低氧运动预适应激活自噬调控Hippo/YAP/TAZ信号通路减轻急性低氧力竭运动大鼠骨骼肌损伤

Hypoxic Exercise Preconditioning Activates Autophagy to Regulate Hippo/YAP/TAZ Signaling Pathway to Alleviate Skeletal Muscle Injury in Acute Hypoxic Exhaustive Exercise Rats

目的:探讨低氧运动预适应激活自噬调控Hippo/YAP/TAZ信号通路对急性低氧力竭运动大鼠骨骼肌保护的机制。方法:50只6周龄SPF级雄性SD大鼠,随机分为空白对照组(Con组,n=10)、单纯急性低氧力竭运动组(HE组,n=10)、低氧预适应+急性低氧力竭运动组(HP+HE组,n=10)、运动预适应+急性低氧力竭运动组(EP+HE组,n=10)、低氧预适应+运动预适应+急性低氧力竭运动组(EP/HP+HE组,n=10)。预适应周期2 w, EP+HE组及EP/HP+HE组进行2次/d常压常氧跑台训练;HP+HE组及EP/HP+HE组进行10 h/d间歇性低氧暴露(FiO213.5%)。预适应后HE组、HP+HE组、EP+HE组及EP/HP+HE组大鼠进行低氧环境跑台力竭训练(FiO211.9%~12.0%)。低氧力竭运动后2 h,尾静脉取血检测血清骨骼肌损伤、炎症反应和氧化应激指标,苏木精-伊红染色观察腓肠肌病理特征,并检测腓肠肌自噬相关蛋白、Hippo/YAP/TAZ信号通路蛋白及mRNA。结果:(1)与HE组比较,预适应组大鼠低氧力竭运动后的腓肠肌结构损伤、炎症反应和氧化应激明显减轻。(2)HE组Atg5、Beclin1蛋白表达显著低于EP+HE组及EP/HP+HE组(P<0.05),EP/HP+HE组Atg5、Beclin1蛋白表达显著高于HP+HE组(P<0.05);HE组LC3-Ⅱ蛋白表达显著高于EP+HE组及EP/HP+HE组(P<0.05),EP+HE组及EP/HP+HE组显著高于HP+HE组(P<0.05)。(3)HE组MST1 mRNA显著高于EP+HE组及EP/HP+HE组(P<0.05),EP+HE组及EP/HP+HE组显著高于HP+HE组(P<0.05);HE组LATS1 mRNA显著低于预适应组(P<0.05);HE组YAP mRNA显著高于预适应组(P<0.05),EP/HP+HE组显著低于HP+HE组(P<0.05);HE组及HP+HE TAZ mRNA显著高于EP+HE组及EP/HP+HE组均显著降低(P<0.05)。(4)HE组MST1、LATS1及其磷酸化水平显著低于EP+HE组及EP/HP+HE组(P<0.05),EP/HP+HE组MST1显著高于HP+HE组(P<0.05);HE组YAP蛋白显著高于EP+HE组及EP/HP+HE组(P<0.05);HE组TAZ蛋白显著高于预适应组均显著降低(P<0.05),EP/HP+HE组均显著低于HP+HE组(P<0.05)。结论:低氧运动预适应可能通过激活骨骼肌自噬抑制Hippo/YAP/TAZ信号通路减轻急性低氧力竭运动大鼠的骨骼肌损伤、炎症反应和氧化应激反应。

Objective:To explore the mechanism of hypoxic exercise preconditioning activating autophagy to regulate Hippo/YAP/TAZ signaling pathway to protect skeletal muscle in rats undergoing acute hypoxic exhaustive exercise.Methods:50 six-week-old SPF male SD rats were randomly divided into blank control group(Con group,n=10),simple acute hypoxic exhaustive exercise group(HE group,n=10),hypoxic preconditioning+acute hypoxic exhaustive exercise group(HP+HE group,n=10),exercise preconditioning+acute hypoxic exhaustive exercise group(EP+HE group,n=10),hypoxic preconditioning+exercise preconditioning+acute hypoxic exhaustive exercise group(EP/HP+HE group,n=10).The preconditioning period was 2 weeks,EP+HE group and the EP/HP+HE group were trained twice a day on the normal pressure and oxygen treadmill;HP+HE group and EP/HP+HE group were exposed to intermittent hypoxia with FiO 213.5 for 10 hours/day.On the next day after preconditioning,rats in HE group,HP+HE group,EP+HE group and EP/HP+HE group were trained to run on the treadmill in a hypoxic environment(FiO 211.9~12.0).Two hours after exercise,tail vein blood was taken to detect serum skeletal muscle injury,inflammatory reaction and oxidative stress indicators,gastrocnemius muscle tissue was taken for HE section staining and autophagy related protein,Hippo/YAP/TAZ signal pathway protein and mRNA were detected.Results:(1)Compared with HE group,the structural damage,inflammatory reaction and oxidative stress of gastrocnemius muscle of rats in preconditioning group were significantly reduced after hypoxic exhaustive exercise.(2)The expression of Atg5 and Beclin1 protein in HE group was significantly lower than that in EP+HE group and EP/HP+HE group(P<0.05),and the expression of Atg5 and Beclin1 protein in EP/HP+HE group was significantly higher than that in HP+HE group(P<0.05);The expression of LC3-Ⅱprotein in HE group was significantly higher than that in EP+HE group and EP/HP+HE group(P<0.05),and that in EP+HE group and EP/HP+HE group was significantly higher than that in HP+HE group(P<0.05).(3)MST1 mRNA in HE group was significantly higher than that in EP+HE group and EP/HP+HE group(P<0.05),and that in EP+HE group and EP/HP+HE group was significantly higher than that in HP+HE group(P<0.05);LATS1 mRNA in HE group was significantly lower than that in preconditioning group(P<0.05);YAP mRNA in HE group was significantly higher than that in preconditioning group(P<0.05),while that in EP/HP+HE group was significantly lower than that in HP+HE group(P<0.05);The mRNA of HE group and HP+HE TAZ was significantly higher than that of EP+HE group and EP/HP+HE group(P<0.05).(4)MST1,LATS1 and their phosphorylation levels in HE group were significantly lower than those in EP+HE group and EP/HP+HE group(P<0.05),and MST1 in EP/HP+HE group was significantly higher than those in HP+HE group(P<0.05);YAP protein in HE group was significantly higher than that in EP+HE group and EP/HP+HE group(P<0.05);TAZ protein in HE group was significantly higher than that in preconditioning group(P<0.05),while that in EP/HP+HE group was significantly lower than that in HP+HE group(P<0.05).Conclusion:Hypoxic exercise preconditioning may inhibit Hippo/YAP/TAZ signal pathway by activating skeletal muscle autophagy,thereby reducing skeletal muscle injury,inflammatory response and oxidative stress response in rats undergoing hypoxic exhaustive exercise.