【作者】 王龙；

【导师】 周刚；

【作者基本信息】 湖南大学 ， 教育学， 2023， 硕士

【摘要】 研究目的:活性氧(reactive oxygen species,ROS)是一类人体代谢过程中产生的一类含氧代谢产物,对人体细胞信号传导和维持体内平衡具有重要作用。一方面,过度生成的ROS是心肌氧化应激反应的重要原因,另一方面,生理范围内ROS的增加可能在氧化还原信号传导中起着重要作用。过去的研究发现,心肌中的ROS的主要来源有线粒体呼吸链途经、黄嘌呤氧化酶途径、NADPH氧化酶(NOX)途径三种途径,而由于运动刺激所产生的ROS主要来源于NOX途径。过氧化物还原酶(peroxiredoxins,Prx)作为心肌中一种重要的抗氧化物还原酶对于心肌抗氧化能力有着不可忽视的作用,但是在心肌长期有氧耐力适应性训练后心肌产生预适应后,Prx蛋白可能作为心肌信号调节因子对下游信号蛋白的表达产生的影响机制尚不清楚。基于此,本研究采用C57BL/6雄性小鼠游泳训练模型,联合NOX抑制剂和Prx抑制剂进行干预,观察长期有氧耐力训练后小鼠心肌中Prx2活性及蛋白表达水平,并推测其与NOX、p38MAPK的上下游信号传导机制,探讨长期有氧耐力运动对心肌过氧化物还原酶的影响以及心肌氧化还原适应性机制。研究方法:实验选取C57BL/6雄性小鼠31只,平均体重25-28g。随机分为安静+安慰剂组(C)6只、长期运动+安慰剂组(E)9只、长期运动+NOX抑制剂组(EA)8只、长期运动+Prx2抑制剂组(EC)8只。E组、EA组和EC组无负重游泳训练1h,1次/天,5天/周,8周。制剂组取样前倒数第3天和第2天在运动前1小时腹腔注射过氧化物氧化还原酶Ⅱ抑制剂conoidinA5mg/kg或NOX抑制剂apocynin 10mg/kg,其他组注射同等体积生理盐水。末次运动结束24h后,10%水合氯醛麻醉,心脏取血,摘取心脏。所有研究过程和实验方案均经湖南大学实验动物伦理委员会审查。采用Western blot法检测心肌Prx2、NOX2、NOX4、p38MAPK、p-p38MAPK蛋白表达,免疫沉淀法测 Prx2-p38MAPK,比色法测血清和心肌SOD活性以及MDA含量、心肌Prx2活性。研究结果:1)长期有氧耐力训练对小鼠血清和心肌MDA含量及SOD活性的影响。在血清MDA水平方面发现,与C组相比,E组MDA水平下降(P<0.05);EA、EC组显著下降(P<0.01);与E组相比,EA组,EC组MDA水平下降(P<0.05);在血清SOD活性结果中发现,与C组相比,EA组SOD活性升高(P<0.05),E组、EC组无明显差异。在小鼠心肌MDA含量结果中发现,与C组相比,E心肌MDA含量下降(P<0.05),EA、EC组MDA含量均下降(P<0.05);在小鼠心肌SOD活性结果中发现,与C组相比,EA、EC组心肌SOD活性显著升高(P<0.01);与E组相比,EA组、EC组心肌SOD活性显著升高(P<0.01)。提示长期有氧训练导致小鼠血液心肌的氧化应激指标SOD和MDA产生适应性改变。2)长期有氧耐力训练后对小鼠心肌Prx2活性及蛋白表达结果显示:与C组相比,E组心肌Prx2活性上升(P<0.05),EA组显著下降(P<0.01),EC组下降(P<0.05);与E组相比,EA组、EC心肌Prx2活性显著下降(P<0.01)。在蛋白表达中发现E组Prx2蛋白较C组显著增加(P<0.05);抑制剂组中Prx2蛋白较C组、E组相比,EA组显著下降(P<0.01),EC组显著下降(P<0.01)。当NOX2被抑制后,Prx2活性与蛋白表达均下降,提示Prx2可能受到上游NOX2的调控,此外Prx2抑制剂可直接抑制心肌Prx2的活性与蛋白表达,对抗运动引起的Prx2激活。3)长期有氧耐力训练后对小鼠心肌NOX2、NOX4蛋白表达结果显示:与C组相比,E组NOX2蛋白表达明显上升(P<0.01);与E组相比,EA、EC组NOX2蛋白表达显著下降(P<0.01)。抑制剂抑制Prx2活性以及蛋白表达后,NOX2蛋白表达显著下降,提示Prx2对NOX2可能存在负反馈调节机制。与C组相比,E组NOX4蛋白表达上升(P<0.05),EA组NOX4蛋白无明显变化,EC组NOX4蛋白表达上升(P<0.05)。4)长期有氧耐力训练后对小鼠心肌p38MAPK、p-p38MAPK蛋白表达结果显示:与C组相比,E组、EA组、EC组p38MAPK蛋白无明显差异。与C组相比,E组p-p38MAPK蛋白表达升高(P<0.01),EA组p-p38MAPK蛋白表达下降(P<0.05),EC组p-p38MAPK蛋白表达下降(P<0.05);与E组相比,EC组p-p38MAPK蛋白表达明显下降(P<0.01)。p-p38MAPK/p38MAPK 比值结果显示:与 C 组相 比,E 组p-p38MAPK/p38MAPK 比值上升(P<0.05),与 E组相比,EA组、EC组p-p38MAPK/p38MAPK 比值显著下降(P<0.01)。5)p38MAPK-Prx2免疫沉淀结果显示:以p38MAPK沉淀物,与C组相比,E组Prx2蛋白与p38MAPK结合水平明显升高(P<0.01),EA组、EC组Prx2蛋白与p38MAPK结合水平下降(P<0.05)。与E组相比,EA组Prx2蛋白与p38MAPK结合水平显著下降(P<0.01),EC组下降(P<0.05)。研究结论:1)长期有氧训练导致小鼠血液心肌的氧化应激指标SOD和MDA产生适应性改变;心肌NOX2和NOX4表达增加,成为训练导致的心肌ROS生成的主要途径。2)长期有氧训练通过NOX/ROS途径增强Prx2活性及蛋白表达。3)长期有氧训练通过激活心肌Prx2来调节p38MAPK的磷酸化水平,从而增强p38MAPK对运动刺激的导致的氧化还原信号的适应。更多还原

【Abstract】 Objective:reactive oxygen species(ROS)is a class of oxygen-containing metabolites produced during human metabolism,which plays an important role in signaling and homeostasis of human cells.On the one hand,overproduction of ROS is an important cause of myocardial oxidative stress response;on the other hand,the increase of ROS on a physiological scale may play an important role in REDOX signaling.Previous studies have found that ROS in myocardium mainly comes from mitochondrial respiratory chain pathway,xanthine oxidase pathway and NADPH oxidase(NOX)pathway,and ROS produced by exercise stimulation mainly comes from NOX pathway.peroxiredoxins(Prx),as an important antioxidant reductase,plays a significant role in the antioxidant capacity of myocarde.However,when the myocardets are preconditioned after long-term aerobic endurance training,The effect of Prx protein on the expression of downstream signal proteins is still unclear.Based on this,this study adopted the swimming training model of C57BL/6 male mice,combined with NOX inhibitors and Prx inhibitors for intervention,to observe the activity and protein expression level of Prx2 in the myocardium of mice after long-term aerobic endurance training,and to speculate the upstream and downstream signal transduction mechanism between Prx2 and NOX and p38MAPK.Objective To explore the effects of long-term aerobic endurance exercise on myocardial peroxidase reductase and the adaptive mechanism of myocardial REDOX.Research methods:Thirty-one male C57BL/6 mice with an average body weight of 25-28g were selected.There were 6 rats in the quiet+placebo group(C),9 rats in the long-term exercise+placebo group(E),8 rats in the long-term exercise+NOX inhibitor group(EA)and 8 rats in the long-term exercise+Prx2 inhibitor group(EC).Group E,EA and EC underwent weightless swimming training for 1h,once a day,5 days a week,8 weeks.The preparation group was intraperitoneally injected with peroxidase oxidoreductase Ⅱ inhibitor conoidinA5mg/kg or NOX inhibitor apocynin 10mg/kg on the penlast day and the second day before sampling 1 hour before exercise,while the other groups were injected with the same volume of normal saline.24h after the end of the last exercise,10%chloral hydrate anesthesia,heart blood,heart extraction.All research procedures and experimental protocols were reviewed by the Experimental Animal Ethics Committee of Hunan University.Western blot method was used to detect myocardial Prx2,NOX2,NOX4,p38MAPK,p-p38MAPK protein expression,immu noprecipitation method to determine Prx2-p38MAPK,colorimetry method to determine serum and myocardial SOD activity and MDA content,myocardial Prx2 activity.Research results:1)Effects of 8-week aerobic training on MDA content and SOD activity in serum and myocardium of mice.The serum MDA level of group E was decreased compared with group C(P<0.05).EA and EC groups were significantly decreased(P<0.01);Compared with group E,MDA level in group EA and EC was decreased(P<0.05).In the results of serum SOD activity,compared with group C,the activity of SOD in group EA was increased(P<0.05),and there was no significant difference among groups C,E and EC.The results showed that compared with group C,MDA content in myocardium of E decreased(P<0.05),and that in both EA and EC groups decreased(P<0.05).Compared with group C,the SOD activity of group EA and EC was significantly increased(P<0.01).Compared with group E,the SOD activity in EC and EA groups was significantly increased(P<0.01).2)The results of myocardial Prx2 activity and protein expression in mice after 8 weeks of aerobic training showed that compared with group C,the myocardial Prx2 activity in group E was increased(P<0.05),significantly decreased in group EA(P<0.01),and decreased in group EC(P<0.05).Compared with E group,Prx2 activity in EC and EA group was significantly decreased(P<0.01).The protein expression of Prx2 in group E was significantly higher than that in group C(P<0.05).The Prx2 protein in the inhibitor group was significantly decreased in the EA group(P<0.01)and the EC group(P<0.01)compared with that in the C and E groups.3)After 8 weeks of aerobic training,the expression of NOX2 and NOX4 protein in myocarpusof mice showed that compared with group C,the expression of NOX2 protein in group E was significantly increased(P<0.01).Compared with E group,NOX2 protein expression in EA and EC groups was significantly decreased(P<0.01).Compared with group C,NOX4 protein expression in group E was increased(P<0.05),in group EA was significantly increased(P<0.01),and in group EC was increased(P<0.05).4)The expression of p38MAPK and p-p38MAPK protein in myocardial of mice after 8 weeks of aerobic training showed that p38MAPK protein in groups E,EA and EC had no significant difference compared with group C.Compared with group C,p-p38MAPK protein expression in group E was increased(p<0.01),p-p38MAPK protein expression in group EA was decreased(P<0.05)and p-p38MAPK protein expression in group EC was decreased(P<0.05).Compared with group E,the expression of p-p38MAPK protein in EC group was significantly decreased(P<0.01).The p-p38MAPK/p38MAPK ratio results showed that compared with group C,the p-p38MAPK/p38MAPK ratio of group E was increased(P<0.05),while the p-p38MAPK/p38MAPK ratio of groups EA and EC was significantly decreased(p<0.01).5)The results of p38MAPK-Prx2 immunoprecipitation showed that compared with group C,the binding level of Prx2 protein and p38MAPK in group E was significantly increased(P<0.01),and the binding level of Prx2 protein and p38MAPK in groups EA and EC was decreased(P<0.05).Compared with group E,the binding level of Prx2 protein to p38MAPK in group EA was significantly decreased(P<0.01),and that in group EC was decreased(P<0.05).Research conclusion:1)Long-term aerobic training resulted in the adaptive changes of the oxidative stress indexes SOD and MDA in the blood myocardium of mice;Increased expression of NOX2 and NOX4 in myocardium becomes the main pathway of ROS production induced by training.2)Long-term aerobic training enhanced Prx2 activity and protein expression through NOX/ROS pathway.3)Long-term aerobic training regulates the phosphorylation level of p38MAPK by activating myocardial Prx2,so as to enhance the adaptation of p38MAPK to REDOX signals caused by exercise stimulation.更多还原