【作者】 王海涛；

【导师】 刘建国；

【作者基本信息】 河北师范大学 ， 体育教育训练学， 2009， 博士

【摘要】 中长跑是典型的周期性耐力项目,有氧氧化供能占主导地位。因此氧的摄取和利用效率是影响骨骼肌产生ATP的重要因素。红细胞中的血红蛋白是运氧的重要载体,骨骼肌中肌红蛋白接受并贮存血红蛋白运载的氧,线粒体是有氧代谢的主要场所,电子传递链中的细胞色素水平影响有氧代谢能力。铁参与了血红蛋白、肌红蛋白和细胞色素等物质的合成,因此机体铁状态可以影响氧的转运及线粒体产生ATP的能力,进而影响运动成绩。对于中长跑运动员来说维持机体铁稳态尤其是骨骼肌的铁含量是十分重要的。目前的研究表明我国优秀运动员仍存在运动引起的铁缺乏,以至影响运动能力。关于运动中铁代谢的变化进行了大量研究,但不同负荷的耐力训练对机体尤其是骨骼肌铁代谢的调节机制尚不明确。最新的研究成果表明一氧化氮（NO）可调节巨噬细胞和人白血病细胞的铁代谢。然而目前尚缺乏直接证据证明在运动中NO对骨骼肌细胞铁代谢究竟有何影响,其调节机制还缺乏深入研究。本研究通过在体和离体实验,研究NO对不同负荷耐力训练过程中大鼠骨骼肌铁代谢的调节机制。观察大鼠不同负荷耐力训练后骨骼肌NO含量、骨骼肌铁代谢变化及其相关关系,通过免疫蛋白印迹（Western Blot）观察骨骼肌相关蛋白表达的变化。通过培养大鼠骨骼肌细胞,检测外源NO对肌细胞摄铁和释铁功能、铁转运蛋白表达的影响,阐明运动中NO对骨骼肌铁代谢的调节机制。研究方法:在体实验:雄性Wistar大鼠18只,体重300±10g,随机分为对照组、适度运动组和过度运动组,每组6只。对照组大鼠不运动,适度运动组和过度运动组大鼠分别进行不同负荷的递增负荷跑台训练。5周后取材,测定血常规、机体铁状态、组织铁含量及腓肠肌铁代谢相关蛋白表达（TfR1、DMT1（IRE）、DMT1（non-IRE）和FPN1）、血清和腓肠肌一氧化氮合酶（NOS）活性及NO含量。离体实验:培养L6大鼠成肌细胞,实验组加入0.5mmol/l的NO供体SNAP,对照组用基本培养基,孵育24小时后用同位素铁⁵⁵Fe测定铁的摄取量与释放量、铁代谢相关蛋白表达（TfR1、DMT1（IRE）、DMT1（non-IRE）和FPN1）。研究结果:（1）不同负荷耐力训练对大鼠体重的影响:经过5周不同负荷递增负荷的运动,过度运动组大鼠体重在第3周开始下降,到第5周末体重与对照组相比下降了19.18%（P<0.01）,适度运动组体重与对照组无显差别。（2）不同负荷耐力训练对大鼠血常规的影响:过度运动组大鼠红细胞（RBC）、血红蛋白（Hb）和红细胞压积（Hct）与对照组比较分别下降了32.39%、32.05%和21.30%（P<0.01）,而适度运动组大鼠RBC、Hb和Hct均高于对照组和过度运动组（P<0.01）。（3）不同负荷耐力训练对大鼠机体铁状态的影响:适度运动组和过度运动组大鼠腓肠肌铁含量均显著高于对照组（P<0.01）,而适度运动组腓肠肌铁含量显著高于过度运动组（P<0.01）。与对照组相比,适度运动组大鼠血清铁含量和转铁蛋白饱合度增加。过度运动组血清铁含量、转铁蛋白饱合度、血清铁蛋白、骨髓铁含量均比对照组和适度运动组降低。各组血清总铁结合力和肝脏铁含量没有变化。（4）不同负荷耐力训练对大鼠腓肠肌铁转运蛋白表达的影响:适度运动组和过度运动组大鼠腓肠肌TfR1和DMT1（IRE）蛋白表达量均比对照组高,而两组FPN1蛋白表达量均低于对照组（P<0.05）。适度运动组与过度运动组之间三种蛋白的表达量差异不具有显著性。各组之间DMT1（non-IRE）蛋白表达量均差异不具有显著性。（5）不同负荷耐力训练对大鼠腓肠肌和血清NO、NOS的影响:过度运动组血清NOS活性和NO含量显著高于对照组和适度运动组（P<0.01）,腓肠肌NOS活性和NO含量显著高于对照组（P<0.01）。适度运动组血清NOS活性和NO含量显著高于对照组（P<0.01）,腓肠肌NO含量高于对照组（P<0.01）,对照组和适度运动组腓肠肌NO含量与腓肠肌铁含量线性相关（r=0.81,P<0.01）。（6）NO对L6细胞铁代谢的影响:同位素测试结果显示实验组L6细胞铁摄入量比对照组高,铁释放量与对照组比较差异不具有显著性。（7）NO对L6细胞铁转运相关蛋白表达的影响:实验组L6细胞TfR1、DMT1（IRE）蛋白表达量比对照组高（P<0.05）,FPN1蛋白表达量比对照组低（P<0.05）,而DMT1（non-IRE）蛋白表达量与对照组比较差异不具有显著性。结论:（1）适度运动可通过增加大鼠体内NO,引起腓肠肌细胞膜上向细胞内转运铁的蛋白（TfR1、DMT1（IRE））表达增加,而向细胞外释放铁的FPN1表达降低,导致肌细胞内铁贮量增加。同时增加Hb含量,保证运动中骨骼肌细胞对氧和铁的需求,促进有氧氧化的顺利进行,使机体对运动产生有益的生理适应。（2）过度运动使大鼠体内NO急剧增加,NO诱发机体贮存铁重新分布,使腓肠肌内铁储量明显增加,但骨髓内铁贮量大幅减少,导致运动性低血色素。同时机体铁储量明显降低。机体大量动员了骨髓中的贮存铁,以保障骨骼肌的铁需求,但并未动员肝脏贮存铁,表明过度运动引发机体铁代谢调控发生紊乱。（3）一定浓度的NO调节TfR1和DMT1蛋白表达增多,骨骼肌摄铁增加,使骨骼肌内铁贮量增加。总之,本实验通过在体和离体实验从细胞和分子水平阐明了运动中NO对骨骼肌铁代谢具有一定的调节作用。这种调节作用与运动负荷关系密切。本研究为合理安排训练,防止运动性低血色素发生提供了一定的理论依据和实验基础。更多还原

【Abstract】 Middle-distance race is a typical periodicity endurance training,and carbohydrate oxidation is the main way to produce energy for exercise.The utilization efficiency of oxygen can affect the production of ATP in skeletal muscle.Hemoglobin（Hb） in the red blood cell can transport oxygen.Myoglobin（Mb） of skeletal muscle can accept and store oxygen transported by Hb.Cytochrome of electron transfer in mitochondrion can affect the ablity of oxidation. Iron is needed for the synthesization of Hb,Mb and Cytochrome.Iron is utilised by the body for oxygen transport and energy production,and therefore iron is essential to athletic performance.Normal iron status in athletes is especially important during exercise.It has been reported that strenuous bouts of prolonged exercise and heavy training were associated with iron deficiency in both animal models and athletes.It is not known whether different load exercise induce iron deficiency.The molecular regulatory mechanism of the changed iron status and iron metabolism in muscle during exercise is not well determined.Some researches reported nitric oxide（NO） may play an important role in iron redistribution in exercise,while the molecular regulatory mechanism of iron metabolism in skeletal muscle was unclear.The present study investigated the effects of NO on iron metabolism in vivo and in vitro in order to illuminate the molecular regulatory mechanism of iron metabolism in exercise.The experiments in vivo:examined the level of iron status,NO,iron storage and the expression of iron transport related proteins in rats’ gastrocnemius after 5-week different load treadmill exercise of progressive loading.The experiments in vitro:examined the effects of SNAP（NO donor） on iron release and intake of L6.We hypothesized that NO plays an important role in iron metabolism in gastrocnemius during exercise.Methods:The experiments in vivo:18 male Wistar rats（300±10g） were randomly divided into three groups:control group（CG）,moderately exercised group（MG） and strenuously exercised group（SG）.The status of blood,serum iron,serum ferrtin,NO content and NOS activity,non-haem iron in different tissue were examined after 5-week different load treadmill exercise of progressive loading.The expression of transferrin receptor 1（TfR1）、divalent metal transporter1（DMTl:DMTl（IRE） and DMT1（non-IRE））and ferroportin 1（FPN1） in gastrocnemius of rats were determined by Western Blot.The experiments in vitro:The L6 cells were treated with SNAP（NO donor）（control group（CG） 0 mM and experimental group（EG） 0.5 mmol/L） for 24 h,and examined the effects of NO on iron release and intake by ⁵⁵Fe.The expression of TfR1、DMT1（IRE）、DMT1（non-IRE） and FPN1 of L6 cells were determined by Western Blot.Results:（1） Effects of different load endurance training on rats’ weight:The body weight of SG was significantly decreased 19.18%compared with CG after 5-week training（P<0.01）.There was no significantly change between CG and MG.（2） Effects of different load endurance training on rats’ blood status:The red blood cell count,haemoglobin and haematocrit of SG were decread 32.39%、32.05%and 21.30%than that of CG（P<0.01）.While the red blood cell（RBC）、Hb and hematocrit（Hct） in MG were increased compared with CG and SG（P<0.01）.（3） Effects of different load endurance training on rats’ serum iron status and non-haem iron distribution in different tissues:The non-haem iron in gastrocnemius of SG and MG were significantly increased compared with CG（P<0.01）.Serum iron,serum ferritin of MG were increased than that of CG.On the contrary,the serum iron,serum ferritin,TS and non-haem iron in bone marrow of SG were significantly decreased compared with CG and MG.（4） Effects of different load endurance training on the expression of TfR1、DMT1（IRE）、DMT1（non-IRE） and FPN1 of rats’ gastrocnemius:Western Blot showed the expression of TfR1 and DMT1（IRE） were increased in MG and SG than that of CG,while the expression of FPN1 in MG and SG was drcreased comared with CG（P<0.05）.There was no significant change of DMT1（non-IRE） among three groups.（5） Effects of different load endurance training on the level of NO and the activity of NOS in serum and gastrocnemius of rats:The level of NO and the activity of NOS in serum of SG were significantly increased than that of CG and MG（P<0.01）.The level of NO and the activity of NOS in gastrocnemius of SG were significantly increased than that of CG（P<0.01）. The level of NO and the activity of NOS in serum of MG were significantly increased than that of CG（P<0.01）,but lower than that of SG（P<0.01）.There is a linearity corelation between the level of NO and iron on rats’ gastrocnemius in physiological range（r=0.81,P<0.01）.（6） Effects of NO on iron release and intake of L6 cells:The iron intake was increased of SNAP cells（P<0.01）.But there was no significant change of iron release between the two groups.（7） Effects of NO on the expression of iron transport proteins of L6 cells:The expression of TfR1 and DMT1（IRE） were increased in EG cells than that of CG cells,while the expression of FPN1 was drcreased comared with control cells（P<0.05）.But there was no significant change of DMT1（non-IRE） of the expression between the two groups.Conclusions:（1）The NO of rats was increased after moderately exercise.The expression of TfR1 and DMT1（IRE） were increased while the expression of FPN1 in gastrocnemius was decreased,in turn the stored iron in gastrocnemius was increased.There was a linearity corelation between the level of NO and iron of rats’ gastrocnemius in physiological range.At the same time,the RBC,Hb content,serum iron and transferrin saturation（TS） were also increased than that of CG and MG,which resulted in the elevated O₂ and iron in the skeletal muscle and the increaed oxidation,which was a physioadaptation to exercise.（2） The NO of rats was rapidly increased after strenuous exercise.Though the non-haem iron of gastrocnemius was increased,the iron in bone marrow was significantly decreased.As a result,the RBC,Hb content,serum iron and TS were significantly decreased,which caused sports hypochrosis.The serum ferritin and the stored iron were decreased.Most of the stored iron in bone marrow was removed to skeletal muscle,while the stored iron was not changed in liver,which showed the iron metabolism was disordered after strenuous exercise.The high load exercise was able to destroy the iron homeostasis.（3） The increased NO induced by exercise might lead to the changed expression of iron-related proteins in gastrocnemius,which resulted in elevated iron absorption and a reduction of iron release.Taken together,these data indicated NO played an important role in iron metabolism of skeletal muscle during exercise,and the regulatory process was related to sports load.Our present results provid the experimental base for the arrangement of training,and preventing exercise-induced hypochrosis.更多还原