低氧经mTOR/4E-BP1和FoxO1/Atrogin-1通路影响骨骼肌蛋白质积累

低氧暴露对骨骼肌蛋白质合成/分解的影响受到广泛关注,但该过程中相关调控通路的研究仍十分有限。本研究拟通过蛋白质相对积累量来研究合成和分解通路的变化。将骨骼肌细胞置于低氧环境中培养,分别在0 h、6 h、12 h和24 h收集细胞,并进行检测。免疫荧光观察肌球蛋白(myosin),翻译表面感应检测蛋白质合成,Western印迹法测试蛋白质合成相关基因(ERK1/2、p-ERK1/2、mTOR、p-mTOR、4E-BP1、p-4E-BP1)、蛋白质分解相关基因(泛素、FoxO1、p-FoxO1、MuRF1和Atrogin-1)表达量。结果发现,随着低氧干预时间延长,肌纤维直径和骨骼肌细胞中蛋白质相对积累量随时间逐渐减小(P<0.01)。与0 h相比,6 h p-4E-BP1/4E-BP1和Atrogin-1的表达显著上调(P<0.05),p-mTOR表达显著高于0 h(P<0.01);6 h和24 h p-mTOR/mTOR的比值显著大于0 h(P<0.05),而p-FoxO1/FoxO1的比值随时间逐渐减小(P<0.01)。上述结果表明,低氧干预能够使骨骼肌细胞直径减少、骨骼肌细胞蛋白质积累减少,并且低氧打破骨骼肌细胞蛋白质合成和分解的平衡,可能是通过调节mTOR/4E-BP1通路活性和FoxO1/Atrogin-1通路的活性实现的。

Hypoxia Affects Skeletal Muscle Protein Accumulation via mTOR/ 4E-BP1 and FoxO1/Atrogin-1 Pathways

The effects of skeletal muscle protein synthesis/decomposition under hypoxic exposure have received extensive attention,but studies on related regulatory pathways in this process are still very limited.This study intends to investigate the changes in protein synthesis and decomposition pathways through the relative accumulation of proteins in the hypoxic environment.L6 cells were cultured in a hypoxic environment,and cells were harvested at 0,6,12,and 24 hours.Myosin was observed by immunofluorescence,and protein synthesis related genes(ERK1/2,p-ERK1/2,mTOR,p-mTOR,4EBP1,p-4E-BP1),proteolytic related genes(FoxO1、p-FoxO1和MuRF1),ubiquitin and puromycin were tested by Western blotting.The results showed that the muscle fiber diameter gradually decreased with the prolongation of hypoxic intervention time(P<0.01).Protein accumulation in skeletal muscle cells gradually decreased(P<0.01).The expression of p-mTOR at 6 h was significantly higher than that at 0 h(P<0.01).And the ratio of p-mTOR/mTOR,6 h and 24 h were significantly higher than that at 0 h(P<0.05).Hypoxia intervention significantly reduces the ratio of p-FoxO1/FoxO1 in skeletal muscle cells.The expression of Atrogin-1 at 6 h(P<0.05)was significantly reduced.The results above indicated that hypoxic intervention reduces the skeletal muscle cell dimension and decreases skeletal muscle protein accumulation.Our results suggest that hypoxia may regulate the activity of the mTOR/4EBP1 and FoxO1/Atrogin-1 pathways.