低氧和低氧复合运动通过ERS-miR-208b-PGC-1α途径调控骨骼肌线粒体生物合成

Hypoxia and Exercise Training in Hypoxia Regulate Mitochondrial Biogenesis in Skeletal Muscle through the ERS-miR-208b-PGC-1α Pathway

目的:观察低氧及低氧复合运动对骨骼肌内质网应激(endoplasmic reticulum stress,ERS)及线粒体生物合成的影响,并探讨相关分子机制。方法:雄性Sprague-Dawley(SD)大鼠随机分为常氧对照组(NC组)、低氧对照组(HC组)和低氧复合运动组(HT组)。低氧干预为常压低氧帐篷,11.3%氧浓度持续暴露6周。运动干预为跑台训练(5°,15 m/min),60 min/d,6 d/周,共6周。低氧暴露结束后即刻处死大鼠,取双下肢股四头肌。紫外分光光度法检测股四头肌羰基化蛋白含量和谷胱甘肽巯基转移酶(GST)活性,荧光定量PCR法检测微小RNA-208b(miR-208b)表达,Western-blot法检测内质网应激相关蛋白葡萄糖调节蛋白78(GRP78)、蛋白激酶R样内质网激酶(PERK)总蛋白及磷酸化水平、真核起始因子2α(eIF2α)总蛋白及磷酸化水平、线粒体生物合成相关蛋白过氧化物酶体增殖物激活受体γ共激活因子-1α(PGC-1α)和细胞色素c氧化酶Ⅳ亚型(COXⅣ)蛋白表达。结果:与NC组比较,HC组骨骼肌羰基化蛋白含量、GRP78、p-PERK/PERK和p-eIF2α/eIF2α表达显著升高(P<0.01),GST活性、miR-208b、PGC-1α和COXⅣ表达显著降低(P<0.05~0.01)。与HC组比较,HT组骨骼肌羰基化蛋白含量、p-PERK/PERK和p-eIF2α/eIF2α表达显著降低(P<0.01),GST活性、miR-208b、PGC-1α和COXⅣ表达显著升高(P<0.01)。结论:低氧复合运动通过上调抗氧化酶抑制低氧活化的ERS,进而通过miR-208b-PGC-1α通路上调骨骼肌线粒体生物合成,提高低氧耐受性。

Objective To observe the effect of hypoxia and exercise training in hypoxia on the endoplasmic reticulum stress(ERS)and mitochondrial biogenesis in the skeletal muscle,and explore its molecular mechanism.Methods Male Sprague-Dawley rats were randomly divided into a normoxia control group(NC),a hypoxia control group(HC),and a hypoxia training group(HT).The hypoxia-treated animals were housed in the normobaric hypoxic tent containing 11.3%oxygen for consecutive 6 weeks.The training animals ran on a motor-driven rodent treadmill at a speed of 15 m/min,5%grade for 60 min/day,6 days per week for 6 weeks.Rats were sacrificed right after the hypoxia exposure,and both quadriceps were removed.The carbonylated protein content and glutathione S-transferase(GST)activity were tested using the spectrophotometry.The MicroRNA-208 b(miR-208 b)expression was detected using the real-time fluorescence quantitative PCR.The ERS related proteins,including 78-kDa glucose-regulated protein(GRP78),total and phosphorylation of protein kinase R-like endoplasmic reticulum kinase(PERK),total and phosphorylation of eukaryotic initiation factor 2α(eIF2α)were detected using Westernblotting.The mitochondrial biogenesis related proteins,including peroxisome proliferator-activated receptor gamma coactivator 1α(PGC-1α)and cytochrome C oxidasesubunits IV(COX IV)were also detected using the same method.Results Compared with NC group,a significant increase was observed in the average carbonylated proteins content and expression of GRP78,p-PERK/PERK,p-eIF2α/eIF2α(P<0.01),but a significant decrease was found in the average expression of miR-208 b,PGC-1αand COXIV of HC group(P<0.05~0.01).Moreover,compared with HC group,a significant decrease was observed in the average carbonylated proteins content and expression of GRP78,p-PERK/PERK,p-eIF2α/eIF2α(P<0.01),but a significant increase was found in the average expression of miR-208 b,PGC-1αand COXIV of HT group(P<0.01).Conclusion Exercise in hypoxia can enhance the tolerance of hypoxia by elevating the antioxidant enzyme and suppressing the hypoxia induced endoplasmic reticulum stress,which improves the mitochondrial biogenesis through upregulation of miR-208 b-PGC-1α.