安仙蓉 管俊欢 杨秀丽 吴先进 梁冰

中图分类号 R965;Q493.4 文献标志码 A 文章编号 1001-0408（2021）14-1703-06

DOI 10.6039/j.issn.1001-0408.2021.14.07

摘 要 目的：研究α-硫辛酸对胰岛素抵抗HepG2细胞糖代谢紊乱的改善作用。方法：采用MTT法测定25～1 000 ?mol/L α-硫辛酸对人肝癌细胞HepG2存活率的影响，以确定α-硫辛酸给药浓度。实验设阴性对照组、胰岛素抵抗组（1×10-7 mol/L胰岛素）、联合抵抗组（30 ?mol/L 亚砷酸钠+1×10-8 mol/L胰岛素）和α-硫辛酸低、中、高浓度组。按分组加入α-硫辛酸作用HepG2细胞12 h后，再分别给予相应浓度的亚砷酸钠或/和胰岛素继续培养24 h。采用葡萄糖氧化酶法检测细胞葡萄消耗量，比色法检测细胞己糖激酶、丙酮酸激酶活性，蒽酮法检测细胞糖原含量，Western blot法检测细胞中葡萄糖转运体4（GLUT4）、磷酸化糖原合成激酶3β（p-GSK3β）、GSK3β蛋白表达水平和磷酸化蛋白激酶B（p-Akt）/Akt、p-GSK3β/GSK3β比值。结果：25、50、100 ?mol/L的α-硫辛酸对细胞存活率无明显影响（P>0.05），且细胞存活率均大于96%，故将其作为后续研究的低、中、高浓度。与阴性对照组比较，胰岛素抵抗组、联合抵抗组细胞葡萄糖消耗量、己糖激酶和丙酮酸激酶活性、糖原含量、GLUT4和p-GSK3β蛋白表达水平、p-Akt/Akt和p-GSK3β/GSK3β比值均显著降低，GSK3β蛋白表达水平均显著升高（P<0.05）。与联合抵抗组比较，α-硫辛酸各浓度组细胞葡萄糖消耗量（α-硫辛酸低浓度组除外）、己糖激酶（α-硫辛酸低、中浓度组除外）和丙酮酸激酶（α-硫辛酸低、中浓度组除外）活性、糖原含量、GLUT4（α-硫辛酸低浓度组除外）、p-GSK3β蛋白表达水平和p-Akt/Akt（α-硫辛酸低、中浓度组除外）、p-GSK3β/GSK3β（α-硫辛酸低浓度组除外）比值均显著升高，GSK3β（α-硫辛酸低、中浓度组除外）蛋白表达水平均显著降低（P<0.05），且α-硫辛酸高浓度组细胞糖原含量、GLUT4蛋白表达水平、p-GSK3β/GSK3β比值和α-硫辛酸中、高浓度组细胞p-GSK3β蛋白表达水平的改善效果更明显（P<0.05）。结论：α-硫辛酸对胰岛素抵抗HepG2细胞的糖代谢紊乱具有一定的改善作用，其机制可能与增加葡萄糖消耗，增强糖代谢相关酶活性，提高糖原含量，上调Akt、GSK3β蛋白的磷酸化水平和GLUT4、p-GSK3β蛋白的表达水平，下调GSK3β蛋白的表达水平有关。

关键词 α-硫辛酸;人肝癌细胞HepG2;亚砷酸鈉;胰岛素抵抗;糖代谢紊乱;磷脂酰肌醇3-激酶/蛋白激酶B

Improvement Effects of α-lipoic Acid on Glucose Metabolism Disorder of Insulin Resistant HepG2 Cells

AN Xianrong1，GUAN Junhuan1，YANG Xiuli1，WU Xianjin1，LIANG Bing1，2（1. Dept. of Pharmacology， School of Basic Medical Sciences， Guizhou Medical University， Guiyang 550025， China; 2. Key Laboratory of Environmental Pollution and Disease Monitoring， Ministry of Education， Guiyang 550025， China）

ABSTRACT   OBJECTIVE： To study the improvement effects of α-lipoic acid on glucose metabolism disorder of insulin resistant HepG2 cells. METHODS： The effects of 25-1 000 ?mol/L α-lipoic acid on survival rate of human hepatoma cell HepG2 were determined by MTT assay so as to determine the concentration of α-lipoic acid. Negative control group， insulin resistance group （1×10-7 mol/L insulin）， combination resistance group （30 ?mol/L sodium arsenite+1×10-8 mol/L insulin）， α-lipoic acid low- concentration， medium-concentration and high-concentration groups were set up. HepG2 cells were treated with α-lipoic acid for 12 h and then cultured with corresponding concentration of sodium arsenite or/and insulin for 24 h. The glucose oxidase method was used to detect the glucose consumption， colorimetric method was used to detect hexokinase activity and pyruvate kinase activity， and anthrone method was used to detect glycogen content. Western blot assay was used to detect the protein expression of GLUT4， p-GSK3β and GSK3β as well as the ratio of p-Akt/Akt and p-GSK3β/GSK3β. RESULTS： 25， 50， 100 ?mol/L α-lipoic acid had no significant effect on the survival rates of HepG2 cells （P>0.05）， and survival rates of HepG2 cells were higher than 96%， so they were used as the low， medium and high concentration for follow-up study. Compared with negative control group， glucose consumption， the activities of hexokinase and pyruvate kinase， glycogen content， protein expression of GLUT4 and p-GSK3β， the ratio of p-Akt/Akt and p-GSK3β/GSK3β were decreased significantly in insulin resistance group and combined resistance group， while the protein expression of GSK3β was increased significantly （P<0.05）. Compared with combination resistance group， the glucose consumption （except for α-lipoic acid low- concentration group）， the activities of hexokinase （except for α-lipoic acid low-concentration and medium-concentration groups） and pyruvate kinase （except for α-lipoic acid low-concentration and medium-concentration groups）， glycogen contents， protein expression of GLUT4 （except for α-lipoic acid low-concentration group） and p-GSK3β， the ratio of p-Akt/Akt （except for α-lipoic acid low-concentration and medium-concentration groups） and p-GSK3β/GSK3β （except for α-lipoic acid low-concentration groups） were increased significantly in α-lipoic acid groups， while protein expression of GSK3β （except for α-lipoic acid low-concentration and medium-concentration groups） was decreased significantly （P<0.05）; glycogen content， protein expression of GLUT4 and the ratio of p-GSK3β/GSK3β in α-lipoic acid high-concentration group as well as the protein expression of p-GSK3β in α-lipoic acid medium-concentration and high-concentration groups were improved significantly（P<0.05）. CONCLUSIONS： α-lipoic acid can improve the disorder of glucose metabolism in insulin resistant HepG2 cells， the mechanism of which may be associated with the increase of glucose consumption， the activities of glucose metabolism related enzymes and  glycogen content， and expression up-regulation of the phosphorylation levels of Akt and GSK3β protein， the expression of GLUT4 and p-GSK3β proteins， down-regulation of the expression of GSK3β protein.

KEYWORDS   α-lipoic acid; Human hepatoma cell HepG2; Sodium arsenite; Insulin resistance; Glucose metabolism disorder; PI3K/Akt

砷是广泛分布于自然界的非金属元素，具有较强的毒性，长期饮用含砷量较高的水会引起慢性砷中毒[1]。已有流行病学调查结果显示，环境砷暴露与糖尿病患病率或发病率增加有关，且砷暴露条件下的糖尿病特征与2型糖尿病相似[2-5]。因此，砷与糖尿病的关系成为砷中毒研究领域的热点问题之一。