Apocynin改善TNF-α诱导肝细胞胰岛素抵抗的机制研究

Mechanism of Apocynin improves insulin resistance induced by TNF-α in HepG2 cells

目的:探讨NADPH氧化酶(NOX)的P47PHOX亚基膜转位的抑制剂Apocynin对TNF-α诱导的胰岛素抵抗以及氧化应激水平的影响。方法:实验分为4组:对照组、TNF-α(4ng/mL)处理组、TNF-α+Apocynin(100μmol/L)处理组和Apocynin(100μmol/L)处理组。蒽酮法检测细胞内糖原合成;用DCFH-DA探针标记,流式细胞术检测细胞内活性氧的水平;Westernblotting检测JNK、p-JNK、IRS1、p-IRS1的表达。结果:与对照组比较,TNF-α刺激HepG2后活性氧水平显著增加,细胞内糖原合成障碍。Apocy-nin能显著降低TNF-α诱导的细胞内活性氧的水平,并促进细胞内糖原合成。TNF-α激活JNK,同时抑制胰岛素信号通路,Apocynin能抑制TNF-α对JNK的激活,并且促进胰岛素信号通路敏感性。单独用Apocynin处理对细胞内活性氧和糖原合成以及下游信号通路均无显著影响。结论:Apocynin能降低TNF-α诱导的细胞内活性氧的水平,改善TNF-α诱导肝细胞胰岛素抵抗状态。

AIM：To investigate the effects of Apocynin on oxidative stress and insulin resistance induced by TNF-α.METHODS：Cells were divided into control group,TNF-α（4 ng/mL）treatment group,TNF-α＋Apocynin（100 μmol/L）treatment group,Apocynin（100 μmol/L）group.The insulin resistance cell model was induced by TNF-α（4 ng/mL）to stimulate human hepatoma carcinoma cell HepG2.The intracellular glycogen was detected using a glucose oxidase assay kit.The level of intracellular reactive oxygen species（ROS） was detected by DCFH-DA fluorescent probe and flow cytometry.The expressions of JNK,p-JNK,IRS1 and p-IRS1 were observed by Western blotting.RESULTS：Compared with the control group,the level of ROS in HepG2 Cells in TNF-α treatment group was significantly increased and the level of intracellular glycogen in cell was decreased,TNF-α activated JNK and inhibited insulin signal pathway,and Apocynin could reverse those effects induced by TNF-α.Apocynin（100 μmol/L）group had no significant effects on ROS,glycogen synthesis and signal pathway.CONCLUSION：Apocynin can decrease the level of ROS in cell and improve insulin resistance condition induced by TNF-α in HepG2 cell.