【作者】 李颖；

【导师】 庄洪；

【作者基本信息】 广州中医药大学 ， 中医骨伤科学， 2009， 博士

【摘要】 目的:从线粒体的角度探讨骨骼肌与骨质疏松的关系,以期为骨骼肌作为治疗靶点提供客观合理的量化依据,同时为中医“肾主骨、脾主肌、脾肾相关”理论治疗骨质疏松症提供科学的依据。方法:一、临床实验选择自2008年5月到2008年12月期间在广州中医药大学第一附属医院因腰椎间盘突出或者腰椎管狭窄症需要行手术治疗的女性骨质疏松患者13例,另选同时期需上述手术治疗的女性非骨质疏松患者13例,总共26例。于术中收集肌肉组织,提取骨骼肌线粒体,进行细胞色素C氧化酶和线粒体通透转换孔的活性检测,并进行相关性分析;二、动物实验选择雌性未孕的SD大鼠52只,按随机数字表法分为手术组36只,空白组16只,手术组行双侧卵巢切除,空白组不切除卵巢。术后3个月,从两组中分别随机选取6只大鼠,进行骨密度检查。在确定造模成功后,将手术组剩下的36只大鼠再随机分为3组,每组10只,分别为模型组、雌激素组、中药组。空白组中剩下的10只大鼠为空白对照组。各组进行灌胃给药治疗,灌胃给药容积为10ml/kg,空白组、模型组灌服蒸馏水;中药组给予中药按4.8g/kg灌胃(相当于临床剂量的10倍),药物以蒸馏水定容至所需浓度,即10ml/kg灌服给药,每日一次;尼尔雌醇组给予尼尔雌醇1mg/kg灌胃,使用前配成混悬液(浓度为0.167mg/ml),1次/周,其余时间给予蒸馏水10ml/kg灌胃。治疗3个月后,收集肌肉组织进行细胞色素C氧化酶、线粒体活性氧和线粒体通透转换孔的活性检测。结果:一、临床实验,①在线粒体通透转换孔在0 min和30 min的OD值比较中,非骨质疏松组在0 min的OD值为0.182±0.047,而在30 min的OD值为0.181±0.046;骨质疏松组在0 min的OD值为0.143±0.071,而在30 min的OD值为0.141±0.071。随着时间的推移,两组的OD值均呈下降的趋势,30 min的OD值与0 min比较,有统计学意义(P＜0.05),表明线粒体通透孔的活性增强;但两组在同一时间点比较,无统计学意义P＞0.05)。②在线粒体通透转换孔活性变化的比较方面,非骨质疏松组的OD比值为0.994±0.007,而骨质疏松组为0.985±0.006,两者比较,有统计学意义(P＜0.05)。③在线粒体细胞色素C氧化酶的活性比较方面,非骨质疏松组为0.338±0.142,骨质疏松组为0.124±0.093,两者比较,有统计学意义(P＜0.05)。④在线粒体通透转换孔与细胞色素C氧化酶的相关性分析中,线粒体通透转换孔的OD比值与细胞色素C氧化酶的活性存在着正相关的关系(r=0.405,P=0.04＜0.05),回归方程如下:Y(细胞色素C氧化酶的活性)=-7.127+7.435X(线粒体通透转换孔的OD值)。二、动物实验①最后纳入研究的动物35只。②在线粒体通透转换孔在0 min、30 min和60min的OD值比较中,各组线粒体通透转换孔的变化,均呈下降趋势。空白对照组在60min时的OD值与30min时比较,有统计学意义(P＜0.05);模型组在60 min时的OD值与0min与30min时比较,差异有统计学意义(P＜0.05),而且30min时的OD值与0min时的比较,也有统计学意义(P＜0.05);而雌激素组在各个时间点上的比较,差异均无统计学意义(P＞0.05);中药组在60min时的OD值;与0min与30min时比较,差异有统计学意义(P＜0.05),但30min时的OD值与0min,时的比较,差异无统计学意义(P＞0.05)。③在线粒体通透转换孔活性比较方面,各组在线粒体通透转换孔活性比较方面,在30min/0minOD比值的比较中,空白对照组的OD值为1.000±0.025,模型组为0.975±0.014,雌激素组为0.993±0.022,中药组为0.990±0.010。雌激素组、空白对照组与模型组比较,差异均有统计学意义(P＜0.05),而中药组与其他各组比较,差异无统计学意义(P＞0.05)。而在60min/0minOD比值的比较中,空白对照组的OD值为1.000±0.047,模型组为0.956±0.016,雌激素组为0.984±0.030,中药组为0.976±0.025,雌激素组和空白对照组与模型组比较,差异均有统计学意义(P＜0.05),而中药组与其他各组比较,差异无统计学意义(P＞0.05)。各组在30min/0min与60min/0minOD比值比较中,除空白对照组的两个比值,差异无统计学意义外(P＞0.05),其余各组的比值,差异均有统计学意义。④在细胞色素C氧化酶比较方面,空白对照组为0.0200±0.0141,模型组为0.0070±0.0051,雌激素组为0.0134±0.0064,中药组为0.0130±0.0092,空白对照组的活性与模型组的比较,差异有统计学意义(P＜0.05)。雌激素组、中药组与模型组比较,差异无统计学意义(P＞0.05);⑤在线粒体活性氧比较方面,空白对照组(1898.89±1078.39、1900.00±1078.76、1911.33±1078.14、1911.33±1077.37)、模型组(2271.22±1349.76、2282.79±1350.78、2297.22±1376.74、2297.00±1375.35)、雌激素组(2227.67±1365.95、2239.89±1363.75、2244.11±1363.55、2242.00±1357.36)和中药组(1536.25±835.51、1576.38±836.50、1550.86±835.27、1550.86±835.27)在15min、20min、30min、45min比较,差异均无统计学意义(P＞0.05),但在这些时间点上,模型组的含量最高,其次为雌激素组和空白对照组,最后为中药组。结论:骨骼肌线粒体变化和骨质疏松存在着互相影响的关系,而通过药物干预后,能有效地调控线粒体的活性变化,抑制肌细胞的凋亡,从而有效地调控骨质疏松的发展。主要结论如下:①在线粒体通透转换孔的通透性方面,骨质疏松骨骼肌的通透性增大,表示有更多的促凋亡的物质刺激或通过该孔,从而更快地诱导细胞的凋亡,也提示骨质疏松骨骼肌的线粒体损伤程度较非骨质疏松的严重。②在细胞色素C氧化酶方面,骨质疏松骨骼肌的细胞色素C氧化酶活性较非骨质疏松低,提示骨质疏松骨骼肌的线粒体外膜完整性较非骨质疏松的差,从而揭示骨质疏松骨骼肌的细胞凋亡速度较非骨质疏松快。③在线粒体通透转换孔的通透性与细胞色素C氧化酶的关系方面,线粒体通透转换孔的OD比值与细胞色素C氧化酶的活性存在着正相关的关系,而线粒体OD比值与通透性是反比的关系,这说明,线粒体通透转换孔的通透性越大(OD比值越小),细胞色素C氧化酶的活性越小。这揭示了只要维持细胞色素C氧化酶的活性,就能有效地防治细胞的过早衰老。从另一个侧面来说,这就为治疗骨质疏松提供一个新的治疗靶点。④在活性氧的活性方面,骨质疏松和非骨质疏松没有明显的差异,可能的原因是活性氧含量的多少对雌激素缺乏大鼠骨骼肌的影响表现不敏感。但随着时间的推移,其含量是不断增加的,这表示细胞在不断积累,当到达一定量时启动细胞的凋亡程序。⑤补肾健脾中药在线粒体通透转换孔、细胞色素C氧化酶和或活性氧这三个指标中,尽管与雌激素相比,未能达到明显的效果,但从其数值的变化来说,认为该中药可以改善线粒体通透转换孔的通透性,提高细胞色素C氧化酶和降低活性氧的含量,是未来研究以骨骼肌为作用靶点的药物之一。更多还原

【Abstract】 ObjectiveInvestigate the relationship between osteoporosis and skeletal muscle from the perspective of mitochondrion,which to provide the objective reasonable quantification basis for the skeletal muscle as a therapeutic target,and provide a scientific basis for chinese medicine theory of "kidney controlling bones,spleen controlling muscle and spleen-renal interrelationship" in the treatment of osteoporosis.MethodThe clinical experiment26 female patients were selected from the first hospital affiliated to Guangzhou university of CM between May 2008 and December 2008,who were diagnosed for lumbar intervertebral disc herniation or lumbar spinal stenosis to need surgery.They were divided into Osteoporosis group(n=13) and non-osteoporosis group(n=13).Mitochondrion were extracted from the muscle tissue that was collecte during the operation,and cytochrome coxidase(COX) and the mitochondrial permeability transition pore(MPPT) were detected. The animal experiment52 female non-pregnant SD rats were randomly divided into normal control and operation group,normal control group were 16,and operation group were 36.The operation group were ovariectomized,while sham operation was performed in normal control group.After 3 monthes,the two groups were randomly selected 10 rats to measured the BMD.After establishing modle,the operation group of 36 were divided into 3 groups:ovariectomized,estrogen supplemented and traditional chinese herbs,the rats of traditional chinese herbs were given the intragastric administration with herbs of reinforcing the kidney and invigorating the spleen according to 4.8g/kg(using the quantity is equal to clinical dosage 10 times),one day at a time.The rats of estrogen supplemented were given nilestriol according to 1mg/kg,once a week.the others were given distilled water,one day at a time.After treating 3 months, mitochondrion were extracted from the muscle tissue that was collecte during the operation,and cytochrome coxidase(COX),the mitochondrial permeability transition pore(MPPT) and reactive oxygen species(ROS) were detected.ResultThe clinical experiment①In the optical delnsity(OD) value of MPPT,OD value of non-osteoporosis group were 0.182±0.047 at 0 min and 0.181±0.046 at 30min.OD value of Osteoporosis group were 0.143±0.071 at 0 min and 0.141±0.071 at 30min. Over time,OD value of the two groups showed a downward trend,there was a statistically significant difference between OD value of 30 min and of 0 min in two group(p＜0.05),that showed MPPT activity,but there was no a statistically significant difference between OD value of two group at the same time(p＞0.05).①In the permeability of MPPT,the ratio of OD value of non-osteoporosis group were 0.994±0.007,osteoporosis group were 0.985±0.006,there was a statistically significant difference between two group(p＜0.05).In the COX, non-osteoporosis group were 0.338±0.142,osteoporosis group were 0.124±0.093,there was a statistically significant difference between two group(p＜0.05).③In the correlation analysis of MPPT and COX,there was positive correlation between the ratio of OD value of MPPT and COX(r=0.405,P=0.04＜0.05),the regression equation was Y(COX)=388.8-647.402X(the ratio of OD value of MPPT).The animal experiment①35 rats were all involved into the result analysis.②In the optical delnsity(OD) value of MPPT,the change of MPPT of each group showed a downward trend.There was a statistically significant difference between OD value of 30 min and of 60 min in normal control group(p＜0.05). There was a statistically significant difference between OD value of 60 min and of other time in ovariectomized group(p＜0.05).There was also a statistically significant difference between OD value of 30 min and of 0 min in ovariectomized group(p＜0.05).There was no a statistically significant difference in all time in estrogen supplemented group(p＞0.05). There was a statistically significant difference between OD value of 60 min and of other time in traditional chinese herbs group(p＜0.05),but there was no a statistically significant difference between OD value of 30 min and of 0 min(p＞0.05).③In the permeability of MPPT,in the ratio of OD value of 30min/0min,the ratio of OD value of normal control group were 1.000±0.025,the ovariectomized group were 0.975±0.014,the estrogen supplemented group were 0.993±0.022 and the traditional chinese herbs group were 0.990±0.010.in 60min/0min,the ratio of OD value of normal control group were 1.000±0.047,the ovariectomized group were 0.956±0.016,the estrogen supplemented group were 0.976±0.025 and the traditional chinese herbs group were 0.976±0.025.There was a statistically significant difference that the ratio of OD value of normal control group,of the estrogen supplemented group respectively compared with the ovariectomized group in 30min/0min and 60min/0min(p＜0.05),and there was no a statistically significant difference between the traditional chinese herbs group and other groups in 30min/0min and 60min/0min(p＞0.05).Compared with the ratio of OD value of 30min/0min and of 60min/0min,There was a statistically significant difference in the normal control group(p＜0.05),there was no a statistically significant difference in the other groups(p＞0.05).④In the COX,the value of normal control group were 0.0200±0.0141,the ovariectomized group were 0.0070±0.0051,the estrogen supplemented group were 0.0134±0.0064 and the traditional chinese herbs group were 0.0130±0.0092。There was a statistically significant difference between the normal control group and the ovariectomized(p＜0.05).There was no a statistically significant difference that the value of the estrogen supplemented group,of the traditional chinese herbs group respectively compared with of the ovariectomized group(p＞0.05).⑤In the ROS,the value of normal control group were 1898.89±1078.39,1900.00±1078.76,1911.33±1078.14 and 1911.33±1077.37,the ovariectomized group were 2271.22±1349.76,2282.79±1350.7,2297.22±1376.74 and 2297.00±1375.35,the estrogen supplemented group were 2227.67±1365.95, 2239.89±1363.75,2244.11±1363.55 and 2242.00±1357.36,the traditional chinese herbs group were 1536.25±835.51,1576.38±836.50,1550.86±835.27 and 1550.86±835.27.There was no a statistically significant difference in all groups in all time,but in these time,the ovariectomized group’s content were highest,next were the estrogen supplemented group and the normal control group,finally were the traditional chinese herbs group.ConclusionThe change of muscle mitochondria and osteoporois have a relationship of mutual influence.Through drug intervention,The change of muscle mitochondria could be effective in controlling,muscle cell apoptosis could be inhibited,and finally the development of osteoporosis could be controlled effectively.The main conclusions are as follows.①In the permeability of MPPT,the permeability of MMPT of osteoporotic muscle is more greater,it means that there is more apoptosis tostimulate or passes MPPT,and induces the cell apoptosis more quickly.It shows that the damage of muscle mitochondria in osteoporosis compares the non-osteoporosis to be serious②In the COX,the enzymatic activity of COX of osteoporotic muscle is worse than non-osteoporosis,that shows mitochondrial outer membrane integrity of osteoporotic muscle is worse than non-osteoporosis,and apoptosis rate of osteoporotic muscle cell is faster than non-osteoporosis.③In the relationship between the ratio of OD value of MPPT and the enzymatic activity of COX,there is positive correlation between the ratio of OD value of MPPT and COX,but the ratio of OD value of MPPT and the permeability are the inverse relationship.It means that the permeability of MPPT is greater,the smaller the enzymatic activity of COX,as long as maintain the enzymatic activity of COX,we could effectively controll cell premature aging.From another side,it provide a new therapeutic target for the treatment of osteoporosis④In the ROS,there is no significant differences in non-osteoporosis and osteoporosis.The possible reason is that ROS is insensitive to rat skeletal muscle tha is lack of estrogen.But over time,the Content of ROS is increasing that means cells continue to accumulate,and start apoptosis when reached a certain amount. ⑤In the MMPT,COX and ROS,herbs of reinforcing the kidney and invigorating fails to achieve significant results,compared with estrogen.But from its value,we believe that herbs of reinforcing the kidney and invigoratin improves the permeability of MPPT and the enzymatic activity of COX,reduces the the content of ROS.So it is one of drug that research the skeletal muscle as a therapeutic target in the future.更多还原