【作者】 刘丰彬；

【导师】 赵斌；

【作者基本信息】 河北师范大学 ， 体育教育训练学， 2009， 博士

【摘要】 随着全世界老龄化国家增多,老龄人口比率加大,人口的老龄化已经成为许多国家都要面对的新的突出的社会问题。如何延长老年人的健康寿命,提高生活质量已成为各国政府和社会各界关注的焦点。对衰老和衰老相关疾病的研究历来就是世界性的医学课题,探索衰老的本质,寻找有效的抗衰老药物和方法,以求得预防衰老过早出现和延缓衰老的进程已成为当前老年医学领域中的研究热点。近年来,老年人骨骼肌功能的衰退受到了科研人员的重视,因为人们发现骨骼肌的衰老会加速人体整体的衰老进程,而骨骼肌功能的恢复对提高老年人的健康水平和生活质量都十分有意义。目前对于预防和延缓衰老主要集中在药物研究上,尽管药物干预有一定的临床效果,但是同时又存在着作用局限、有毒副作用等不足。运动是生命活动的基础,而营养则是生命活动直接的保障,如何将运动和营养干预这两种更为经济和安全的方式结合起来,使身体各器管、各系统的功能产生良好的效应,以对抗机体衰老,特别是骨骼肌的衰老,值得深入研究和探讨。针对运动干预衰老的研究,国内外已有相关的报道,但多数研究是对人体整体机能的调节和影响,指标也较为单一,缺少针对某一器官和组织,尤其是缺少运动抗骨骼肌衰老的机理及综合指标的研究,选择形式也以有氧运动或短期力量训练为主,对骨骼肌刺激更明显的长期抗阻力量训练或负重训练的研究较少,作用效果也不明确。国内外针对营养干预衰老的研究也较多,相关的营养补剂产品也层出不穷,但从整体、组织、基因不同层面入手,观察长期补充大豆天然提取物大豆多肽对骨骼肌衰老的影响并探讨其机制,国内外还没有见到相关文献的报道。本研究就是运用衰老研究中常用的D-半乳糖皮下注射的方法,复制大鼠亚急性骨骼肌衰老模型,并在造模过程中和造模成功后都进行负重训练和补充大豆多肽的干预。通过观察一般指标、骨骼肌组织切片和相应的生化指标,判定两种干预方式对预防骨骼肌衰老过早出现和延缓骨骼肌衰老的作用效果,同时测量血清及骨骼肌超氧化物歧化酶(SOD)和丙二醛(MDA)、血清睾酮(T)、皮质醇(C)、生长激素(GH)和胰岛素样生长因子- I (IGF-I)、骨骼肌总蛋白和脂褐素、骨骼肌肌动蛋白(α-actin)、IGF-I和生长分化因子-8(GDF-8)mRNA等指标,初步探讨两种干预方式在预防和延缓骨骼肌衰老中的作用机制,为进一步进行人群干预试验积累一定的理论和实验基础,为提供更有针对性的运动方式和研制、开发合理有效的营养补剂,以对抗中老年人骨骼肌的衰老,开辟一条新思路和新途径。同时,也为指导中老年人科学的健身,合理进行慢性病的康复,提高健康水平和生活质量,提供一定的帮助和借鉴。实验分为两部分:预防衰老实验和延缓衰老进程实验一、预防衰老实验3月龄SD雄性大鼠56只,随机分为7组:成年组(C)、D-半乳糖组(M)、D-半乳糖小负重组(S)、D-半乳糖大负重组(B)、D-半乳糖补肽组(P)、D-半乳糖补肽小负重组(PS)、D-半乳糖补肽大负重组(PB)。所有大鼠在6周D-半乳糖造模的过程中进行相应的负重训练和补充大豆多肽的干预。6周末处死,比较各组一般指标、骨骼肌组织切片、血清和骨骼肌组织生化以及分子生物学指标,结果如下:1.一般指标:所有造模组大鼠均出现了衰老的外观特征,体重呈现自然增长趋势,双侧腓肠肌总重量、腓肠肌相对重量有下降的趋势,各干预组大鼠以上变化幅度趋缓;所有组大鼠每日摄食量都有下降的趋势,造模组趋势更为明显。提示:两种干预方式可以有效改善衰老的外部特征。2.骨骼肌组织切片:光镜下观察C组大鼠为正常骨骼肌组织学特征,所有造模组大鼠均出现不同程度的肌细胞萎缩或退行性改变特征,以M组变化最为明显。提示:两种干预方式可以有效预防骨骼肌衰老过早的出现。3.血清学指标:与C组相比,M组大鼠血清中SOD活力及SOD/MDA、T含量及T/C、GH含量显著下降,血清MDA含量、C含量显著上升,(P<0.01或P<0.05)血清IGF-I含量有下降的趋势,但无显著性差异;负重训练或补充大豆多肽干预可以有效的逆转以上趋势,均以小负重组表现更为明显,并且两种干预方式均具有显著的交互作用。提示:两种干预方式可以减轻D-半乳糖造成的机体脂质过氧化和激素水平的紊乱。4.骨骼肌组织生化指标:与C组相比,M组大鼠骨骼肌SOD活力、MDA含量、脂褐素含量显著升高(P<0.01或P<0.05),SOD/MDA有降低的趋势,但无显著性差异;骨骼肌总蛋白含量显著下降(P<0.05);负重训练或补充大豆多肽干预除可以进一步提高骨骼肌SOD活力外,均可以逆转其他的趋势,多数以小负重组表现更为明显,并且以上情况两种干预方式均具有显著的交互作用。提示:两种干预方式可以减轻D-半乳糖造成的骨骼肌组织脂质过氧化和肌蛋白合成的减少。5.骨骼肌组织分子生物学指标:与C组相比,M组大鼠骨骼肌组织中α-actin mRNA和IGF-I mRNA表达显著下降(P<0.01);GDF-8 mRNA表达显著升高(P<0.01);负重训练或补充大豆多肽干预可以有效的逆转以上的趋势,均以小负重组表现更为明显,并且两种干预方式均具有显著的交互作用。提示:两种干预方式可以升高D-半乳糖造成的骨骼肌组织α-actin mRNA和IGF-I mRNA的低表达,降低GDF-8 mRNA的高表达。二、延缓衰老进程实验3月龄SD雄性大鼠60只,随机分为8组:6周安静对照组(C6)、6周模型组(M6)、12周模型组(M12)、造模后大负重组(B12)、造模后小负重组(S12)、造模后补肽组(P12)、造模后补肽大负重组(PB12)、造模后补肽小负重组(PS12),14月龄SD雄性大鼠8只作为自然衰老组(OC)。除C6组、M6组、M12组和OC组,其余各组在6周D-半乳糖造模后进行相应的负重训练和补充大豆多肽的干预。分别于6周末和12周末处死相应组大鼠,测试相关指标,结果如下:1. 6周组指标比较:与C6相比,M6组大鼠体重升高,腓肠肌总重量及相对重量均有降低的趋势,但均未达到统计学差异;血清SOD活力显著降低(P<0.05),血清MDA和骨骼肌脂褐素含量均显著升高(P<0.01);骨骼肌切片均出现不同程度的肌细胞萎缩或退行性改变特征。提示:造模成功。2. 12周一般指标:所有造模组和OC组大鼠均出现了衰老的外观特征,各干预组大鼠衰老特征较轻;所有组大鼠体重呈现自然增长趋势,各干预组大鼠其幅度明显趋缓(P<0.05),两种干预方式具有显著的交互作用;两种干预方式使双侧腓肠肌总重量显著降低(P<0.05和P<0.01),有显著的交互作用,腓肠肌相对重量有降低的趋势,无显著的交互作用;各组大鼠每日摄食量呈现平稳增长的趋势,两种干预方式使增长幅度趋缓,无显著的交互作用。提示:造模成功后,再进行两种方式的干预,可以有效延缓衰老进程中的外部特征。3. 12周骨骼肌组织切片:光镜下观察各组大鼠肌细胞形态有着不同程度的萎缩或退行性改变,以M12组和OC组变化最为明显,提示:造模成功后,再进行两种方式的干预,可以有效延缓骨骼肌衰老的进程。4. 12周血清学指标:与OC组相比,M12组大鼠血清SOD活力、MDA含量以及SOD/MDA、血清T含量、C含量以及T/C、血清GH和IGF-I含量均无显著性差异。负重训练或补充大豆多肽可以显著提高血清SOD活力以及SOD/MDA、T含量以及T/C、GH和IGF-I含量,降低血清MDA含量和C含量,多数以小负重组表现更为明显,并且两种干预方式都具有显著的交互作用。(P<0.05或P<0.01)提示:造模成功后,再进行两种方式的干预,可以减轻机体脂质过氧化和激素水平的紊乱。5. 12周骨骼肌组织生化指标:与OC组相比,M12组大鼠骨骼肌SOD活力、MDA含量及SOD/MDA、总蛋白含量和脂褐素含量均无显著性差异。负重训练或补充大豆多肽可以显著提高骨骼肌SOD活力及SOD/MDA、总蛋白含量,降低骨骼肌MDA含量和脂褐素含量,多数以小负重组表现更为明显,并且两种干预方式都具有显著的交互作用。(P<0.05或P<0.01)提示:造模成功后,再进行两种方式的干预,可以减轻骨骼肌组织脂质过氧化和肌蛋白合成的减少。6. 12周骨骼肌组织分子生物学指标:与OC组相比,M12组大鼠骨骼肌α-actin mRNA、IGF-I mRNA和GDF-8 mRNA表达均无显著性差异。负重训练或补充大豆多肽干预可以提高骨骼肌α-actin mRNA和IGF-I mRNA的表达,降低GDF-8 mRNA的表达,均以小负重组表现更为明显,并且两种干预方式都具有显著的交互作用。(P<0.05或P<0.01)提示:造模成功后,再进行两种方式的干预,可以升高骨骼肌组织α-actin mRNA和IGF-I mRNA的低表达,降低GDF-8 mRNA的高表达。结论1. 6周D-半乳糖皮下注射,可以成功复制大鼠亚急性骨骼肌衰老模型。可能的作用机制为:①增多血液中自由基;②增强骨骼肌氧化应激以及脂质过氧化;③增加骨骼肌脂褐素的沉积;④促使激素及相关因子的代谢紊乱;⑤降低骨骼肌α-actin和IGF-I mRNA的表达,提高骨骼肌GDF-8 mRNA的表达。2.负重训练或补充大豆多肽均可以有效的预防6周D-半乳糖造模过程中大鼠骨骼肌衰老过早的出现,以小负重训练效果最好,而且两种方式联合运用效果更为明显。3. 6周负重训练或补充大豆多肽的干预均可以对6周D-半乳糖造模后大鼠骨骼肌衰老有积极的延缓作用,以小负重训练效果最好,而且两种方式联合运用效果更为明显。4.负重训练和补充大豆多肽预防和延缓大鼠骨骼肌衰老的效果可能通过下列机制发挥作用:①减少血液中自由基;②减轻骨骼肌氧化应激以及脂质过氧化;③减少骨骼肌脂褐素的沉积;④纠正激素及相关因子的代谢紊乱;⑤提高骨骼肌α-actin和IGF-I mRNA的表达,降低骨骼肌GDF-8 mRNA的表达。更多还原

【Abstract】 With the increasing of the world aging country and the proportion of old people, many countries face with the new and projecting problem of aging population. The governments and societies focus on how to improve people’s healthy life-span and life quality. The research in senescence and its correlated disease has been taking the worldwide medical subject invariably. At present, explore the essential of aging, search the effective antiaging agents and methods to prevent and postpone aging have become the hotspot study in the area of geriatric medicine. Recent years, the scientists pay more attention to the issue of decline of muscle function, because it can influence old people’s health. It is close correlative between the muscle function and senescence.At present, most research about preventing and postponing aging focus on drugs. Although drugs have certain effect, their disadvantages of limitation and side effect are also obvious. Exercise is the basis of vital movement, and nutrition is the immediate guarantee of it. How to combine the exercise and nutrition together in an economic and safe way, make the organs and systems in a satisfactory condition, in order to confront the aging especially the muscle aging , it is worth to be studied thoroughly.There are some correlative reports of exercise interference in aging. Although, most research are about the influence of whole function, the parameteres are limited and lack of the study in certain organ and tissue ,especially be short of the research in mechanism and comprehensive parameter of exercise interference in aging. Also, the exercise styles are in aerobic and short term strength training mostly, the patterns in long term strength and weight training which are more effective to the skeletal muscle are less, the affect are indefinite either. There are many studies of nutrition interference in aging in and out doors, the relative nutritional supplements are also to come out one after the other. Though, there is no correlated reports about the mechanism and effect of the long term soy polypeptide supplement in skeletal muscle aging from the aspects of whole body, tissue and genes.The present research copys the rat subacute skeletal muscle aging model by the method of D-galactose hypodermic which is common in senescence study. And take the interference way in weight training and soy polypeptide supplement in and after the process of model making. The research aims to observe the general parameter, skeletal muscle tissue slice and biochemical parameter, in order to judge the prevention and postpone effect in skeletal muscle aging by the two interference ways. and to discuss the probable mechanisms by measuring serum & skeletal muscle superoxide dismutase (SOD) and malondealdehyde (MDA), serum testosterone(T), cortisol(C), growth hormone(GH) and insulin-like growth factor-1(IGF-I), skeletal muscle total protein level and lipofuscin, skeletal muscleα-actin,IGF-I and growth and differential factor-8(GDF-8) mRNA. With the purpose of accumulating the theory and experiment foundation in further human test, opening a new thinking and way to be against skeletal muscle aging in middle-old aged people by providing more direct exercise way and developing effective nutritional supplement. At the same time, it can provide some help of middle-old aged people in the direction of scientific body building, the reasonable recovery from chronic disease and the elevation of health level and quality of life.The experiment is consisted of two parts, prevention aging part and delaying senility part.Part I Prevention aging experimentFifty-six three month male Sprague-Dawley (SD) rats were randomly assigned into seven groups: adult group (C), D-galactose group (M), D-galactose small load exercise group(S), D-galactose big load exercise group (B), D-galactose peptide group (P), D-galactose peptide and small load exercise group(PS), D-galactose peptide and big load exercise group (PB). All the rats were taken the interference way in weight training and soy polypeptide supplement in the process of 6w D-galactose hypodermic. By the end of 6th week, all rats were killed and serum and skeletal muscle parameters were evaluated.1. General parameter: All the model rats showed up the aging appearances, the body weight were in normal increase trend, di-gastrocnemius &relative weight were in the decrease trend ,and all the rats in interference group could slow down the above trend; All the rats quotidie ingestion were in the decrease trend, the model groups were in obvious way. Results indicate that two interference way can improve the aging appearances effectively. 2. Skeletal muscle tissue slice: Under light microscope, the rats in group C were in normal skeletal muscle histologic characteristics, All the model rats showeg up muscle cell atrophy or regressive changes in a different level, especially the rats in group M. Results indicate that two interference way can prevent the premature appearance in skeletal muscle aging.3. Serum parameter: Compare with group C, the serum SOD, SOD/MDA, T, T/C and GH in group M were lower, MDA and C were higher.(significant or very significant difference, P<0.05 or P<0.01), but serum IGF-I was in decrease trend and had no statistic difference; Weight training or soy polypeptide supplement could reverse the trend, the groups of small load all had more obvious appearance, and they had notable interaction. Results indicate that two interference way can alleviate organism lipid peroxidation and hormone abnormality due to D-galactose.4. Skeletal muscle biochemical parameter: Compare with group C, the skeletal muscle SOD, MDA and Lipofuscin in group M were higher (significant or very significant difference, P<0.05 or P<0.01), but SOD/MDA was in decrease trend and had no statistic difference (P>0.05), skeletal muscle total protein level was lower(significant difference, P<0.05); Weight training or soy polypeptide supplement could make skeletal muscle SOD in a further increase and reverse the rest trend, the groups of small load had more obvious appearance in majority, also they had notable interaction. Results indicate that two interference way can alleviate skeletal muscle lipid peroxidation and low myoprotein synthesis due to D-galactose.5. Skeletal muscle molecular biological parameter:Compare with group C, the skeletal muscleα-actin mRNA and IGF-I mRNA in group M were lower,but GDF-8 mRNA was higher(very significant difference, P< 0.01); Weight training or soy polypeptide supplement could reverse the trend, the groups of small load all had more obvious appearance, and they had notable interaction. Results indicate that two interference way can increase the low expression ofα-actin mRNA and IGF-I mRNA and decrease the high of GDF-8 mRNA due to D-galactose.Part II Delaying senility experimentSixty three month male SD rats were randomly assigned into eight groups: 6w control group (C6), 6w model group (M6), 12w model group (M12), small load exercise group(S12), big load exercise group (B12), peptide group (P12), peptide and big load exercise group (PB12),peptide and small load exercise group(PS12)), eight fourteen month male SD rats were taken the natural aging group(OC). Besides the group C6, M6, M12 and OC, all the other rats were taken the interference way in weight training and soy polypeptide supplement after 6w D-galactose hypodermic. By the end of 6th week and 12th week, the corresponding rats were killed and serum and skeletal muscle parameters are evaluated.1.Comparison between group C6 and M6: Compare with group C6, the di-gastrocnemius &relative weight in group M6 were in the decrease trend, though they had no statistic difference; the serum SOD in group M6 was lower (significant difference, P<0.05), serum MDA and skeletal muscle lipofuscin were higher (very significant difference P<0.01), All the skeletal muscle tissue slices showed up muscle cell atrophy or regressive changes in a different level. These results indicate that the skeletal muscle aging model is successful.2. General parameter of 12 week: All the model rats and group OC showed up the aging appearances, the rats in interference group could slow down the above trend; The body weight in all groups were in normal increase trend, the rats in interference group could slow down it obviously(significant difference, P<0.05),and two interference way had notable interaction; two interference way could make di-gastrocnemius &relative weight in the decrease trend(significant or very significant difference, P<0.05 or P<0.01), and maked it relative weight in a decrease trend; All the rats quotidie ingestion were in the increase trend, two interference way could make it in a reverse way, but had no notable interaction .Results indicate that two interference way can improve the aging appearances effectively after the process of model.3. Skeletal muscle tissue slice of 12 week: Under light microscope, All the model rats showed up muscle cell atrophy or regressive changes in a different level, especially the rats in group M12 and OC. Results indicate that two interference way can postpone the course in skeletal muscle aging after the process of model.4.Serum parameter of 12 week: Compare with group OC, the serum SOD, MDA, SOD/MDA, T, C, T/C, GH and IGF-I in group M12 had no statistic difference. Weight training or soy polypeptide supplement could increase the content of serum SOD, SOD/MDA, T, T/C, GH and IGF-I, decreased the content of serum MDA and C, the groups of small load had more obvious appearance in majority, and they had notable interaction. (significant or very significant difference, P<0.05 or P<0.01).Results indicate that two interference way can alleviate organism lipid peroxidation and hormone abnormality after the process of model.5. Skeletal muscle biochemical parameter of 12 week: Compare with group OC, the skeletal muscle SOD, MDA, SOD/MDA, total protein level and lipofuscin in group M12 had no statistic difference. Weight training or soy polypeptide supplement could increase the content of skeletal muscle SOD, SOD/MDA and total protein level, decreased the content of skeletal muscle MDA and lipofuscin, the groups of small load had more obvious appearance in majority, and they had notable interaction. (significant or very significant difference, P<0.05 or P<0.01). Results indicate that two interference way can alleviate skeletal muscle lipid peroxidation and low myoprotein synthesis after the process of model.6. Skeletal muscle molecular biological parameter of 12 week:Compare with group OC, the skeletal muscleα-actin mRNA and IGF-I mRNA in group M12 had no statistic difference. Weight training or soy polypeptide supplement could increase the expression ofα-actin mRNA and IGF-I mRNA, decreased the expression of GDF-8 mRNA, the groups of small load all had more obvious appearance, and they had notable interaction. Results indicate that two interference way can increase the low expression ofα-actin mRNA and IGF-I mRNA , decrease the high of GDF-8 mRNA after the process of model.Conclusions1. Rat skeletal muscle aging model can be copied successfully by 6 week D-galactose hypodermic and the probable pathogenesy are as follows:①increase the blood free radical;②increase skeletal muscle oxidative stress and lipid peroxidation;③increase the deposition of lipofuscin in skeletal muscle;④promote the metabolic disorder of hormone and correlation factors;⑤decrease the expression of skeletal muscleα-actin mRNA and IGF-I mRNA, increase the expression of skeletal muscle GDF-8 mRNA.2. Weight training or soy polypeptide supplement can prevent the premature appearance in skeletal muscle aging resulted from 6 week D-galactose model effectively, the exercise of small load has the best result, and the two interference way united can have more obvious effect.3. 6 week weight training or soy polypeptide supplement can have an active postpone effect of skeletal muscle aging after the 6 week D-galactose model, the exercise of small load has the best result, and the two interference way united can have more obvious effect.4. The mechanisms of preventive and postponed effect of weight training and soy polypeptide supplement in skeletal muscle aging are probably as follows:①decrease the blood free radical;②decrease skeletal muscle oxidative stress and lipid peroxidation;③decrease the deposition of lipofuscin in skeletal muscle;④correct the metabolic disorder of hormone and correlation factors;⑤i ncrease the expression of skeletal muscleα-actin mRNA and IGF-I mRNA, decrease the expression of skeletal muscle GDF-8 mRNA.更多还原