补肾益气方联合有氧运动对衰老大鼠骨骼肌线粒体生物合成相关信号分子表达的影响
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摘要
目的观察补肾益气方及有氧运动对D-半乳糖所致衰老大鼠骨骼肌线粒体生物合成相关信号分子丝裂原活化蛋白激酶(p38MAPK)、钙调蛋白激酶Ⅱ(Ca MKⅡ)、过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1α)、心肌细胞增强因子2C(MEF2C)及细胞色素氧化酶(COXⅣ)表达的影响。方法将40只雄性SD大鼠随机分为5组,分别为正常组、模型组、中药组、运动+中药组、美雄酮组。除正常组外,其余各组每天颈背部皮下注射10%的D-半乳糖(100μg/g)溶液,制作"拟衰老"大鼠模型。模型大鼠分别经补肾益气方药、有氧跑台运动、美雄酮干预10周后,采用ELISA法测定大鼠血清β-半乳糖苷酶(β-Galase)含量,采用甲基百里香酚蓝法(MTB)检测大鼠腓肠肌线粒体钙含量,采用Western blotting法检测大鼠腓肠肌线粒体生物合成信号通路分子p38MAPK、Ca MKⅡ、PGC-1α、MEF2C及COXⅣ蛋白表达变化。结果与正常组比较,模型组大鼠衰老生物学标志物β-Galase、线粒体钙含量显著增高,Ca MKⅡ、MEF2C、PCG-1α蛋白表达明显下降,而P38MAPK蛋白表达显著升高(P<0.05,P<0.01)。与模型组比较,中药组可显著降低血清β-GALase蛋白含量、线粒体钙含量(P<0.01);运动+中药组可显著降低线粒体钙含量,上调Ca MKⅡ、PGC-1α、MEF2C及COXⅣ蛋白表达;美雄酮组显著降低线粒体钙含量,上调PCG-1α、COXIV蛋白表达(P<0.05,P<0.01)。结论衰老大鼠骨骼肌功能减退可能与线粒体生物合成相关信号转导通路分子Ca MKⅡ、p38 MAPK、PCG-1α、MEF2C及能量代谢相关酶蛋白基因(COXIV)表达异常变化有关;补肾益气方联合有氧运动可一定程度改善上述指标的异常变化,延缓骨骼肌衰老。
Objective To observe the influence of Bushen Yiqi Decoction and aerobic exercise on the expression of mitochondria biosynthesis related signaling molecules p38 MAPK,Ca MKⅡ,PGC-1α,MEF2 C and COXⅣ in D- galactose-induced aging rats' skeletal muscle. Methods 40 male SD rats were randomly divided into five groups: normal group,model group,traditional Chinese medicine group,exercise and traditional Chinese medicine group and methandienone group. The rats were received a subcutaneous injection of 10% D-galactose solution( 100 mg·kg- 1,once a day) for 10 weeks to make the aging model except normal group rats. Bushen Yiqi Decoction,aerobic exercise and methandienone were used to the corresponding experimental groups to intervene for 10 weeks. β-Galase of blood serum were tested by ELISA; Calcium content of gastrocnemius mitochondria were tested by Methyl thyme phenol blue( MTB); The expression of mitochondrial biosynthesis related signal transduction molecules( p38 MAPK,Ca MKⅡ,PGC-1α,MEF2 C and COXⅣ) were tested by Western blot respectively.Results Compared with normal group,model group rats' aging biology markers β-Galase,calcium content in mitochondria increased significantly. The protein expression( protein) of model rats' Ca MKⅡ,MEF2 C,PCG-1 alpha decreased obviously,and the protein expression of P38 MAPK increased significantly. The Bushen Yiqi Decoction combined with aerobic exercise could obviously decrease calcium content,and increase the protein expression of Ca MK Ⅱ,PGC-1α,MEF2 C and COXⅣ of the model aging rats. The methandienone could obviously increase the protein expression of PCG-1 α and COXIV of the model aging rats,decrease calcium content obviously. Conclusion The decline of aging model rats' skeletal muscle function may related to expression changes of mitochondria biosynthesis related signal transduction pathways molecular Ca MK Ⅱ,p38 MAPK,PCG-1 alpha,MEF2 C and energy metabolism related genes( COXIV). The Bushen Yiqi Decoction combined with aerobic exercise can delay age-related degradation of the skeletal muscle in some degree through improving the abnormal changes of the above index.
Objective To observe the influence of Bushen Yiqi Decoction and aerobic exercise on the expression of mitochondria biosynthesis related signaling molecules p38 MAPK,Ca MKⅡ,PGC-1α,MEF2 C and COXⅣ in D- galactose-induced aging rats' skeletal muscle. Methods 40 male SD rats were randomly divided into five groups: normal group,model group,traditional Chinese medicine group,exercise and traditional Chinese medicine group and methandienone group. The rats were received a subcutaneous injection of 10% D-galactose solution( 100 mg·kg- 1,once a day) for 10 weeks to make the aging model except normal group rats. Bushen Yiqi Decoction,aerobic exercise and methandienone were used to the corresponding experimental groups to intervene for 10 weeks. β-Galase of blood serum were tested by ELISA; Calcium content of gastrocnemius mitochondria were tested by Methyl thyme phenol blue( MTB); The expression of mitochondrial biosynthesis related signal transduction molecules( p38 MAPK,Ca MKⅡ,PGC-1α,MEF2 C and COXⅣ) were tested by Western blot respectively.Results Compared with normal group,model group rats' aging biology markers β-Galase,calcium content in mitochondria increased significantly. The protein expression( protein) of model rats' Ca MKⅡ,MEF2 C,PCG-1 alpha decreased obviously,and the protein expression of P38 MAPK increased significantly. The Bushen Yiqi Decoction combined with aerobic exercise could obviously decrease calcium content,and increase the protein expression of Ca MK Ⅱ,PGC-1α,MEF2 C and COXⅣ of the model aging rats. The methandienone could obviously increase the protein expression of PCG-1 α and COXIV of the model aging rats,decrease calcium content obviously. Conclusion The decline of aging model rats' skeletal muscle function may related to expression changes of mitochondria biosynthesis related signal transduction pathways molecular Ca MK Ⅱ,p38 MAPK,PCG-1 alpha,MEF2 C and energy metabolism related genes( COXIV). The Bushen Yiqi Decoction combined with aerobic exercise can delay age-related degradation of the skeletal muscle in some degree through improving the abnormal changes of the above index.
引文
[1]宋雅芳,王晓燕,刘友章,等.健脾益气中药对脾虚大鼠骨骼肌、胃粘膜线粒体超微结构的影响[J].中药药理与临床,2009,25(2):6-8.
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[16]Chung J,Lee HS,Chung HY,et al.Salicylideneamino-2-thiophenol inhibits inflammatory mediator genes(RANTES,MCP-1,IL-8 and HIF-1α)expression induced by tert-butyl hydroperoxide via MAPK pathways in rat peritoneal macrophages[J].Biotechnol Lett,2008,30(9):1553-1558.
[17]Scarpulla RC.Transcriptional paradigms in mammalian mitochondrial biogenesis and function[J].Physiol Rev,2008,88(2):611-638.
[18]Alaynick WA.Nuclear receptors,mitochondria and lipid metabolism[J].Mitoch ondrion,2008,8(4):329-337.
[19]崔玉娟,张龙飞,平政,等.红景天苷对力竭大鼠心肌线粒体生物发生关键调控因子的影响[J].解放军医药杂志,2014,26(11):6-10.
[20]臧莎莎,刘颐轩,宋光耀,等.津力达对胰岛素抵抗大鼠骨骼肌脂质沉积及线粒体功能的影响[J].中成药,2014,36(7):1371-1376.
[21]Brand NJ.Myocyte enhancer factor 2(MEF2)[J].Int J Biochem Cell Biol,1997,29(12):1467-1470.
[22]Srinivasan S,Avadhani NG.Cytoehrome C oxidase dysfunction in oxidative stress[J].Free Radic Biol Med,2012,53(6):1252-1263.
[23]Wu H,Kanatous SB,Thurmond FA.Regulation of mitochondrial biogenesis in skeletal muscle by Ca MK[J].Science,2002,296(5566):349-352.
[24]Fan M,Rhee J,St-Pierre J,et al.Suppression of mitochondrial respiration through recruitment of p160 myb binding protein to PGC-1α:modulation by p38 MAPK[J].Genes Dev,2004,18(3):278-289.
[2]张茂林,张六通,邱幸凡.补肾防衰方对老年大鼠肾、肝、心肌、骨骼肌线粒体超微结构的影响[J].中国中医药科技,2002,9(2):98-99.
[3]黄红,黄宏兴,李颖,等.补肾健脾活血方对骨质疏松骨骼肌线粒体通透转换孔调控的影响[J].新中医,2010,42(6):113-115.
[4]刘萍,丁玉琴,王爱梅,等.六味地黄汤对D-半乳糖致衰老小鼠运动能力的影响[J].中国医药导报,2008,5(11):16-18.
[5]徐划萍,吴志坤,金国琴,等.补肾益气方药对衰老大鼠骨骼肌能量代谢的影响[J].上海中医药杂志,2015,49(2):74-77.
[6]Kumar A,Prakash A,Dogra S.Naringin alleviates cognitive impairment,mitochondrial dysfunction and oxidative stress induced by D-galactose in mice[J].Food Chem Toxicol,2010,48(2):626-632.
[7]Bedford TG,Tipton CM,Wilson NC,et al.Maximum oxygen consumption of rats and its changes with various experimental procedures[J].J Appl Physiol Respir Environ Exerc Physiol,1979,47(6):1278-1283.
[8]Siu PM,Bryner RW,Martyn JK,et al.Apoptotic adaptations from exercise training in skeletal and cardiac muscles[J].FASEB J,2004,18(10):1150-1152.
[9]Dimri GP,Lee X,Basile G,et al.A biomarker that identifies senescent human cells in culture and in aging skin in vivo[J].Proc Natl Acad Sci USA,1995,92(20):9363-9367.
[10]温宝书,张敏.牛磺酸的细胞保护作用[J].中国药学杂志,1995,30(8):451.
[11]Beckman KB,Ames BN.The free radical theory of aging matures[J].Physiol Rev,1998,78(2):547-581.
[12]Hurne AM,Chai CL,Moerman K,et al.Influx of calcium through a redox-sensitive plasma membrane channel in thymocytes causes early necrotic cell death induced by the epipolythiodioxopiperazine toxins[J].J Biol Chem,2002,277(35):31631-31638.
[13]张君,于嘉屏,曹雪涛.钙离子/钙调素依赖性蛋白激酶-Ⅱ及其抑制剂[J].国外医学:临床生物化学与检验学分册,2002,23(4):237-239.
[14]Mylabathula DB,Rice KM,Wang Z,et al.Age-associated changes in MAPK activation in fast and slow twitch skeletal muscle of the F344/NNia HSD X Brown Norway/Bi Nia rat model[J].Exp Gerontol,2006,41(2):205-214.
[15]Williomson D,Gallgher P,Harber M,et al.Mitogen-activated protein kinase(MAPK)pathway activation:effects of age and acute exercise on human skeletal muscle[J].J Physiol,2003,547(Pt 3):977-987.
[16]Chung J,Lee HS,Chung HY,et al.Salicylideneamino-2-thiophenol inhibits inflammatory mediator genes(RANTES,MCP-1,IL-8 and HIF-1α)expression induced by tert-butyl hydroperoxide via MAPK pathways in rat peritoneal macrophages[J].Biotechnol Lett,2008,30(9):1553-1558.
[17]Scarpulla RC.Transcriptional paradigms in mammalian mitochondrial biogenesis and function[J].Physiol Rev,2008,88(2):611-638.
[18]Alaynick WA.Nuclear receptors,mitochondria and lipid metabolism[J].Mitoch ondrion,2008,8(4):329-337.
[19]崔玉娟,张龙飞,平政,等.红景天苷对力竭大鼠心肌线粒体生物发生关键调控因子的影响[J].解放军医药杂志,2014,26(11):6-10.
[20]臧莎莎,刘颐轩,宋光耀,等.津力达对胰岛素抵抗大鼠骨骼肌脂质沉积及线粒体功能的影响[J].中成药,2014,36(7):1371-1376.
[21]Brand NJ.Myocyte enhancer factor 2(MEF2)[J].Int J Biochem Cell Biol,1997,29(12):1467-1470.
[22]Srinivasan S,Avadhani NG.Cytoehrome C oxidase dysfunction in oxidative stress[J].Free Radic Biol Med,2012,53(6):1252-1263.
[23]Wu H,Kanatous SB,Thurmond FA.Regulation of mitochondrial biogenesis in skeletal muscle by Ca MK[J].Science,2002,296(5566):349-352.
[24]Fan M,Rhee J,St-Pierre J,et al.Suppression of mitochondrial respiration through recruitment of p160 myb binding protein to PGC-1α:modulation by p38 MAPK[J].Genes Dev,2004,18(3):278-289.