毛蕊花苷对递增负荷运动小鼠骨骼肌损伤的保护作用
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摘要
本研究采用小鼠跑台训练模型,应用免疫共沉淀等方法,研究毛蕊花苷对递增负荷运动小鼠骨骼肌损伤的保护作用及对谷胱甘肽的影响。90只小鼠随机分为:正常对照组(A)、正常+毛蕊花苷组(B)、单纯运动组(C)、运动+毛蕊花苷低剂量组(D)、运动+毛蕊花苷中剂量组(E)、运动+毛蕊花苷高剂量组(F).结果表明:与C组比较,D、E、F组小鼠骨骼肌损伤程度依次减轻,F组小鼠骨骼肌形态基本正常。F组小鼠血浆CK水平、骨骼肌组织GSSG含量分别低于C、D、E组;但CK水平高于A、B组,均P<0.01;GSSG含量与A、B组比较,差异无显著性,P>0.05。F组小鼠骨骼肌组织GSH含量、GSH/GSSG比值、GCL和GR酶活性、RyR1复合物中GSH表达水平分别高于C、D、E组,但低于A、B组,均P<0.01。研究结果提示毛蕊花苷能降低递增负荷运动小鼠血浆CK的活性,保护运动鼠骨骼肌的形态;其机理与其提高运动鼠骨骼肌组织GSH含量和GCL、GR酶的活性,降低GSSG/GSH比值;提高RyR1复合物中GSH表达水平有关。
Using the training model of mice running and the methods of immunoprecipitation and immunoblotting,this study aimed to investigate the protective effects of verbascoside on skeletal muscle damage by incremental load exercise and glutathione of mice. Ninety mice were divided into six groups including:the normal group(A),the normal + verbascoside treated group(B),the exercise group(C),the exercise + verbascoside treated by the low dosage(D),the exercise + verbascoside treated by the moderate dosage(E),the exercise + verbascoside treated by the high dosage(F). The results showed that the degree of skeletal muscle damage decreased in turn in the three groups of exercise +verbascoside(D、E、F) compared with group C. The skeletal muscle morphology of the mice in group F was almost normal. The plasma CK levels and skeletal muscle tissue GSSG content in group F were lower than those of groups of C、D、E(P < 0. 01). The plasma CK levels in group F were higher than those of groups of A and B(P < 0. 01),but there is no significant difference in the skeletal muscle tissue GSSG content among group F、A and B(P > 0. 05). The GSH contents,GSH/GSSG ratios,the enzyme activities of GCL and GR in skeletal muscle tissue,the levels of protein expression of GSH in RyR1 complexes of mice in group F were higher than those of groups C、D、E,but were lower than those of group A and B(P < 0. 01). The results suggested that verbascoside can reduce the levels of plasma CK,protect the morphology of the skeletal muscle of mice exercised by incremental load. Its mechanism was related to verbascoside increasing of GSH contents and the enzyme activities of GCL and GR in skeletal muscle,improving the levels of protein expression of GSH in RyR1 complexes and decreasing the ratios of GSSG/GSH in skeletal muscle of mice exercised by incremental load.
Using the training model of mice running and the methods of immunoprecipitation and immunoblotting,this study aimed to investigate the protective effects of verbascoside on skeletal muscle damage by incremental load exercise and glutathione of mice. Ninety mice were divided into six groups including:the normal group(A),the normal + verbascoside treated group(B),the exercise group(C),the exercise + verbascoside treated by the low dosage(D),the exercise + verbascoside treated by the moderate dosage(E),the exercise + verbascoside treated by the high dosage(F). The results showed that the degree of skeletal muscle damage decreased in turn in the three groups of exercise +verbascoside(D、E、F) compared with group C. The skeletal muscle morphology of the mice in group F was almost normal. The plasma CK levels and skeletal muscle tissue GSSG content in group F were lower than those of groups of C、D、E(P < 0. 01). The plasma CK levels in group F were higher than those of groups of A and B(P < 0. 01),but there is no significant difference in the skeletal muscle tissue GSSG content among group F、A and B(P > 0. 05). The GSH contents,GSH/GSSG ratios,the enzyme activities of GCL and GR in skeletal muscle tissue,the levels of protein expression of GSH in RyR1 complexes of mice in group F were higher than those of groups C、D、E,but were lower than those of group A and B(P < 0. 01). The results suggested that verbascoside can reduce the levels of plasma CK,protect the morphology of the skeletal muscle of mice exercised by incremental load. Its mechanism was related to verbascoside increasing of GSH contents and the enzyme activities of GCL and GR in skeletal muscle,improving the levels of protein expression of GSH in RyR1 complexes and decreasing the ratios of GSSG/GSH in skeletal muscle of mice exercised by incremental load.
引文
1 Liao F,Zheng RL,Gao JJ,et al.Retardation of skeletal muscle fatigue by the two phenylpropanoid glycosides:Verbascoside and martynoside from Pedicularis plicata Maxim[J].Phytother Res,1999,13:621-623.
2 Zhu MJ(朱梅菊),Tan NH(谭宁华),Tan NH(朱洪竹),et al.Effects of verbascoside and martynoside on exercise-induced fatigue and their effects on free radical metabolism in the rats[J].Chin J Sports Med(中国运动医学杂志),2009,28(3):310-312.
3 Zeng FX(曾凡星).Research development of mechanism of exercise induced muscle damage[J].J Jingzhou Teachers Coll,Nat Sci(荆州师专学报,自科版),1994,17(5):73-76.
4 Gou X,Yu TY,Zhou X,et al.Review of the mechanism of muscle fatigue and injury[J].CJTCMP((中华中医药杂志(原中国医药学报)),2016,31:2720-2724.
5 Hua N(华楠),Zhang H(张华),Zhang Y(张缨).Changes of glutathione redox in skeletal muscles of AMPKα2 overexpression mice after hypoxia training[J].J Beijing Sport Univer(北京体育大学学报),2013,36(12):73-76,81.
6 Xi W(汶希),Pan HS(潘华山),Feng YC(冯毅翀).Establishment of a rat model of exercise-Induced fatigue[J].Acta Laboratorium Animalis Sci Sinica(中国实验动物学报),2009,17:368-372.
7 Zhou Y(周越),Li Y(李扬),Wang Y(王瑞元),et al.Comparation of evaluating indicators for exercise-induced muscle damage[J].Chin J Sports Med(中国运动医学杂志),2008,27:206-207.
8 Cao QL(曹庆雷),He JW(何建伟),Yang JL(杨金龙).Effects of far infrared ceramic microspheres on indexes CK,CK-MM,LDH of the skeletal muscles of rats going through one time exhaustive exercising[J].J Physical Education(体育学刊),2013,20:125-128.
9 Polotow TG,Vardaris CV,Mihaliuc AR,et al.Astaxanthin supplementation delays physical exhaustion and prevents redox imbalances in plasma and soleus muscles of Wistar rats[J].Nut,2014,6:5819-5838.
10 Boyarskaya LA,Turchaninov DV,Efremenko ES,et al.Study of glutathione and enzymes of its metabolism under the influence of enriched dairy products for prophylactic use in conditions of oxidative[J].Gig Sanit,2015,94(8):52-56.
11 Peternelj TT,Marsh SA,Strobel NA,et al.Glutathione depletion and acute exercise increase O-Glc NAc protein modification in rat skeletal muscle[J].Mol Cell Biochem,2015,400:265-275.
12 Carfagna S,Napolitano G,Barone D,et al.Dietary supplementation with the microalga Galdieria sulphuraria(Rhodophyta)reduces prolonged exercise-induced oxidative stress in rat tissues[J].Oxid Med Cell Longev,2015,2015:732090.
13 Golbidi S,Botta,Gottfred,et al.Glutathione administration reduces mitochondrial damage and shifts cell death from necrosis to apoptosis in ageing diabetic mice hearts during exercise[J].Br J Pharmacol,2014,171:5345-5360.
14 Li Q(李靖).Effects of sprint training on glutathione expression in liver and skeletal muscle[J].J Yichun College(宜春学院学报),2012,34:124-126.
15 Jain SK,Micinski D.Vitamin D upregulates glutamate cysteine ligase and glutathione reductase and GSH formation and decreases ROS and MCP-1 and IL-8 secretion in high-glucose exposed U937 monocytes[J].Biochem Bioph Res Co,2013,437(1):7-11.
16 Xiong Y,Xiong Y,Zhou S,et al.Inhibition of glutathione synthesis induced by exhaustive running exercise via the decreased influx rate of L-cysteine in rat erythrocytes[J].Cell Physiol Biochem,2016,40:1410-1421.
17 Aydin C1,Sonat F,Sahin SK,et al.Long term dietary restriction ameliorates swimming exercise-induced oxidative stress in brain and lung of middle-aged rat[J].Indian J Exp Biol,2009,47(1):24-31.
18 Leeuwenburgh C,Hollander J,Leichtweis S,et al.Adaptations of glutathione antioxidant system to endurance training are tissue and muscle fiber specific[J].Am J Physiol,1997,272:363-369.
19 Mei Y,Xu L,Kramer HF,et al.Stabilization of the skeletal muscle ryanodine receptor ion channel-FKBP12 complex by the 1,4-benzothiazepine derivative S107[J].PLo S One,2013,8:e54208.
20 Bellinger AM,Reiken S,Dura M,et al.Remodeling of ryanodine receptor complex causes“leaky”channels:A molecular mechanism for decreased exercise capacity[J].PNAS,2008,105:2198-2202.
21 Li JX,Xin D,Li H,et al.Effect of verbascoside on decreasing concentration of oxygen free radicals and lipid peroxidation in skeletal muscle[J].Acta Phar Sin,1999,20:126-130.
22 Wan Y,Zou B,Zeng H,et al.Inhibitory effect of verbascoside on xanthine oxidase activity[J].Int J Biol Macromol,2016,93 (Pt A):609-614.
23 Qiusheng Z,Yuntao Z,Rongliang Z,et al.Effects of verbascoside and luteolin on oxidative damage in brain of heroin treated mice[J].Pharmazie,2005,60:539-543.
24 Wang PF,Kang JH,Zheng RL,et al.Scavenging effects of phenylpropanoid glycosides from Pedicularis on superoxide anion and hydroxyl radical by the spin trapping method(95)02255-4[J].Biochem Pharm,1996,51:687-691.
2 Zhu MJ(朱梅菊),Tan NH(谭宁华),Tan NH(朱洪竹),et al.Effects of verbascoside and martynoside on exercise-induced fatigue and their effects on free radical metabolism in the rats[J].Chin J Sports Med(中国运动医学杂志),2009,28(3):310-312.
3 Zeng FX(曾凡星).Research development of mechanism of exercise induced muscle damage[J].J Jingzhou Teachers Coll,Nat Sci(荆州师专学报,自科版),1994,17(5):73-76.
4 Gou X,Yu TY,Zhou X,et al.Review of the mechanism of muscle fatigue and injury[J].CJTCMP((中华中医药杂志(原中国医药学报)),2016,31:2720-2724.
5 Hua N(华楠),Zhang H(张华),Zhang Y(张缨).Changes of glutathione redox in skeletal muscles of AMPKα2 overexpression mice after hypoxia training[J].J Beijing Sport Univer(北京体育大学学报),2013,36(12):73-76,81.
6 Xi W(汶希),Pan HS(潘华山),Feng YC(冯毅翀).Establishment of a rat model of exercise-Induced fatigue[J].Acta Laboratorium Animalis Sci Sinica(中国实验动物学报),2009,17:368-372.
7 Zhou Y(周越),Li Y(李扬),Wang Y(王瑞元),et al.Comparation of evaluating indicators for exercise-induced muscle damage[J].Chin J Sports Med(中国运动医学杂志),2008,27:206-207.
8 Cao QL(曹庆雷),He JW(何建伟),Yang JL(杨金龙).Effects of far infrared ceramic microspheres on indexes CK,CK-MM,LDH of the skeletal muscles of rats going through one time exhaustive exercising[J].J Physical Education(体育学刊),2013,20:125-128.
9 Polotow TG,Vardaris CV,Mihaliuc AR,et al.Astaxanthin supplementation delays physical exhaustion and prevents redox imbalances in plasma and soleus muscles of Wistar rats[J].Nut,2014,6:5819-5838.
10 Boyarskaya LA,Turchaninov DV,Efremenko ES,et al.Study of glutathione and enzymes of its metabolism under the influence of enriched dairy products for prophylactic use in conditions of oxidative[J].Gig Sanit,2015,94(8):52-56.
11 Peternelj TT,Marsh SA,Strobel NA,et al.Glutathione depletion and acute exercise increase O-Glc NAc protein modification in rat skeletal muscle[J].Mol Cell Biochem,2015,400:265-275.
12 Carfagna S,Napolitano G,Barone D,et al.Dietary supplementation with the microalga Galdieria sulphuraria(Rhodophyta)reduces prolonged exercise-induced oxidative stress in rat tissues[J].Oxid Med Cell Longev,2015,2015:732090.
13 Golbidi S,Botta,Gottfred,et al.Glutathione administration reduces mitochondrial damage and shifts cell death from necrosis to apoptosis in ageing diabetic mice hearts during exercise[J].Br J Pharmacol,2014,171:5345-5360.
14 Li Q(李靖).Effects of sprint training on glutathione expression in liver and skeletal muscle[J].J Yichun College(宜春学院学报),2012,34:124-126.
15 Jain SK,Micinski D.Vitamin D upregulates glutamate cysteine ligase and glutathione reductase and GSH formation and decreases ROS and MCP-1 and IL-8 secretion in high-glucose exposed U937 monocytes[J].Biochem Bioph Res Co,2013,437(1):7-11.
16 Xiong Y,Xiong Y,Zhou S,et al.Inhibition of glutathione synthesis induced by exhaustive running exercise via the decreased influx rate of L-cysteine in rat erythrocytes[J].Cell Physiol Biochem,2016,40:1410-1421.
17 Aydin C1,Sonat F,Sahin SK,et al.Long term dietary restriction ameliorates swimming exercise-induced oxidative stress in brain and lung of middle-aged rat[J].Indian J Exp Biol,2009,47(1):24-31.
18 Leeuwenburgh C,Hollander J,Leichtweis S,et al.Adaptations of glutathione antioxidant system to endurance training are tissue and muscle fiber specific[J].Am J Physiol,1997,272:363-369.
19 Mei Y,Xu L,Kramer HF,et al.Stabilization of the skeletal muscle ryanodine receptor ion channel-FKBP12 complex by the 1,4-benzothiazepine derivative S107[J].PLo S One,2013,8:e54208.
20 Bellinger AM,Reiken S,Dura M,et al.Remodeling of ryanodine receptor complex causes“leaky”channels:A molecular mechanism for decreased exercise capacity[J].PNAS,2008,105:2198-2202.
21 Li JX,Xin D,Li H,et al.Effect of verbascoside on decreasing concentration of oxygen free radicals and lipid peroxidation in skeletal muscle[J].Acta Phar Sin,1999,20:126-130.
22 Wan Y,Zou B,Zeng H,et al.Inhibitory effect of verbascoside on xanthine oxidase activity[J].Int J Biol Macromol,2016,93 (Pt A):609-614.
23 Qiusheng Z,Yuntao Z,Rongliang Z,et al.Effects of verbascoside and luteolin on oxidative damage in brain of heroin treated mice[J].Pharmazie,2005,60:539-543.
24 Wang PF,Kang JH,Zheng RL,et al.Scavenging effects of phenylpropanoid glycosides from Pedicularis on superoxide anion and hydroxyl radical by the spin trapping method(95)02255-4[J].Biochem Pharm,1996,51:687-691.