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

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）₂D₃ 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）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^－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）₂D₃ group ［given 0.4 mmol·L^－1 PA for 24 h and 100 nmol·L^－1 1，25（OH）₂D₃ for 24 h］， and PA+APS+1，25（OH）₂D₃ group （given 0.4 mmol·L^－1 PA for 24 h and then 100 mg·L^－1APS and 100 nmol·L^－1 1，25（OH）₂D₃ 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<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）₂D₃ and PA+APS+1，25（OH）₂D₃ groups 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）₂D₃ group were lower than those in PA+APS and PA+1，25（OH）₂D₃ groups （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）₂D₃ group （P<0.05）. Conclusion 1，25（OH）₂D₃ in 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.

Key words: Skeletal muscle cells, Insulin resistance, 1，25-dihydroxycholecalciferol, Astragalus polysaccharide, Oxidative stress