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目的:探讨补充氢水对反复力竭运动大鼠海马氧化应激损伤及细胞自噬的影响。方法:选取雄性SD大鼠48只,随机分为安静组(C),力竭运动组(EX),力竭运动+维生素E组(EX+E),力竭运动+氢水组(EX+H),每组12只。C组常规安静饲养。EX组、EX+E组和EX+H组均进行4周反复力竭运动,4周运动强度为(15 m/min×5 min→20 m/min×5 min→25 m/min×5 min→30 m/min×5 min→35 m/min,直至力竭,5 d/周),记录大鼠运动至力竭时间。EX组每次运动前30 min纯水灌胃4 mL,EX+E组维生素E溶液灌胃4 mL(20 mg),EX+H组氢水灌胃4 mL。训练结束后24 h取材。透射电子显微镜观察海马超微结构变化。海马组织抗氧化指标测定:硫代巴比妥(TBA)法测定丙二醛(MDA)含量,比色法测定总抗氧化能力(T-AOC)水平,黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性;Western blot检测海马组织自噬相关蛋白,包括雷帕霉素靶蛋白(mTOR)、p-mTOR和微管相关蛋白1轻链3B(LC3B)表达。结果:EX+H组大鼠力竭时间较EX组显著延长(P<0.05);EX组海马神经元结构水肿严重,核内出现亮区,内质网、线粒体严重肿胀。EX+E组和EX+H组神经元结构水肿及内质网和线粒体肿胀程度明显减轻;EX组海马组织MDA、T-AOC水平及SOD活性均显著高于C组(P<0.01)。EX+H组MDA含量较EX组显著降低(P<0.05),T-AOC水平及SOD活性均显著升高(P<0.01,P<0.05)。EX+H组T-AOC水平较EX+E组显著升高(P<0.05);EX组海马组织mTOR、p-mTOR及LC3B蛋白表达均显著高于C组(P<0.01,P<0.05,P<0.01)。EX+H组mTOR、p-mTOR表达显著高于EX组(P<0.05)及EX+E组(P<0.01)。EX+H组LC3B表达显著低于EX组(P<0.05)。EX+H组LC3B表达与EX+E组比较无显著差异。结论:补充氢水可有效抑制反复力竭运动造成的海马组织超微结构、氧化应激损伤及细胞自噬现象,且补充氢水较传统抗氧化剂维生素E效果更显著。
Objective: To investigate the effects of hydrogen peroxide on oxidative stress injury and autophagy in hippocampus of rats after repeated exhaustive exercise. Methods: Forty-eight male Sprague-Dawley rats were randomly divided into three groups: C group, EX group, EX + E group, EX + H), 12 in each group. C group routine quiet feeding. The rats in EX group, EX + E group and EX + H group were subjected to repeated exhaustive exercise for 4 weeks. The exercise intensity for 4 weeks was (15 m / min × 5 min → 20 m / min × 5 min → 25 m / min × 5 min → 30 m / min × 5 min → 35 m / min until exhaustion, 5 d / week). The rats were sacrificed and exhausted. In EX group, 4 mL pure water was infused 30 min before each exercise. Vitamin E solution in EX + E group was intragastrically administered 4 mL (20 mg) and in EX + H group, 4 mL hydrogen gas was infused. 24 h after the training drawing. The changes of ultrastructure in hippocampus were observed by transmission electron microscope. The anti-oxidative index of hippocampus was determined by the method of thiobarbituric acid (TBA), the total antioxidant capacity (T-AOC) by colorimetry, the superoxide dismutase (SOD). Western blot was used to detect the expression of autophagy-related proteins including mTOR, p-mTOR and LC3B in the hippocampus. Results: Exhaustive time of EX + H group was significantly longer than that of EX group (P <0.05). The edema of hippocampal neurons in EX group was severe, and bright area, endoplasmic reticulum and mitochondria were severe swelling in EX group. The edema of neurons and the swelling of the endoplasmic reticulum and mitochondria in EX + E and EX + H groups were significantly reduced. The levels of MDA, T-AOC and the activity of SOD in hippocampus of EX group were significantly higher than those in C group (P <0.01). The content of MDA in EX + H group was significantly lower than that in EX group (P <0.05), the level of T-AOC and the activity of SOD were significantly increased (P <0.01, P <0.05). The level of T-AOC in EX + H group was significantly higher than that in EX + E group (P <0.05). The protein expression of mTOR, p-mTOR and LC3B in hippocampus of EX group was significantly higher than that in group C (P <0.01, P <0.01). The expressions of mTOR and p-mTOR in EX + H group were significantly higher than those in EX group (P <0.05) and EX + E group (P <0.01). The expression of LC3B in EX + H group was significantly lower than that in EX group (P <0.05). The LC3B expression in EX + H group was not significantly different from EX + E group. CONCLUSION: Hydrogen supplementation can effectively inhibit hippocampal ultrastructure, oxidative stress injury and autophagy caused by repeated exhaustive exercise. Hydrogen supplementation is more effective than traditional antioxidant vitamin E supplementation.