摘要
目的探讨α-硫辛酸(alpha-lipoic acid,α-LA)是否通过激活核因子E2相关因子2(nuclear factor erythroid 2-related factor 2,Nrf2)信号通路改善HepG2细胞氧化应激,进而缓解胰岛素抵抗(insulin resistance,IR)。方法通过高糖(0.03 mol/L)联合胰岛素(10-6mol/L)诱导HepG2细胞建立IR模型。采用CCK-8法检测细胞活性,葡萄糖氧化酶法检测细胞葡萄糖消耗量,比色法检测细胞己糖激酶(hexokinase,HK)、丙酮酸激酶(pyruvate kinase,PK)活性及甘油三酯(triglyceride,TG)、游离脂肪酸(free fatty acid,FFA)含量,蒽酮硫酸法检测细胞糖原含量,比色法检测超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性和丙二醛(malondialdehyde,MDA)含量,荧光探针法检测活性氧(reactive oxygen species,ROS)含量,Western blot法检细胞总Nrf2、核Nrf2(Nuc-Nrf2)、血红素加氧酶-1(heme oxygenase-1,HO-1)、NAD(P)H醌氧化还原酶1[NAD(P)H quinone oxidoreductase 1,NQO1]和超氧化物歧化酶2(superoxide dismutase 2,SOD2)蛋白表达水平,通过免疫荧光法和siRNA技术证明α-LA通过Nrf2改善细胞氧化应激和胰岛素抵抗。结果α-LA显著提高IR-HepG2细胞葡萄糖消耗量、糖原含量以及HK和PK酶活性(P<0.05),并显著降低TG和FFA的含量(P<0.05)。在α-LA干预下,IR-HepG2细胞氧化应激水平显著降低,包括ROS和MDA减少、抗氧化酶SOD和GSH-Px的活性增强(P<0.05),以及Nrf2通路上抗氧化蛋白表达水平(Total-Nrf2、Nuc-Nrf2、HO-1、NQO1、SOD2)显著提高(P<0.05)。沉默细胞中Nrf2基因后,α-LA改善IR-HepG2细胞氧化应激和胰岛素抵抗的能力被显著削弱。结论α-LA可能通过降低细胞氧化应激水平改善HepG2细胞胰岛素抵抗,其机制与Nrf2信号轴的激活相关。
Objective To investigate whetherα-lipoic acid(α-LA)alleviates insulin resistance(IR)in HepG2 cells by reducing oxidative stress through activation of the nuclear factor erythroid 2-related factor 2(Nrf2)signaling pathway.Methods An IR model was established in HepG2 cells by induction with high glucose(0.03 mol/L)combined with insulin(10-6 mol/L).Cell viability was measured using the CCK-8 assay.Glucose consumption was determined by the glucose oxidase method.Hexokinase(HK)and pyruvate kinase(PK)activity,as well as triglyceride(TG)and free fatty acid(FFA)levels,were assessed using colorimetric assays.Glycogen content was detected by the anthrone-sulfuric acid method.The activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),along with the content of malondialdehyde(MDA),were assessed using colorimetric methods.Reactive oxygen species(ROS)levels were measured using fluorescent probes.Western blot was performed to analyze the protein expression levels of Total-Nrf2,nuclear Nrf2(Nuc-Nrf2),heme oxygenase-1(HO-1),NAD(P)H quinone oxidoreductase 1(NQO1),and superoxide dismutase 2(SOD2).Immunofluorescence and siRNA techniques were employed to confirm thatα-LA alleviates oxidative stress and insulin resistance via the Nrf2 pathway.Resultsα-LA significantly increased glucose consumption,glycogen content,and HK/PK enzymes activity(P<0.05),while markedly reducing TG and FFA levels in IR-HepG2 cells(P<0.05).Underα-LA treatment,oxidative stress in IR-HepG2 cells was significantly alleviated,as evidenced by decreased ROS and MDA levels,enhanced activities of SOD and GSH-Px enzymes(P<0.05),and upregulated expression of Nrf2 pathway-related antioxidant proteins,such as Total-Nrf2,Nuc-Nrf2,HO-1,NQO1,and SOD2(P<0.05).After silencing the Nrf2 gene in cells,the ability ofα-LA to ameliorate oxidative stress and IR in IR-HepG2 cells was significantly attenuated.Conclusionα-LA may ameliorate IR in HepG2 cells by reducing cellular oxidative stress levels,and the mechanism is associated with the activation of the Nrf2 signaling axis.
作者
覃川
侯成香
王鋆柯
陈梦玲
李昊羽
魏绍峰
梁冰
QIN Chuan;HOU Chengxiang;WANG Junke;CHEN Mengling;LI Haoyu;WEI Shaofeng;LIANG Bing(Department of Pharmacology,School of Basic Medicine,Guizhou Medical University,Guian New District 561113,Guizhou,China;Key Laboratory of Environmental Pollution Monitoring and Disease Control,Ministry of Education,Guizhou Medical University,Guian New District 561113,Guizhou,China)
出处
《贵州医科大学学报》
2025年第8期1132-1143,共12页
Journal of Guizhou Medical University
基金
国家自然科学基金项目(82460643)
贵州省科技计划项目[黔科合基础-ZK-(2024)一般155]
贵州省卫生健康委员会科技基金项目(gzwkj2021-426)。
