力竭运动诱导的大鼠红细胞氧化应激表现及发生机制的研究

发布时间：2018-06-04 18:00

本文选题：力竭运动 + 红细胞　；参考：《重庆大学》2014年博士论文

【摘要】：合理适度的运动锻炼能够增强机体的免疫力，而力竭、缺氧等极端条件下的运动通常伴随着多种心血管疾病的增加，甚至猝死。已有的研究表明：运动性休克、猝死等疾病往往伴随着严重的心血管功能障碍的发生。其中，心室纤颤是导致运动性猝死最常见的并发症，而这种运动诱导的致命性心律失常的发病机制仍不甚明确。一般认为，动脉粥状硬化患者心肌组织供血障碍或血栓形成及运动性冠状动脉痉挛是导致运动性心肌局部供血不足，进而诱发致命性心律紊乱的重要原因。然而，作为循环系统主要组分的红细胞，其在不同运动条件下对机体微循环畅通和组织供氧活动的影响仍有待研究。因此，研究不同运动条件下RBC抗氧化、清除自由基能力的改变，自身受到氧化损伤程度的变化，以及氧化应急条件下红细胞力学性质和携氧功能的改变将对运动诱发的休克、缺氧损伤、红细胞凋亡等多种生理和病理现象的探索提供重要的理论依据。机体在运动过程中会产生大量的活性氧簇（ROS），这些ROS在组织生成后会迅速的释放的血浆中。尽管红细胞拥有非常完善的抗氧化机制，能够迅速的清除来自胞内和胞外的ROS。但是当机体处于氧化应激状态时，持续、大量产生的ROS被释放到血浆之中，使红细胞的氧化-还原平衡被打破。本课题中，对不同运动组大鼠红细胞抗氧化指标SOD、CAT和GSH检测结果表明：力竭运动后大鼠红细胞抗氧化酶SOD和CAT的活性显著增加，与此同时，胞内主要的非酶类抗氧化物GSH含量出现显著降低。这表明，力竭运动使大鼠红细胞抗氧化系统进入应急状态，以抵御运动诱导的自由基爆发。通过TBA法检测膜脂质TBARS水平，结果表明：力竭运动导致大鼠红细胞膜脂质TBARS水平显著升高。与此同时，胞内高铁血红蛋白和膜结合高铁血色原的含量也出现了显著增加。红细胞膜蛋白含有丰富的SH，它们对维系RBC膜上众多酶类和功能蛋白的正常结构功能起到重要作用。与对照组相比，力竭运动后红细胞膜蛋白SH含量也出现显著下降。由此可见，力竭运动诱导大鼠红细胞出现严重的氧化应激损伤。在运动引发的多种心脑血管疾病中，心肌、脑组织等局部供血不足被认为是重要的并发症。从红细胞的角度看，红细胞变形性和携氧功能的维系对组织微循环畅通和有效携氧起着重要的作用。本课题组，不同运动条件下大鼠红细胞变形能力和携氧功能检测分析结果表明：在力竭运动过程中，由膜脂质过氧化，，膜蛋白交联等氧化应激诱导的红细胞损伤，红细胞膜流动性下降，导致细胞变形能力降低。对RBC携氧-释氧热力学和动力学曲线分析表明：在力竭运动过程中，大鼠红细胞携氧热力学曲线右移，P50值显著升高；与此同时，红细胞氧压衰减曲线左移T50指标都出现了显著下降。这意味着，在力竭环境下大鼠红细胞结合-释放氧的胞内环境以及相关的膜蛋白组分都受到了不同程度的影响或损伤。红细胞中以Band3蛋白为中心的蛋白复合体在维系红细胞膜结构完整性和稳定性，以及调控红细胞能量代谢和气体转运的过程中都起到重要作用。这个大的Band3复合体的主要功能有：作为跨膜蛋白与骨架网路的连接枢纽，维持膜结构的稳定；与糖酵解酶类相互作用，调控糖代谢速率和途径；与碳酸酐酶（CAII）等相互作用，形成代谢区室，形成以Band3为核心的O2/CO2交换中心。因此，为了进一步探讨力竭运动诱导的红细胞变形和携氧功能障碍的分子机制，本文对运动性氧化应激反应诱导的红细胞膜Band3蛋白表达和分布情况及其调控的阴离子通道活性进行了分析。Western Blotting和免疫荧光检测的结果都表明：力竭运动导致包括Band3蛋白在内的膜蛋白交联，导致红细胞Band3蛋白结构和功能性损伤。Band3蛋白的损伤将进一步诱导红细胞携氧和变形能力的下降，成为运动相关疾病的潜在致病因素。为了进一步探讨力竭运动诱导的红细胞氧化损伤与运动过程中血液氧含量的下降之间的关系。本文在体外条件下模拟不同氧分压环境，并以次黄嘌呤/黄嘌呤氧化酶体系构建了体外氧化模型。通过对不同氧分压环境下红细胞受氧化损伤程度和抗氧化能力的变化，初步探讨了环境氧分压与红细胞氧化损伤的关系。结果表明：RBC氧化损伤和抗氧化力受环境氧分压条件的影响。在不同氧分压环境下，红细胞血红蛋白构象在R态和T态之间的转变会间接地导致胞内糖代谢途径的改变，从而对红细胞氧化-还原代谢途径产生影响。论文还对力竭恢复阶段大鼠红细胞氧化应激表现及红细胞凋亡和再生过程进行了初步的探讨。结果表明，在力竭恢复初期大鼠红细胞仍受到严重的氧化应激损伤。伴随着恢复时间的增加，受损伤严重的红细胞逐渐衰老、凋亡。与此同时，体内的造血系统被动员，产生大量的新鲜红细胞以补充循环中受损伤清除的红细胞。综上所述，本课题主要得出以下结论：力竭运动过程中，机体进入氧化应激状态。诱导大鼠红细胞膜脂质过氧化、膜蛋白聚簇化和血红蛋白氧化在内的氧化损伤。膜蛋白氧化损伤，尤其是Band3蛋白聚簇化和磷酸化程度的增加，导致力竭运动后大鼠红细胞携氧功能和变形能力受到严重影响，使红细胞在循环过程中气体转运效率下降，成为组织局部供血不足等运动性疾病的潜在致病因素。
[Abstract]:Reasonable exercise can enhance the immunity of the body, and the exercise under extreme conditions such as exhaustion and hypoxia is usually accompanied by the increase of a variety of cardiovascular diseases and even sudden death. The most common complication of sudden motor death, and the pathogenesis of the fatal arrhythmia induced by exercise is still not clear. It is generally believed that the blood supply disorder or thrombosis and motor coronary spasm in atherosclerotic patients lead to the insufficiency of the local blood supply of the motor myocardium, and then induce the fatal arrhythmia. However, as the main component of the circulation system, the influence of the red blood cells on the microcirculation and oxygen supply activities of the body under different sports conditions remains to be studied. Therefore, the study of the changes in the antioxidant capacity of RBC, the ability to scavenge free radicals, the changes in the degree of oxidative damage, and the oxidation emergency strip under different sports conditions are still to be studied. The changes in the mechanical properties and oxygen carrying functions of the red cells will provide important theoretical basis for the exploration of many physiological and pathological phenomena such as shock induced shock, hypoxia injury, erythrocyte apoptosis and so on.
While the body produces a large number of active oxygen clusters (ROS) during the process of movement, the ROS can be released rapidly after tissue formation. Although red cells have a very perfect antioxidant mechanism, it can quickly remove ROS. from both intracellular and extracellular, but when the body is in oxidative stress state, a large amount of ROS is released to the body. In the plasma, the oxidation-reduction balance of red blood cells was broken. In this study, the results of SOD, CAT and GSH of erythrocyte antioxidant indices of rats in different sports groups showed that the activity of antioxidant enzyme SOD and CAT increased significantly after exhaustive exercise, while the GSH content of the main non enzyme antioxidants in the cells decreased significantly. This shows that the exhaustion movement makes the rat erythrocyte antioxidant system enter the emergency state to resist the free radical outbreak induced by movement. The lipid TBARS level of the membrane is detected by TBA method. The results show that the level of lipid TBARS in the erythrocyte membrane of rats is significantly increased by the exhaustion movement. The original content also increased significantly. The erythrocyte membrane protein contains rich SH, which plays an important role in maintaining the normal structural functions of many enzymes and functional proteins on the RBC membrane. Compared with the control group, the content of the erythrocyte membrane protein SH after exhaustive exercise also decreased significantly. Severe oxidative stress damage.
In a variety of cardio cerebral vascular diseases caused by exercise, the insufficiency of local blood supply, such as myocardium and brain tissue, is considered as an important complication. From the point of view of the red cell, the deformability of red blood cells and the maintenance of oxygen carrying function play an important role in the smooth and effective oxygen carrying of tissue. The results of detection and analysis of capacity and oxygen carrying function showed that during the exhaustion movement, erythrocyte damage induced by lipid peroxidation, membrane protein crosslinking and other oxidative stress induced the decrease of erythrocyte membrane fluidity and the decrease of cell deformability. The analysis of the thermodynamics and kinetic curves of RBC oxygen carrying oxygen showed that during the exhaustion movement, it was large At the same time, the left shift T50 index of the red blood cell oxygen pressure attenuation curve decreased significantly. This means that the intracellular environment of erythrocyte binding oxygen release and the related membrane protein components in the exhausted environment were affected or damaged in varying degrees under the environment of exhaustion.
The Band3 protein complex in red cells plays an important role in maintaining the structural integrity and stability of the erythrocyte membrane, as well as in regulating the energy metabolism and gas transport of red blood cells. The major functions of the large Band3 complex are as the junction of the transmembrane protein and the skeleton network to maintain the membrane structure. Stability, interacting with glycolytic enzymes, regulating the rate and pathway of glycometabolism, interacting with carbonic anhydrase (CAII), forming a metabolic zone and forming a O2/CO2 exchange center with Band3 as the core. Therefore, in order to further explore the molecular mechanism of red cell deformation and oxygen carrying dysfunction induced by exhaustion movement, this article is on the movement oxidation. The expression and distribution of Band3 protein in erythrocyte membrane induced by stress reaction and its regulated anion channel activity were analyzed. The results of.Western Blotting and immunofluorescence detection showed that the exhaustion movement led to the membrane protein crosslinking including the Band3 protein, resulting in the structural and functional damage of the erythrocyte Band3 protein to.Band3 protein. The damage will further induce the decrease of oxygen carrying capacity and deformability of erythrocytes and become a potential pathogenic factor of sports related diseases.
In order to further investigate the relationship between the oxidative damage induced by exhaustive movement and the decrease of blood oxygen content during exercise, this paper simulates the different oxygen partial pressure environment under the conditions of in vitro, and constructs an in vitro oxidation model with the system of hypoxanthine / xanthine oxidase. The relationship between the environmental oxygen partial pressure and the oxidative damage of red cells was preliminarily discussed. The results showed that the oxidative damage and antioxidant capacity of RBC were affected by the conditions of environmental oxygen pressure. In different oxygen partial pressure conditions, the transformation of the erythrocyte hemoglobin conformation between the R state and the T state would indirectly lead to the intracellular sugar metabolism. The change of pathway will affect the redox pathway of RBC.
The results show that red blood cells still suffer severe oxidative stress in the early period of exhaustion recovery. With the increase of recovery time, the damaged red blood cells gradually aging and apoptosis. The hematopoietic system in the body is mobilized to produce a large amount of fresh red blood cells to supplement the damaged red cells in circulation.
To sum up, the following conclusions are drawn as follows: in the process of exhaustion movement, the body enters oxidative stress, induces lipid peroxidation in erythrocyte membrane, membrane protein clustering and oxidation of hemoglobin, and the oxidative damage of membrane protein, especially the increase of Band3 protein clustering and phosphorylation, resulting in exhaustion The function of oxygen carrying and deformability of red blood cells in rats is seriously affected, and the efficiency of gas transport in the cycle of erythrocytes is reduced, and it is a potential pathogenic factor of sports diseases such as the lack of local blood supply in the tissue.
【学位授予单位】：重庆大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R87

【共引文献】