细胞无明显脂滴。结论 PI3K抑制剂LY294002能抑制小鼠前脂肪细胞分化及C/EBPα和PPARγ的表达,提示IRSs/PI3K信号通路可通过调节PPARγ和C/EBPα的表达在3T3-L1前脂肪细胞的分化中发挥重要作用。

Objective This study examined the effects of PI3K inhibitor LY294002 on the differentiation of mouse preadipocytes and the expression of CCAAT enhancer binding protein α（C/EBPα） and peroxisome proliferation activated receptor γ（PPARγ）,in order to study the possible roles of insulin receptor substrate（IRSs）/PI3K signal pathway in the differentiation of preadipocytes.Methods The mouse 3T3-L1 cells were cultured normally and divided into experimental and control groups.3T3-L1 cells in the experimental group were treated with PI3K inhibitor LY294002（25 μmol/L） and those in the control group were treated with DMSO culture medium.3-isobutyl-1-methylxanthine（IBMX）（0.5 mmol/L）,dexamethasone（10-6 mol/L） and insulin（5 μg/mL） were used to induce the differentiation of 3T3-L1 preadipocytes in both groups.Before culture,and 2,4 and 8 days after culture,the cells were collected to detect the expression of C/EBPα and PPARγ by real-time PCR and Western blot assays.The lipid droplets of 3T3-L1 preadipocytes were observed by oil-red O staining.Results PI3K inhibitor LY294002 did not affect the expression of C/EBPα and PPARγ in un-induced 3T3-L1 preadipocytes（P0.05）,but decreased the expression of C/EBPα and PPARγ during the in vitro induced differentiation of 3T3-L1 preadipocytes compared with the control group（P0.05 or 0.01）.The lipid droplets count was greatly reduced by LY294002.Conclusions PI3K inhibitor LY294002 can inhibit the differentiation of mouse 3T3-LI preadipocytes and the expression of C/EBPα and PPARγ in the differentiation of 3T3-LI preadipoeytes,suggesting that IRSs/PI3K signal pathway may play an important role in the differentiation of 3T3-L1 preadipocytes by regulating the expression of C/EBPα and PPARγ.