【作者】 刘丽霞；

【导师】 陈骐； 吴秀琴；

【作者基本信息】 福建师范大学 ， 生物学， 2018， 博士

【摘要】 背景:运动性疲劳,如果得不到及时恢复,将极大限制锻炼和训练的效果,并可造成机体损伤,影响人们的健康、工作和生活。因此,依据运动性疲劳的氧化应激、炎症反应、线粒体损伤等发生机制,探索新型抗运动性疲劳营养因子,开发安全、高效的抗疲劳功能食品,对提高运动员竞技水平和促进大众健身效果具有重要意义。而吡咯喹啉醌(pyrroloquinoline quinone,PQQ)抗氧化、抗炎、维持线粒体正常功能等多样的生物学功能,提示其在运动性疲劳防护方面具有重要的研发价值,但迄今为止,国内外尚未见到相关报道。目的:探讨PQQ在体内外运动性疲劳模型中,对提高运动能力、抗氧化应激、抗组织损伤、抗NF-κB(Nuclear factorκB)介导的炎症反应等作用效果,并阐明PQQ抗运动性疲劳作用的靶点和机制,从而为将其开发成高效的运动营养补剂提供有力的科学依据。方法:1.电刺激肌管建立运动性疲劳细胞模型,通过检测肌管ROS(reactive oxygen species)生成、培养基LDH(lactate dehydrogenase)含量、细胞活力、NF-κB蛋白表达、细胞因子和凋亡相关基因表达,探讨PQQ在运动性疲劳细胞模型中,对细胞损伤、NF-κB介导的炎症反应、细胞凋亡相关基因等的调控作用。2.通过小鼠反复力竭游泳方式,建立运动性疲劳动物模型;通过力竭时间、CK(creatine kinase)、LDH、cTnI(cardiac troponin I)、MDA(Malonaldehyde)含量、SOD(superoxide dismutase)活性、GSH-Px(glutathione peroxidase)活性、NF-κB、IL-6(interleukin-6)、interleukin 1β(IL-1β)、CXCL10(interferon-inducible protein-10)、Bcl-2(B-cell lymphoma-2)、Bax(Bcl-2 associated X protein)及Caspase-3等参数指标的测试,研究PQQ在该模型中提高运动能力、抗氧化应激、抗组织损伤、抗NF-κB介导的炎症反应等作用。3.通过对线粒体形态染色、线粒体膜电位、线粒体呼吸功能以及ATP(adenosine-triphosphate)合成的检测,探究PQQ在运动性疲劳细胞模型中,对维持线粒体正常形态和功能的作用;并通过相应抑制剂的使用,探讨PQQ发挥抗运动性疲劳效应的可能靶点。4.采用色谱-质谱联用的代谢组学技术,测试反复力竭游泳干预及补充一定剂量PQQ后小鼠血清代谢物的变化情况,通过原始数据预处理、PCA分析(principal component analysis)、OPLS-DA分析(orthogonal partial Least square-iscriminate Analysis)、差异代谢物的筛选以及相关代谢通路分析,寻找关键的潜在标志物,阐述PQQ作用的代谢通路和调控机制,进一步验证其可能的作用靶点。5.通过对本课题研究结果的综合分析,推测PQQ预防与延缓运动性疲劳的作用机制。结果:1.PQQ在运动性疲劳的细胞模型中,能够抑制ROS的生成,降低培养基的LDH活力,同时,能够抑制转录因子NF-κB的过表达,降低IL-6、IL-1β及CXCL10等促炎介质以及凋亡相关基因的表达。2.PQQ在运动性疲劳的动物模型中,能够延长力竭运动的时间,抑制心肌和肝组织中SOD和GSH-Px活性的降低,减少血清、肝脏和心脏中MDA的生成;能够降低血清CK、LDH活力及cTnI水平,明显改善肝组织病理学异常形态;能够抑制心肌和肝组织中NF-κB的过表达和活化,减少TNF-α、IL-1β、IL-6、CXCL-10等炎症介质的表达和释放;还能够上调心肌和肝组织中抗凋亡因子Bcl-2和促凋亡因子Bax的比例,抑制凋亡关键执行分子Caspase-3的过表达。3.PQQ在运动性疲劳细胞模型中,具有维持线粒体正常形态和功能的作用;线粒体复合物Ⅰ为PQQ发挥抗运动性疲劳效应的可能位点。4.运动性疲劳会导致机体代谢紊乱,而PQQ在运动疲劳中具有维持正常血清代谢水平的重要作用,这种作用与其对柠檬酸、L-脯氨酸、木糖醇、N-乙酰-L-苯丙氨酸等标志物及所在代谢通路的调节密切相关。结论:1.证实PQQ能够延缓运动性疲劳的发生,提高运动能力;在体内、外运动性疲劳模型中,发挥抗氧化应激、抗NF-κB介导的炎症反应、抗组织损伤等多种生物学效应。2.PQQ在运动性疲劳模型中,具有维持正常线粒体形态和功能、正常血清代谢水平的重要作用。3.线粒体复合物Ⅰ为PQQ发挥抗运动性疲劳效应的可能位点。4.推测PQQ通过直接调节线粒体复合物Ⅰ的活性,从而稳定了线粒体功能,增加了ATP的合成,保证了机体的正常代谢和能量供应;与此同时,抑制了ROS的生成,下调了ROS介导的氧化应激,减轻了NF-κB参与的炎症反应,减少细胞凋亡,进而预防与延缓了运动性疲劳的发生。更多还原

【Abstract】 Background:Fatigue has a significant impact on exercise outcome,and affects human health,work and life.Thus,it draws a lot of interest to explore new anti-fatigue nutritional factors and develop safe and efficient anti-fatigue functional supplements for improving exercise ability and promoting human health.Pyrroloquinoline quinone(PQQ)has a variety of biological functions,such as anti-oxidation,anti-inflammatory,and maintaining normal mitochondrial function,suggesting that it may have important research and development value in anti-fatigue injury induced by over-exercise,but so far,no relevant research has been reported.Objective:The present study assessed the potential protective effects and mechanism of PQQ against the fatigue,oxidative stress,tissue injury,inflammation and apoptosis caused by over-exercise.The study will provide a theoretical support for the development of PQQ as an efficient supplement in anti-fatigue injury induced by over-exercise.Design:1.Cell and animal models for over exercise-induced fatigue were established,and the parameters including TTE(the time to exhaustion),ROS(reactive oxygen species),CK(creatine kinase),LDH(lactate dehydrogenase),cTnI(cardiac troponin I),SOD(superoxide dismutase),GSH-Px(glutathione peroxidase)activity levels,and the expression of NF-κB(nuclear factor kappa B),IL-6(interleukin-6),IL-1β(interleukin1β),CXCL10(interferon-inducible protein-10),Bcl-2(B-cell lymphoma-2),Bax(Bcl-2associated X protein)and Caspase-3 were measured to evaluate the function of PQQ in the models.2.The mitochondrial function was studied by testing changes on mitochondrial morphology,membrane potential,mitochondrial respiratory function,and adenosine triphosphate(ATP)levels.Moreover,mitochondrial complex I inhibitor was used to identify its possible targets.3.The overall changes of serum metabolic profiles were analyzed by GC-MS metabolomics.Furthermore,principal component analysis(PCA)and orthogonal partial least square-iscriminate analysis(OPLS-DA)were used to identify key potential markers,elaborate the metabolic pathways and regulatory mechanisms of PQQ,and explore its possible targets.Result:1.PQQ was able to inhibit the production of ROS,the LDH activity of the medium,the over-expression of transcription factor NF-kappa B,as well as to decrease m RNA expression of IL-6,IL-1β,interferon-inducible protein-10(CXCL10)and Caspase-3 in the cell model of exercise-induced fatigue.2.PQQ was able to prolong the time of exhaustive exercise,decrease the activity of CK,LDH and cTnI in the serum,increase the activity of SOD and GSH-Px in the heart and liver,reduce the expression and release of inflammatory mediators such as TNF-a,IL-1β,IL-6,CXCL-10,up-regulate the proportion of Bcl-2 and Bax,and inhibit the over-expression and activation of NF-κB(p65)and caspase-3.3.PQQ is able to maintain the normal morphology and function of mitochondria in over exercise-induced fatigue model.Moreover,mitochondrial complex I is likely a possible target of PQQ.4.Over exercise-induced fatigue could lead to metabolic disorders,and suggest that PQQ may play an important role in maintaining normal serum metabolic level.The levels of citric acid,L-proline,xylitol,N-acetyl-L-phenylalanine were changed,and their related metabolic pathways were affected.Conclusion:We first demonstrate that PQQ can delay over exercise-induced fatigue and improve exercise ability.Furthermore,in the cell and animal models for over exercise-induced fatigue,PQQ can significantly protect mice from the oxidation,inflammation,tissue damage and mitochondrial dysfunction and so on.The data suggest that PQQ may be used as a nutritional supplement to improve exercise outcome.更多还原