【摘要】 目的：探讨骨化三醇[1,25（OH）₂D₃]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）₂D₃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）₂D₃(1、10、100和1 000 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^-1PA作用24 h)、PA+APS组(给予0.4 mmol·L^-1PA作用24 h后再给予100 mg·L^-1APS作用24 h)、PA+1,25（OH）₂D₃组[给予0.4 mmol·L^-1PA作用24 h后再给予100 nmol·L^-11,25（OH）₂D₃作用24 h]、PA+APS+1,25（OH）₂D₃组[给予0.4 mmol·L^-1PA作用24 h后再给予100 mg·L^-1APS和100 nmol·L^-11,25（OH）₂D₃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）₂D₃和PA+APS+1,25（OH）₂D₃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）₂D₃组细胞中ROS水平、p-P65和p38MAPK蛋白表达水平均低于PA+APS和PA+1,25（OH）₂D₃组（P<0.05）,InsR、IRS-1、p-IRS-1和GLUT4蛋白表达水平均高于PA+APS和PA+1,25（OH）₂D₃组（P<0.05）。结论：1,25（OH）₂D₃联合APS可有效减轻IR，其机制可能是通过抑制氧化应激通路以及炎性因子的释放来实现的，且联合应用效果优于单独应用。更多还原

【Abstract】 Objective :To investigate the improvement effect of 1,25-dihydroxycholecalciferol[1,25（OH）₂D₃] 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）₂D₃and 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,and200 mg·L^-1),and the optimal dose of 1,25（OH）₂D₃(1,10,100,and 1 000 nmol·L^-1). The skeletal muscle cells were divided into control group（without any treatment）,PA group(given 0. 4 mmol·L^-1PA for 24 h),PA+APS group(given 0. 4 mmol·L^-1PA for 24 h and 100 mg·L^-1APS for 24 h),PA+1,25（OH）₂D₃group[given 0. 4 mmol·L^-1PA for 24 h and 100 nmol·L^-11,25（OH）₂D₃for 24 h],and PA+APS+1,25（OH）₂D₃group(given 0. 4 mmol·L^-1PA for 24 h and then 100 mg·L^-1APS and100 nmol·L^-11,25（OH）₂D₃for 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<0. 05）;compared with PA group,the levels of ROS,IL-6,MCP-1,TNF-α,and the expression levels of P65,p-P65,p38MAPK,and TLR4 proteins in the skeletal muscle cells in PA+APS,PA+1,25（OH）₂D₃and PA+APS+1,25（OH）₂D₃groups were decreased（P<0. 05）,and the levels of IL-10,and the expression levels of IRS-1,p-IRS-1 and GLUT4 proteins were increased（P<0. 05）. The level of ROS,and the expression levels of p-P65 and p38MAPK proteins in the skeletal muscle cells in PA+APS+1,25（OH）₂D₃group were lower than those in PA+APS and PA+1,25（OH）₂D₃groups（P<0. 05）,and the expression levels of InsR,IRS-1,p-IRS-1 and GLUT4 protein were higher than those in PA+APS and PA+1,25（OH）₂D₃group（P<0. 05）. Conclusion:1,25（OH）₂D₃in combination with APS can effectively reduce the IR,which may be achieved by inhibiting the release of oxidative stress pathways and inflammatory factors,and the the effect of combined application is better than that of single application.更多还原