黄芪散有效部位群对胰岛素抵抗HepG2细胞SREBP-1c及其靶基因表达的影响

目的:观察黄芪散有效部位群(HQS)对胰岛素抵抗HepG2细胞固醇调控元件结合蛋白-1c(SREBP-1c)及其靶基因表达的影响,并探讨其可能的作用机制。方法:噻唑蓝(MTT)比色法检测HQS对细胞活性的影响,采用高糖高胰岛素诱导HepG2细胞建立胰岛素抵抗模型,造模同时予以不同浓度HQS进行干预,并以二甲双胍(MET)作为阳性药,另设空白组。采用荧光D-葡萄糖同系物2-脱氧-D-葡萄糖(2-NBDG)检测细胞糖摄取量,测定细胞内甘油三酯(TG),游离脂肪酸(FFA)含量,油红O染色法观察细胞脂质沉积情况,实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞内SREBP-1c,乙酰辅酶A羧化酶1(ACC1),脂肪酸合成酶(FAS),硬脂酰辅酶A去饱和酶(SCD1)基因的表达。结果:依据MTT实验结果,选择25,50,100 mg·L-1分别作为HQS低、中、高质量浓度值(HQS-L,HQS-M,HQS-H),处理时间为24 h。与空白组比较,模型组细胞糖摄取显著减少(P0.01),细胞内TG,FFA含量显著升高(P0.01),胞浆内可见大量红色的小泡性脂滴,SREBP-1c,ACC1,FAS,SCD1 mRNA的表达显著上调(P0.01)。与模型组比较,不同质量浓度HQS均可显著升高细胞糖摄取量(P0.05,P0.01),HQS-H,HQS-M可显著降低细胞中TG,FFA含量(P0.01),减少细胞内脂滴数量,HQS-L亦可降低FFA含量(P0.05),此外,HQS-H可显著下调SREBP-1c,ACC1,FAS,SCD1 mRNA的表达(P0.05,P0.01),HQS-M亦可下调SREBP-1c,FAS,SCD1 mRNA的表达(P0.05),对ACC1 mRNA表达具有一定程度的抑制。结论:HQS可能通过抑制SREBP-1c的表达,减少脂质合成,改善HepG2细胞胰岛素抵抗。

Effect of Effective Fractions of Huangqisan on mRNA Expression of SREBP-1c and Its Target Genes in Insulin-resistant HepG2 Cells

Objective: To observe the effect of effective fractions of Huangqisan (HQS) on mRNA expression of sterol regulatory element binding protein-1c (SREBP-1c) and its target genes in insulin-resistant HepG2 cells. Method: The effect of HQS on the activity of HepG2 cells was detected by methye thiazolye telrazlium(MTT) method. The model of insulin-resistant HepG2 cells was induced and established by high glucose and high insulin. While inducing the model, HepG2 cells were treated with different concentrations of HQS, and metformin (MET) was used for positive control. The glucose uptake of cells was detected by fluorescent D-glucose homologue (2-NBDG), and the contents of total triglyceride(TG) and free fatty acids(FFA) in cells were also detected by assay kits. Meanwhile, intracellular lipid droplets were observed by oil red O staining. Besides,Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) method was used to detect mRNA expressions of SREBP-1c, acetyl CoA carboxylase 1 (ACC1), fatty acid synthase (FAS) and stearoyl-CoA desaturase 1(SCD1). Result: According to the findings of MTT experiment, three different concentrations 25 mg·L^-1 (HQS-L), 50 mg·L^-1 (HQS-M) and 100 mg·L^-1 (HQS-H) of HQS were selected for treating cells, and the treating time was 24 h. Compared with control group, the glucose uptake of cells significantly decreased in model group (P<0.01), and the contents of TG and FFA markedly increased (P<0.01). Besides,a large number of red vesicular lipid droplets were also observed in cytoplasm, and the mRNA expressions of SREBP-1c, ACC1, FAS and SCD1 all apparently up-regulated (P<0.01). Compared with model group, different concentrations of HQS all significantly increased the glucose uptake (P<0.05, P<0.01). HQS-H and HQS-M reduced the contents of TG and FFA in cells (P<0.01) and the number of intracellular lipid droplets. Meanwhile, HQS-L decreased FFA content (P<0.05). Besides, HQS-H apparently down-regulated the mRNA expressions of SREBP-1c, ACC1, FAS and SCD1 (P<0.05, P<0.01), and HQS-M also down-regulated the mRNA expressions of SREBP-1c, FAS and SCD1 (P<0.05). Although HQS-M could also inhibit ACC1 mRNA expression, there was no statistically significant difference. Conclusion: HQS may alleviate insulin resistance in HepG2 cells by inhibiting SREBP-1c mRNA expression and reducing lipid synthesis.