荭草素抑制3T3-L1前脂肪细胞分化及其改善胰岛素抵抗的作用研究

Effects of orientin on insulin resistance in 3T3-L1 adipocytes and its mechanism

目的研究荭草素对3T3-L1前脂肪细胞分化及对脂肪细胞胰岛素抵抗的影响,并探讨其作用机制。方法传统鸡尾酒法诱导分化3T3-L1前脂肪细胞,MTT法检测荭草素对前脂肪细胞活力的影响,油红O染色法检测脂质积累,酶法检测细胞内甘油三酯(triglyceride,TG)的含量。地塞米松诱导成熟的脂肪细胞,建立胰岛素抵抗模型,荧光标记2-脱氧葡萄糖(2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose,2-NBDG)摄入法观察脂肪细胞对葡萄糖的摄取能力;Western blot检测腺苷酸活化蛋白激酶(adenosine monophosphate activated protein kinase,AMPK)、乙酰辅酶A羧化酶(acetyl Co A carboxylase,ACC)的磷酸化水平及葡萄糖转运体4(glucose transporter type 4,GLUT4)的表达水平,免疫荧光法检测GLUT4的向膜转位能力。结果荭草素可浓度依赖性地减少细胞脂滴的积累及细胞内TG的含量,但对细胞活力无明显的影响(P>0.05)。胰岛素抵抗状态下,荭草素明显增加脂肪细胞对2-NBDG的摄取(P<0.05),明显上调AMPK、ACC的磷酸化水平(P<0.05),促进GLUT4的向膜转位及表达。结论荭草素抑制前脂肪细胞的分化,同时,荭草素通过上调AMPK/GLUT4信号途径相关蛋白的表达,促进了细胞对葡萄糖的摄取,达到改善胰岛素抵抗的作用。

Aim To investigate the effect of orientin on proliferation and differentiation of 3T3-L1 pre-adipocytes and on insulin resistance（ IR） in 3T3-L1 adipocytes and the possible mechanisms. Methods MTT assay and oil red O staining were applied to investigate the proliferation and the differentiation of 3T3-L1 preadipocytes,respectively. The intracellular triglyceride（ TG） contents were detected by enzymatic analysis.IR model was induced with dexamethasone. A fluorescent glucose analogue,2-NBDG,was used to measure the rate of glucose uptake. Western blot was used to detect the protein level of GLUT4 and phosphorylation of AMPK and ACC. The GLUT4 translocation was measured by fluorescent-immunohistochemistry. Results Orientin decreased the formation of lipid droplets and intracellular TG contents（ P〈0. 01） in a concentration-dependent manner（ P〈0. 05）,but it had no obvious effects on the cell vitality. Under the IR state,orientin significantly increased 3T3-L1 adipocytes glucose uptake（ P〈0. 05）. Meanwhile,orientin up-regulated the protein expression of p-AMPK, pACC,and enhanced GLUT4 translocation and its expression. Conclusion Orientin can effectively inhibit the differentiation of 3T3-L1 pre-adipocytes and increase insulin sensitivity due to the activation of AMPK/GLUT4 signal pathway.