低氧暴露经FoxO1介导的蛋白质分解途径降低肌管直径

Proteolysis Pathways via FoxO1 Reduce the Diameter of Myotube under Hypoxia Exposure

高原/低氧环境诱导骨骼肌萎缩被认为是一个多系统多器官相互作用的结果,但该过程中低氧对肌细胞的直接作用机制并不明确。本研究拟观察低氧暴露对骨骼肌蛋白质分解相关途径蛋白质的影响,并探究低氧影响肌管直径改变的可能机制。将骨骼肌细胞(L6细胞)分化成肌管后,在1%氧浓度下暴露0 h、6 h、12 h和24 h,使用免疫荧光(IF)观察肌球蛋白重链(myosin)水平,计算肌管直径的改变;Western印迹检测肌球蛋白、低氧诱导因子-1α(HIF-1α)、叉头框蛋白O1(FoxO1)、泛素蛋白酶途径(MuRF1和Atrogin1)和自噬溶酶体途径相关蛋白质(p62、Beclin1、LC3)的水平;IF检测LC3并计算积分光密度值(IOD)。结果发现,低氧干预后,6 h和12 h肌管直径显著减小,同时6 h肌球蛋白的水平显著降低(P<0.05);6 h HIF-1α和FoxO1蛋白质水平显著增加(P<0.05);与0 h相比,低氧干预各时长MuRF1含量都显著增加(P<0.05),Atrogin1水平无显著性变化;与0 h相比,低氧干预各时长p62水平均显著降低,6 h Beclin1水平和LC3 IOD值显著升高,LC3Ⅱ/Ⅰ比值在6 h显著增加,在12 h和24 h显著降低(P<0.05)。本研究提示,低氧暴露(1%氧浓度)诱导L6骨骼肌细胞肌管直径减小,可能机制是HIF-1α调控FoxO1介导的泛素蛋白酶途径和自噬溶酶体途径相关蛋白质造成。

The high altitude/hypoxic environment induced skeletal muscle atrophy is considered to be the interaction of multi-system and multi-organ,but the direct mechanism of hypoxia on muscle cells in this process is not clear.This study intended to investigate the effects of hypoxia exposure on proteins in ubiquitin and autophagy pathways,and explored the possible mechanism of hypoxia induced change of myotube diameter.The expression of myosin,hypoxia inducible factor-1α(HIF-1α),forkhead box protein O1(FoxO1),and ubiquitin protease pathway(MuRF1 and Atrogin1)and autophagy lysosomal pathway(p62,Beclin1,LC3)related proteins were detected by Western blot;The integrated optical density(IOD)of Myosin and LC3 was detected by IF.The results showed that the diameters of myotube at 6 h and 12 h were significantly reduced,and the expression of myosin was significantly reduced at 6 h after hypoxia exposure(P<0.05);the protein levels of HIF-1α and FoxO1 were significantly increased at 6 h(P<0.05);The expression of MuRF1 in each time points of hypoxia was significantly higher than 0 h(P<0.05),but no difference of Atrogin1 expression was detected;Compared with 0 h,the expression of p62 was reduced significantly in response to hypoxia.The protein expression of Beclin1 and the IOD of LC3 was increased significantly at 6 h,and the LC3Ⅱ/Ⅰ ratio was significantly higher at 6 h,but significantly lower at 12 h and 24 h(P<0.05).The results above indicated that the reduction of the myotube diameter of L6 skeletal muscle cells was induced by hypoxia exposure(1%O2),possibly via HIF-1αregulation of FoxO1-mediated ubiquitin protease pathway and autophagy-lysosomal pathway-related proteins.