Study On The Effect Of H₂O₂ Induced Oxidantive Stress On Deiodinase Activity And The Interventional Effect Of α-lipoic Acid In MIN6 Cell

Objectives: Diabetes is a metabolic disease resulted from insulin secretion deficiency and insulin dyfunction. The nonthyroid illness syndrome(NTIS) is often accompanied by the occurrence of diabetes, with decreased serum total T3 and free T3 level, but normal total T4, free T4 and TSH level. All these studies indicate that the conversion of T4 to T3 may be disrupted in diabetics. Deiodinases family mediate the catabolism of 5,3′,5′-tetraiodothyronine(T4) to 3,5,3′-triiodothyronine(T3), by which the major part of T3 is produced in peripheral tissues. This shows that the function of deiodinases may be injured in diabetes patents. Besides, α-lipoic acid(α-LA) is a common antioxidant that reported to play a protective role in islet β cell function. In the present study, we established oxidative damage model of MIN6 cell induced by H2O2 to investigate the possible influence of oxidative damage and α-LA on deiodinase activity in MIN6 cells and its potential mechanism.Methods: Cells were pretreated with 100, 200, 400 μM H2O2 for 6 h to establish oxidative damage model, and received different experimental protocols as following groups: control group, H2O2 group, H2O2+T3 group, H2O2+T4 group, α-LA group and H2O2+T4+α-LA group. At the end of experimental program, cell viability and ROS level were determined by MTT and DCFH-DA assay respectively, the activity of deiodinase were measured by RIA(Radioimmunoassay) methods, the antioxidant index(GSH-Px, GSH/GSSG, T-AOC) and MDA level were determined by using the reagent kits, T3 and autocrine insulin levels were measured by ELISA method, and the mRNA expression of antioxidant genes(Nrf2, HO-1, NQO-1), anti- and pro-apoptotic genes(BCL-2, BAX), deiodinase and its regulatory genes(Dio2 、 Dio3 、 SHH 、 PKA 、 Gli-2 、 WSB-1 、p38MAPK), as well as insulin secretion genes(INS-2、PDX-1、MafA、GLUT-2、GCK) were determined by RT-PCR.Results: H2O2 significantly decreased MIN6 cell viability, GSH-Px activity, T-AOC, GSH/GSSG ratio and mRNA level of Nrf2, NQO-1, HO-1, while increased cellular ROS and MDA level, suggesting that H2O2 induced cell oxidative damage. 10-9、10-7、10-6 M T3 could improve the cell viability, antioxidant enzymes and mRNA levels of genes related to antioxidant. But high concentrations of T3 could have pro-oxidant effects. 100 μM H2O2 increased D2 activity and D3 activity, decreased T3 content; 400 μM H2O2 inhibited D2 activity and increased D3 activity, both leading to decreased T3 content. Besides, oxidative damage could also induce abnormal Dio2, Dio3 mRNA levels. α-LA administration rescued D2, D3 activity and Dio2, Dio3 mRNA levels, increased T3 content.; regulated Dio2, Dio3 mRNA levels, increased GSH-Px activity, T-AOC, GSH/GSSG, upregulated Nrf-2, NQO-1, HO-1, BCL-2, INS2, PDX-1, MafA, GLUT-2 and GCK mRNA expression, improved cell viability and promoted insulin secretion as well.Conclusions: H2O2 induced oxidative damage in MIN6 cells. D2, D3 activity were increased in low oxidative damage group, increased T3 removal; D2 activity was inhibited and D3 activity was increased in high oxidative damage group that decreased T3 production, and increased T3 removal at the same time, both may ultimately lead to decreased T3 content. Besides, oxidative damage induced anormal Dio2, Dio3 mRNA levels. α-LA significantly alleviated these abnormalities about D2, D3, improved the cell redox status, decreased apoptosis and improved insulin and secretion. Furthermore, the regulatory effect of α-LA on deiodinases in MIN6 cells is likely to be related with SHH, PKA and p38 MAPK pathways.