富氢水对骨骼肌运动性氧化应激损伤与选择性抗氧化作用机制研究
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摘要
大量研究证实急性大强度运动中体内氧自由基增加，导致脂质过氧化物蓄积、亚硝酸盐浓度升高及抗氧化酶活力降低等，引起细胞脂质过氧化损伤、蛋白质损伤与DNA损伤，致使肌肉疲劳、肌肉损伤、运动能力降低及过度训练等。许多研究发现补充生物抗氧化剂可减轻氧化应激损伤，提高运动能力，延长运动寿命。2007年国际上首次报道，富氢水是一种优质的抗氧化剂，其抗氧化特性具有选择性，对缺血再灌注等氧化应激损伤具有显著的防治作用。然而，有关富氢水对运动性氧化应激损伤的防治研究至今仍是一处空白，其在体选择性抗氧化作用与内在机制也尚不清楚。
研究目的：
本研究从最易受氧化攻击的动力源性器官—骨骼肌入手，采用运动动物实验，研究富氢水对骨骼肌运动性氧化应激损伤、抗氧化防御系统及其信号转导分子、运动能力等的影响，并通过测定ROS水平，探寻富氢水选择性抗氧化的在体依据及可能机制。
研究方法：
80只健康雄性SD大鼠，随机分成安静对照组（EC组、C组）、运动对照组（EE组、E组）、运动前注射富氢水组（EH组、H组）与运动前后联合注射富氢水组（EM组、M组）8组，每组10只。实验前一周，除EC组与C组外，其余各组每天进行1次适应性低强度跑台运动，坡度0°，速度10m·min~（-1），持续10min。正式实验时，EH组与H组于运动前1min腹腔注射10ml·kg~（-1）体重富氢水，EM组、M组于运动前1min及运动后即刻腹腔注射各10ml·kg~（-1）体重富氢水，EE组与E组腹腔注射同体积生理盐水。EE组、EH组与EM组采用Marra方案进行一次性大强度力竭跑台运动，E组、H组与M组进行定量跑台运动（其持续时间根据力竭跑台运动实验结果确定），于运动后3h，取E组、H组、M组与C组后肢腓肠肌，用Elisa法测定骨骼肌3-NT、8-OHdG及PKC含量，用化学比色法测定MDA、SOD、GSH、T-AOC及O_2~-、H_2O_2、ONOO~-、 OH、iNOS与NO水平。
研究结果：
1.富氢水可显著延长大鼠运动至力竭的持续时间，具有显著的抗疲劳作用。
2.运动导致骨骼肌ROS水平显著升高，SOD与GSH显著降低，T-AOC显著下降，PKC激活，iNOS活性升高，NO过量生成，3-NT、MDA与8-OHdG显著升高，氧化应激损伤加剧。
3.富氢水能显著阻抑O_2~-、H_2O_2、NO、ONOO~-与OH等的运动性加剧，在体清除强效细胞毒性物质ONOO~-与OH，提高SOD与GSH，增强T-AOC，抑制PKC激活与iNOS活性升高，抑制NO过量生成，减轻NO介导的细胞毒性，改善微循环，减轻细胞损伤，对骨骼肌运动性氧化应激损伤具有显著保护作用；运动前后联合注射富氢水对运动性氧化应激损伤防治具有协同作用。
4.富氢水对运动性氧化应激损伤保护作用的基础是富氢水的选择性抗氧化作用；其可能机制在于：通过富氢水的选择性抗氧化作用，在体清除ONOO~-与OH等的运动性加剧，正反馈抑制了细胞信号调控分子PKC的激活与NO的过量生成，减轻了NO介导的细胞毒性，减少了氧化损伤介质的产生，增强了骨骼肌的抗氧化能力，减缓了氧化损伤引起的运动能力下降。
研究结论：
1.富氢水具有抗疲劳效果。
2.富氢水对运动性氧化应激损伤具有保护作用，其保护作用是通过多靶点进行的，其基础在于富氢水的选择性抗氧化作用。
3.从细胞内信号转导机制层面看，富氢水对运动性骨骼肌氧化应激损伤的保护作用可能是通过PKC/NO途径介导的。
Extensive researches confirmed that the acute intensive exercise, resulting inoxygen radicals increase, LPO accumulation, nitrite rise and antioxidant enzymesreduce, always caused LPO injury, protein and DNA damage, muscular fatigue, muscledamage, exercise impairment, excessive training and so on. So, large numbers ofadditional antioxidants were suggested to be supplemented to relieve oxidative damage,enhance competitive ability and extend sports life. In2007, an international study firstindicated that hydrogen-rich water, a fairly better antioxidant characterized by selectiveantioxidation, could significantly treat ischemia/reperfusion oxidative injuries.However,up to now, the study of hydrogen-rich water on exercise-induced oxidative stressdamage was still left a blank, especially, of which in vivo selective antioxidation andinherent mechanism remained unclear.
Objective:
This research was designed, with skeletal muscle, the organ of power supplyvulnerable to attack by ROS, to study the effects of hydrogen-rich water onexercise-induced oxidative stress damage, in such respects as ROS level, signaltransduction molecules, antioxidant defense system, exercise ability and so on,especially, to explore its in vivo selective antioxidation and probable mechanism by themethods of ROS determination.
Methods:
Eighty healthy SD male rats were equally and randomly divided into eight groups：sedentary control group（EC,C），exercise control group（EE,E）, intraperitoneal injectionof hydrogen-rich water before exercis（eEH,H）and combined intraperitoneal injection ofhydrogen-rich water before﹠after exercise（EM,M）.One week before experiment, each group, except EC and C, exercised for adaptation on a treadmill at low speed of10m·min~（-1）with0°slope for10min once a day.During formal experiment, intraperitonealinjection of hydrogen-rich water（10ml·kg~（-1））were performed1min before exercise forgroup EH and H, while for group EM and M, intraperitoneal injection of hydrogen-richwater （10ml·kg~（-1））were performed1min before exercise and immediately afterexercise,and the same amount of normal saline was intraperitoneally injected to groupEE and E1min before exercise. The three groups, EE, EH and EM participated inexhaustive exercise at the speed of28m·min~（-1）with0°slope. While, the other threegroups, E,H and M devoted to quantitative exercise（,the endurance time was defined bythe results of exhaustive exercise）,3hours after exercise,the gastrocnemius weredrawn from group E,H, M and C to determine the density of3-nitrotyrosine（3-NT）,8-hydroxy deoxyguanosine （8-OhdG） and protein kinase C （PKC） by Elisameasurement,and malonaldehyde（MDA）,superoxide dismutase（SOD）,glutathione（GSH）, total antioxidative capacity （T-AOC）,superoxide anion radical（O_2~-）,hydrogen peroxid（eH_2O_2）, peroxynitrite（ONOO~-）, hydroxyl radical（OH）,induciblenitric oxide synthase （iNOS）and nitric oxide （NO）by chemical colorimetry.
Results:
1.Hydrogen-rich water had marked anti-fatigue role by significantly extendingendurance time to exhaustion without a time-and dose-dependent manner.
2.Exercise induced oxidative stress in skeletal muscle including increases of ROS,3-NT,MDA and8-OHdG,and weakened effects of SOD,GSH and T-AOC, and activationof PKC, iNOS,also, excessive generating of NO.
3.Hydrogen-rich water had a strong protective effect on exercise-induced oxidativedamage, which was achieved by inhibiting O_2~-、H_2O_2、NO、ONOO~-and OH, especially,clearing away such powerful toxic substances as ONOO~-and OH in vivo, anddeveloping the effects of SOD, GSH and T-AOC, repressing the activation of PKC andiNOS, also, the excessive generating of NO, which relieved NO-induced toxicitydamage, as well as improved microcirculation of skeletal muscle cells. These resultssuggested that combined injection of hydrogen-rich water both before and after exercisehave syn-ergetic protective effect on exercise-induced oxidative stress damage.
4.The basis of its protective effects of hydrogen-rich water on exercise-inducedoxidative stress damage was its selective antioxidation;And, as a probable mechanismdue to the role that hydrogen-rich water inhibited the activation of signal transductionmolecules PKC and the excessive generating of NO resulting from a positive feedbackof its selectively cleared away such powerful toxic ROS as ONOO~-and OH, whichrelieved NO-induced toxicity, also, reduced oxidative stress medium, enhancedantioxidation capacity of skeletal muscle, inhibited a reduced ability to exercise byoxidative damage.
Conclusions:
1.Hydrogen-rich water had a function to anti-fatigue.
2.Hydrogen-rich water had a strong protective effect on exercise-induced oxidativestress damage, which was achieved from multi-targets; and, the basis of which was itsselective antioxidation.
3.In terms of intracellular signal transduction mechanism, the protective effect ofhydrogen-rich water on exercise-induced oxidative damage may be modulated byPKC-NO pathway.
研究目的：
本研究从最易受氧化攻击的动力源性器官—骨骼肌入手，采用运动动物实验，研究富氢水对骨骼肌运动性氧化应激损伤、抗氧化防御系统及其信号转导分子、运动能力等的影响，并通过测定ROS水平，探寻富氢水选择性抗氧化的在体依据及可能机制。
研究方法：
80只健康雄性SD大鼠，随机分成安静对照组（EC组、C组）、运动对照组（EE组、E组）、运动前注射富氢水组（EH组、H组）与运动前后联合注射富氢水组（EM组、M组）8组，每组10只。实验前一周，除EC组与C组外，其余各组每天进行1次适应性低强度跑台运动，坡度0°，速度10m·min~（-1），持续10min。正式实验时，EH组与H组于运动前1min腹腔注射10ml·kg~（-1）体重富氢水，EM组、M组于运动前1min及运动后即刻腹腔注射各10ml·kg~（-1）体重富氢水，EE组与E组腹腔注射同体积生理盐水。EE组、EH组与EM组采用Marra方案进行一次性大强度力竭跑台运动，E组、H组与M组进行定量跑台运动（其持续时间根据力竭跑台运动实验结果确定），于运动后3h，取E组、H组、M组与C组后肢腓肠肌，用Elisa法测定骨骼肌3-NT、8-OHdG及PKC含量，用化学比色法测定MDA、SOD、GSH、T-AOC及O_2~-、H_2O_2、ONOO~-、 OH、iNOS与NO水平。
研究结果：
1.富氢水可显著延长大鼠运动至力竭的持续时间，具有显著的抗疲劳作用。
2.运动导致骨骼肌ROS水平显著升高，SOD与GSH显著降低，T-AOC显著下降，PKC激活，iNOS活性升高，NO过量生成，3-NT、MDA与8-OHdG显著升高，氧化应激损伤加剧。
3.富氢水能显著阻抑O_2~-、H_2O_2、NO、ONOO~-与OH等的运动性加剧，在体清除强效细胞毒性物质ONOO~-与OH，提高SOD与GSH，增强T-AOC，抑制PKC激活与iNOS活性升高，抑制NO过量生成，减轻NO介导的细胞毒性，改善微循环，减轻细胞损伤，对骨骼肌运动性氧化应激损伤具有显著保护作用；运动前后联合注射富氢水对运动性氧化应激损伤防治具有协同作用。
4.富氢水对运动性氧化应激损伤保护作用的基础是富氢水的选择性抗氧化作用；其可能机制在于：通过富氢水的选择性抗氧化作用，在体清除ONOO~-与OH等的运动性加剧，正反馈抑制了细胞信号调控分子PKC的激活与NO的过量生成，减轻了NO介导的细胞毒性，减少了氧化损伤介质的产生，增强了骨骼肌的抗氧化能力，减缓了氧化损伤引起的运动能力下降。
研究结论：
1.富氢水具有抗疲劳效果。
2.富氢水对运动性氧化应激损伤具有保护作用，其保护作用是通过多靶点进行的，其基础在于富氢水的选择性抗氧化作用。
3.从细胞内信号转导机制层面看，富氢水对运动性骨骼肌氧化应激损伤的保护作用可能是通过PKC/NO途径介导的。
Extensive researches confirmed that the acute intensive exercise, resulting inoxygen radicals increase, LPO accumulation, nitrite rise and antioxidant enzymesreduce, always caused LPO injury, protein and DNA damage, muscular fatigue, muscledamage, exercise impairment, excessive training and so on. So, large numbers ofadditional antioxidants were suggested to be supplemented to relieve oxidative damage,enhance competitive ability and extend sports life. In2007, an international study firstindicated that hydrogen-rich water, a fairly better antioxidant characterized by selectiveantioxidation, could significantly treat ischemia/reperfusion oxidative injuries.However,up to now, the study of hydrogen-rich water on exercise-induced oxidative stressdamage was still left a blank, especially, of which in vivo selective antioxidation andinherent mechanism remained unclear.
Objective:
This research was designed, with skeletal muscle, the organ of power supplyvulnerable to attack by ROS, to study the effects of hydrogen-rich water onexercise-induced oxidative stress damage, in such respects as ROS level, signaltransduction molecules, antioxidant defense system, exercise ability and so on,especially, to explore its in vivo selective antioxidation and probable mechanism by themethods of ROS determination.
Methods:
Eighty healthy SD male rats were equally and randomly divided into eight groups：sedentary control group（EC,C），exercise control group（EE,E）, intraperitoneal injectionof hydrogen-rich water before exercis（eEH,H）and combined intraperitoneal injection ofhydrogen-rich water before﹠after exercise（EM,M）.One week before experiment, each group, except EC and C, exercised for adaptation on a treadmill at low speed of10m·min~（-1）with0°slope for10min once a day.During formal experiment, intraperitonealinjection of hydrogen-rich water（10ml·kg~（-1））were performed1min before exercise forgroup EH and H, while for group EM and M, intraperitoneal injection of hydrogen-richwater （10ml·kg~（-1））were performed1min before exercise and immediately afterexercise,and the same amount of normal saline was intraperitoneally injected to groupEE and E1min before exercise. The three groups, EE, EH and EM participated inexhaustive exercise at the speed of28m·min~（-1）with0°slope. While, the other threegroups, E,H and M devoted to quantitative exercise（,the endurance time was defined bythe results of exhaustive exercise）,3hours after exercise,the gastrocnemius weredrawn from group E,H, M and C to determine the density of3-nitrotyrosine（3-NT）,8-hydroxy deoxyguanosine （8-OhdG） and protein kinase C （PKC） by Elisameasurement,and malonaldehyde（MDA）,superoxide dismutase（SOD）,glutathione（GSH）, total antioxidative capacity （T-AOC）,superoxide anion radical（O_2~-）,hydrogen peroxid（eH_2O_2）, peroxynitrite（ONOO~-）, hydroxyl radical（OH）,induciblenitric oxide synthase （iNOS）and nitric oxide （NO）by chemical colorimetry.
Results:
1.Hydrogen-rich water had marked anti-fatigue role by significantly extendingendurance time to exhaustion without a time-and dose-dependent manner.
2.Exercise induced oxidative stress in skeletal muscle including increases of ROS,3-NT,MDA and8-OHdG,and weakened effects of SOD,GSH and T-AOC, and activationof PKC, iNOS,also, excessive generating of NO.
3.Hydrogen-rich water had a strong protective effect on exercise-induced oxidativedamage, which was achieved by inhibiting O_2~-、H_2O_2、NO、ONOO~-and OH, especially,clearing away such powerful toxic substances as ONOO~-and OH in vivo, anddeveloping the effects of SOD, GSH and T-AOC, repressing the activation of PKC andiNOS, also, the excessive generating of NO, which relieved NO-induced toxicitydamage, as well as improved microcirculation of skeletal muscle cells. These resultssuggested that combined injection of hydrogen-rich water both before and after exercisehave syn-ergetic protective effect on exercise-induced oxidative stress damage.
4.The basis of its protective effects of hydrogen-rich water on exercise-inducedoxidative stress damage was its selective antioxidation;And, as a probable mechanismdue to the role that hydrogen-rich water inhibited the activation of signal transductionmolecules PKC and the excessive generating of NO resulting from a positive feedbackof its selectively cleared away such powerful toxic ROS as ONOO~-and OH, whichrelieved NO-induced toxicity, also, reduced oxidative stress medium, enhancedantioxidation capacity of skeletal muscle, inhibited a reduced ability to exercise byoxidative damage.
Conclusions:
1.Hydrogen-rich water had a function to anti-fatigue.
2.Hydrogen-rich water had a strong protective effect on exercise-induced oxidativestress damage, which was achieved from multi-targets; and, the basis of which was itsselective antioxidation.
3.In terms of intracellular signal transduction mechanism, the protective effect ofhydrogen-rich water on exercise-induced oxidative damage may be modulated byPKC-NO pathway.
引文
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