【作者】 高超；

【导师】 姚平；

【作者基本信息】 华中科技大学 ， 营养与食品卫生学， 2014， 博士

【摘要】 目的：1、探讨槲皮素对剧烈运动小鼠心肌、肝脏氧化损伤的保护作用；2、探讨槲皮素对剧烈运动小鼠心肌线粒体氧化应激和功能紊乱的拮抗作用；3、探讨槲皮素对剧烈运动小鼠肝脏炎性反应的干预效应及对NF-xB/IxBa介导炎症的调节作用。方法：48只成年雄性BALB/c小鼠,随机分为正常对照组、运动对照组、运动+槲皮素20mg/(kg-d)组、运动+槲皮素50mg/(kg-d)组、运动+槲皮素100mg/(kg-d)组和槲皮素100mg/(kg-d)组。根据Marra法并进行适当修改建立急性剧烈运动损伤动物模型。槲皮素干预四周,在干预期后10天,运动组先进行2天适应性训练,休息2天后,开始正式运动。初始速度为10m/min,在10min内逐渐增加负荷至28m/min,坡度为5度,持续90min,采用以上运动方式连续运动7天,末次运动完成后即刻处死小鼠。结果：1、与正常对照组相比,高剂量槲皮素干预明显降低剧烈运动导致的小鼠血清CK-MB (P<0.01)、AST (P<0.01)和ALT (P<0.01)水平上升；槲皮素干预明显抑制剧烈运动导致的小鼠心肌和肝脏的氧化应激以及超微病理结构异常。2、槲皮素干预明显拮抗了剧烈运动导致的小鼠心肌线粒体氧化应激以及心肌线粒体膜电位(P<0.01)和呼吸控制率(RCR)(P<0.05)的下降。3、剧烈运动导致小鼠血清TNF-α (P<0.01)、IL-1β (P<0.01)、IL-6(P<0.01)和IL-10(P<0.05)水平显著升高,槲皮素干预明显降低剧烈运动小鼠血清TNF-α (P<0.05). IL-1β (P<0.05)和IL-6(P<0.05)水平。4、与正常对照组相比,剧烈运动小鼠肝脏IL-1β (P<0.01)、IL-6(P<0.01)和IL-10(P<0.01) mRNA水平显著升高,槲皮素干预降低了运动小鼠肝脏IL-1β (P<0.05)和IL-6(P<0.01) mRNA水平。5、与正常对照组相比,剧烈运动导致小鼠肝脏iNOS (P<0.01)、COX-2(P<0.01)和ICAM-1(P<0.01) mRNA表达分别增加3.2倍、1.6倍和0.7倍,槲皮素干预明显抑制了运动小鼠肝脏iNOS (P<0.01)、COX-2(P<0.05)和ICAM-1(P<0.05)mRNA水平升高。槲皮素干预抑制了剧烈运动导致的小鼠肝脏iNOS、COX-2和ICAM-1蛋白表达增加。6、采用免疫荧光双标技术,运用激光共聚焦显微镜观察到槲皮素干预明显抑制剧烈运动导致的小鼠肝细胞NF-κB转位入核。7、与正常对照组相比,剧烈运动导致肝脏IκKBα蛋白表达降低0.59倍(P<0.05), P-IκBα蛋白表达增加1.7倍(P<0.01)；槲皮素干预提高IKBa蛋白表达(P<0.05),同时抑制P-IκBα蛋白表达(P<0.05),表明槲皮素对剧烈运动导致的IκBα磷酸化有一定抑制作用。剧烈运动小鼠肝脏P-IKKα表达比正常对照组增加9.9倍(P<0.01),槲皮素明显抑制P-IKKα的高表达(P<0.01)。8、剧烈运动小鼠肝细胞核内NF-κB与DNA结合能力提高115%(P<0.01),槲皮素干预明显抑制运动小鼠肝细胞核内NF-κB与DNA的结合活性(P<0.01)。结论：1、槲皮素对剧烈运动导致的小鼠心肌和肝脏氧化损伤具有明显保护作用；2、槲皮素对剧烈运动小鼠心肌线粒体超微结构损伤和功能紊乱的拮抗作用,可能与其抗氧化作用有关；3、槲皮素可能通过调节P-IKKα/IκBα/P-IκBα信号通路而抑制NF-κB转录激活,发挥良好的抗炎保护作用,减轻了剧烈运动导致的肝脏损伤。更多还原

【Abstract】 Objective:1. To investigate the protection of quercetin against intense exercise-induced oxidative stress focused on myocardium and liver.2. To investigate the protective effect of quercetin on intense exercise-induced oxidative stress and dysfunction focused on myocardial mitochondria.3. To evaluate whether overexpression of inflammatory mediators induced in the liver by intense exercise could be prevented by quercetin and whether the protective effect of quercetin was related with inhibition of NF-κB activation.Methods:All adult male mice were randomly divided into six groups of eight animals each:rested controls (Ct), intense exercise (Ex), Ex+dietary quercetin20mg/(kg·d)(Ex+Qu20), Ex+dietary quercetin50mg/(kg-d)(Ex+Qu50), Ex+dietary quercetin100mg/(kg-d)(Ex+Qu100) and rested plus dietary quercetin supplementation (Qu). Quercetin was administrated for4weeks and intense exercise program was performed as described by Marra with some modifications at the fourth week. Simply, the mice were acclimated to running on a motor-driven treadmill for2successive days, beginning at10m/min on a5°slope for10min/day, and then submitted to daily sessions of intense exercise for7consecutive days following a two-day rest. Mice were run at28m/min on a5°slope for90min while a10-min warm-up was maintained. Immediately upon final exercise, the mice were sacrificed. Results:1. Compared to normal control mice, Qu100treatment decreased intense exercise-induced the leakage of creatine kinase-MB (P<0.01), aspartate aminotransferase (P<0.01), and alanine aminotransferase (P<0.01). Intense exercise resulted in sustained oxidative stress and ultrastructural malformation on myocardium and liver which was evidently reversed by quercetin intervention.2. Quercetin pretreatment partially normalized intense exercise-induced myocardial mitochondrial oxidative stress and decline of mitochondrial membrane potential (P<0.01) and respiratory control ratio (RCR)(P<0.01).3. Significant increases postexercise were measured for serum TNF-a (P<0.01), IL-1β (P<0.01), IL-6(P<0.01), and IL-10(P<0.05) levels compared with normal control mice, quercetin pretreatment to exercise mice substantially decreased TNF-a (P<0.05), IL-1β (P<0.05), and IL-6(P<0.05) levels but not IL-10.4. Compared to normal control mice, exercise coursed with a significant rise in hepatic IL-1β (P<0.01), IL-6(P<0.01), and IL-10(P<0.01) mRNA levels. These effects of IL-1β (P<0.05) and IL-6(P<0.01) were significantly reduced by quercetin.5. Compared to normal control mice, intense exercise resulted in a significant increase of hepatic iNOS (P<0.01), COX-2(P<0.01), and ICAM-1(P<0.01) mRNA levels by3.2fold,1.6fold and0.7fold, respectively, and iNOS (P<0.01), COX-2(P<0.05), and ICAM-1(P<0.05) mRNA levels were partially blocked by quercetin administration compared with exercised mice. Intense exercise increased hepatic protein levels of iNOS, COX-2and ICAM-1which were evidently reversed by quercetin intervention.6. Following acute exercise, NF-κB was evident and colocalized with many of the nuclei. Comprared with exercised mice, quercetin pretreatment partially reduced the transfer of NF-κB into the nuclei. 7. Intense exercise resulted in a significant decrease of the nonphosphorylated form (-0.59fold)(P<0.05), while phosphorylated IkBα protein level was markedly increased (+1.7fold) compared with normal control (P<0.01). These effects were partially blocked by quercetin prevention (P<0.05). Protein level of P-IKKa was up-regulated in exercised mice (+9.9fold)(P<0.01), and this effect was obviously suppressed by quercetin intervention (P<0.01).8. Compared to normal control mice, intense exercise induced a marked activation of NF-κB (115%)(P<0.01) that was evidently blocked in liver of animals treated with quercetin (P<0.01).Conclusion:1. Quercetin exhibited protective role on intense exercise-induced oxidative stress injuries of myocardium and liver.2. Quercetin may protect mouse myocardium from intense exercise injury, including ultrastructural damage and mitochondrial dysfunction, through its antioxidative effect on mitochondria.3. Quercetin protects mouse liver against intense exercise damage, and impaired production of noxious mediators involved in P-IKKα/IκBα/P-IκBα inflammatory process and down-regulation of the NF-κB signal transduction pathway appear to contribute to the beneficial anti-inflammatory effects of quercetin.更多还原