1,25(OH)_(2)D_(3)联合黄芪多糖对体外骨骼肌细胞胰岛素抵抗的改善作用及其机制  

Improvement effect of 1,25(OH)_(2)D_(3) combined with astragalus polysaccharide on insulin resistance of skeletal muscle cells in vitro and its mechanism

作　　者：李浩 刘东阁 闫姝琪 任淑萍[1] LI Hao;LIU Dongge;YAN Shuqi;REN Shuping(Department of Public Health,School of Labor Health and Environmental Health,Jilin University,Changchun 130021,China)

机构地区：[1]吉林大学公共卫生学院劳动卫生与环境卫生学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2022年第6期1411-1421,共11页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科研项目(20210204024YY)。

摘　　要：目的:探讨骨化三醇[1,25(OH)_(2)D_(3)]联合黄芪多糖(APS)对骨骼肌细胞胰岛素抵抗(IR)的改善作用及其作用机制,为采用1,25(OH)_(2)D_(3)和APS缓解IR提供依据。方法:采用CCK-8法和己糖激酶法确定棕榈酸(PA)溶液(0.2、0.4、0.6、0.8和1.0 mmol·L^(-1))的最佳作用剂量和最佳作用时间、APS(25、50、100和200 mg·L^(-1))最佳作用剂量及1,25(OH)_(2)D_(3)(1、10、100和1000 nmol·L^(-1))最佳作用剂量。将骨骼肌细胞分为对照组(不进行任何处理)、PA组(给予0.4 mmol·L^(-1) PA作用24 h)、PA+APS组(给予0.4 mmol·L^(-1) PA作用24 h后再给予100 mg·L^(-1) APS作用24 h)、PA+1,25(OH)_(2)D_(3)组[给予0.4 mmol·L^(-1) PA作用24 h后再给予100 nmol·L^(-1)1,25(OH)_(2)D_(3)作用24 h]、PA+APS+1,25(OH)_(2)D_(3)组[给予0.4 mmol·L^(-1) PA作用24 h后再给予100 mg·L^(-1) APS和100 nmol·L^(-1)1,25(OH)_(2)D_(3)作用24 h]。酶联免疫吸附试验(ELISA)法检测各组细胞培养上清中白细胞介素6(IL-6)、白细胞介素10(IL-10)、单核细胞趋化蛋白1(MCP-1)和肿瘤坏死因子α(TNF-α)水平,流式细胞术检测各组细胞中活性氧(ROS)水平,Western blotting法检测各组细胞中胰岛素受体(InsR)、胰岛素受体底物1(IRS-1)、磷酸化胰岛素受体底物1(p-IRS-1)、葡萄糖转运蛋白4(GLUT4)、P65、磷酸化P65(p-P65)、P38丝裂原活化蛋白激酶(p38MAPK)和Toll样受体4(TLR4)蛋白表达水平。结果:与对照组比较,PA组骨骼肌细胞中ROS、IL-6、TNF-α、MCP-1水平和P65、p-P65、p38MAPK及TLR4蛋白表达水平均升高(P<0.05),IL-10水平和InsR、IRS-1、p-IRS-1及GLUT4蛋白表达水平均降低(P<0.05);与PA组比较,PA+APS、PA+1,25(OH)_(2)D_(3)和PA+APS+1,25(OH)_(2)D_(3)组细胞中ROS、IL-6、MCP-1和TNF-α水平及P65、p-P65、p38MAPK及TLR4蛋白表达水平均降低(P<0.05),IL-10水平和InsR、IRS-1、p-IRS-1及GLUT4蛋白表达水平均升高(P<0.05)。PA+APS+1,25(OH)_(2)D_(3)组细胞中ROS水平、p-P65和p38MAPK蛋白表达水平均低于PA+APS和PObjective:To investigate the improvement effect of 1,25-dihydroxycholecalciferol[1,25(OH)_(2)D_(3)]combined with astragalus polysaccharide(APS)on the insulin resistance(IR)in the skeletal muscle cells and its mechanism,and to provide the basis for alleviating IR by 1,25(OH)_(2)D_(3) and APS.Methods:CCK-8 method and hexokinase method were used to determine the optimal dose and duration of PA(0.2,0.4,0.6,0.8,and 1.0 mmol·L^(-1)),the optimal dose of APS(25,50,100,and 200 mg·L^(-1)),and the optimal dose of 1,25(OH)_(2)D_(3)(1,10,100,and 1000 nmol·L^(-1)).The skeletal muscle cells were divided into control group(without any treatment),PA group(given 0.4 mmol·L^(-1) PA for 24 h),PA+APS group(given 0.4 mmol·L^(-1) PA for 24 h and 100 mg·L^(-1) APS for 24 h),PA+1,25(OH)_(2)D_(3) group[given 0.4 mmol·L^(-1) PA for 24 h and 100 nmol·L^(-1)1,25(OH)_(2)D_(3) for 24 h],and PA+APS+1,25(OH)_(2)D_(3) group(given 0.4 mmol·L^(-1) PA for 24 h and then 100 mg·L^(-1)APS and 100 nmol·L^(-1)1,25(OH)_(2)D_(3) for 24 h).The levels of interleukin-6(IL-6),interleukin-10(IL-10),monocyte chemoattractant protein-1(MCP-1),and tumor necrosis factor-α(TNF-α)in the cell culture supernatant in various groups were detected by enzyme-linked immunosorbent assay(ELISA)method,and the levels of intracellular reactive oxygen species(ROS)in the cells in various groups were detected by flow cytometry.Western blotting method was used to detect the expression levels of insulin receptor(InsR),insulin receptor substrate 1(IRS-1),phosphorylated insulin receptor substrate 1(p-IRS-1),glucose transporter 4(GLUT4),P65,phosphorylated P65(p-P65),P38 mitogen-activated protein kinase(p38MAPK),and Toll-like receptor 4(TLR4)proteins in the cells in various groups.Results:Compared with control group,the levels of ROS,IL-6,TNF-α,MCP-1,and the expression levels of P65,p-P65,p38MAPK,and TLR4 in the skeletal muscle cells in PA group were increased(P<0.05),and the levels of IL-10,and the expression levels of InsR,IRS-1,p-IRS-1,and GLUT4 proteins were decreased(P<

关 键 词：骨骼肌细胞 胰岛素抵抗 骨化三醇 黄芪多糖 氧化应激 

分 类 号：R994.6[医药卫生—毒理学]