当归和川芎对尾部悬吊大鼠比目鱼肌的影响及其机制研究
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摘要
研究背景:大量的研究数据和实验资料表明,在失重和模拟失重条件下,骨骼肌会出现明显的废用性萎缩,不仅对航天员返回时的救生构成严重威胁,而且影响航天员返回地面后的再适应能力,因此,寻找有效的预防和治疗肌萎缩的药物,是航天医学领域亟待解决的重要课题之一。
在失重或模拟失重状态下,由于重力作用的减少或消失,肌梭的传入放电减少,肌梭出现明显的退行性改变,这可能是失重性肌萎缩发生的重要原因之一。增加肌梭传入冲动的措施,则可对抗或减缓骨骼肌萎缩。另一方面,失重或模拟失重造成的肌肉萎缩会诱导骨骼肌产生“血瘀样”症状,并且两者互相影响,形成恶性循环。由此,我们认为那些能兴奋肌梭的活血类药物很有可能成为预防和治疗失重性肌萎缩的理想药物。本研究选用前期实验筛选出的可兴奋肌梭的经典活血类药物——当归,以大鼠尾部悬吊建立模拟失重动物模型,观察其对比目鱼肌的形态结构、肌纤维类型的影响,并从血液流变性以及肌梭结构等方面探讨其萎缩机制。此外,本课题组前期已经证实川芎嗪有抗失重性肌萎缩的作用,但是它是否是川芎中抗失重性肌萎缩的主要成分?本次研究将对川芎抗失重性肌萎缩的药效作用进行研究,探讨其主要有效成分是否为川芎嗪。以期为失重性肌萎缩的药物防护提供基础实验资料。
目的:观察当归和川芎对模拟失重大鼠比目鱼肌萎缩的影响,并对其影响机制进行初步探讨。检验川芎中抗肌萎缩的主要成份是否为川芎嗪。
方法:以大鼠尾部悬吊法建立模拟失重模型。(1)在大鼠尾部悬吊的同时,给予大鼠当归或川芎灌胃,利用光镜及免疫组化等方法分别观察并比较当归和川芎对尾部悬吊大鼠比目鱼肌(soleus,SOL)一般形态结构和肌纤维横截面积(crosssectional area,CSA)的影响;(2)应用免疫组织化学方法,观察当归和川芎对模拟失重大鼠比目鱼肌快肌肌球蛋白重链(myosin heavy chain,MHC)表达水平的影响;(3)通过测定全血粘度、血浆粘度、红细胞压积、红细胞变形能力等血液流变学指标,观察当归和川芎对尾部悬吊大鼠血液流变性的影响;(4)利用透射电镜观察并比较当归和川芎对尾部悬吊大鼠比目鱼肌肌梭超微结构的影响。
结果:
1.当归和川芎对尾部悬吊大鼠比目鱼肌形态结构的影响:
1)与吊尾灌溶媒组(TS+W)相比,当归或川芎治疗后,大鼠比目鱼肌肌纤维显微结构紧凑,形态恶化得到改善;2)吊尾同期给予当归治疗,可明显缓解尾部悬吊大鼠比目鱼肌肌纤维横截面积的下降。而川芎的缓解作用有限,提示川芎中对抗失重性肌萎缩的主要有效成分可能是川芎嗪。
2.当归和川芎对大鼠比目鱼肌肌球蛋白重链的表达水平和肌纤维类型的影响:
1)吊尾灌溶媒组(TS+W)的比目鱼肌中快肌肌球蛋白增加显著,而慢肌肌球蛋白亦明显减少;给予当归和川芎治疗后,与溶媒组相比大鼠比目鱼肌中慢肌肌球蛋白重链表达水平显著增加,而快肌肌球蛋白重链表达水平显著减少。2)与吊尾灌溶媒组相比,给药组比目鱼肌中Ⅰ型肌纤维比例均显著升高,Ⅱ型肌纤维比例均显著降低。
3.当归和川芎对尾部悬吊大鼠血液流变性的影响:
尾部悬吊14d后,大鼠的全血粘度、血浆粘度、红细胞刚性指数均明显增高,红细胞压积明显下降,提示模拟失重使大鼠的红细胞质量下降,血粘度提高,出现明显的“血淤症”。给予当归或川芎治疗后,全血粘度、血浆粘度、红细胞刚性指数的增高和红细胞压积的下降均得到明显抑制。提示,当归和川芎抗失重性肌萎缩作用的机制之一可能是通过提高红细胞的质量和改善血粘度而实现。
4.当归和川芎对尾部悬吊大鼠比目鱼肌肌梭超微结构的影响:
与正常组相比,吊尾灌溶媒组大鼠比目鱼肌肌梭超微结构松散,发生退行性变化。与吊尾灌溶媒组(TS+W)相比,当归或川芎治疗后大鼠比目鱼肌肌梭超微结构紧凑,形态恶化得到改善。提示,当归和川芎抗失重性肌萎缩作用的另一机制可能是通过改善肌梭结构而实现的。
结论:
(1)当归和川芎对模拟失重所致的大鼠比目鱼肌显微结构的改变均有明显的对抗作用,且当归对尾部悬吊大鼠肌纤维横截面积的减小有明显对抗作用。川芎对尾部悬吊大鼠肌纤维横截面积的减小仅有对抗趋势。因此,川芎嗪可能是川芎中对抗肌萎缩的主要有效成分。
(2)当归和川芎对模拟失重所致大鼠比目鱼肌快肌肌球蛋白重链表达水平的升高均有明显的抑制作用。
(3)当归和川芎对模拟失重所致大鼠血液粘度、血浆粘度、红细胞压积和红细胞刚性指数等血液流变性的改变有明显的抑制作用。
(4)当归和川芎对模拟失重所致大鼠比目鱼肌肌梭超微结构的退行性改变有抑制作用。
Background
A great deal of scientific data and experiments have indicated weightlessness andsimulated microgravity can lead to apparent muscle atrophy. Muscle atrophy affects astronaut performance both in- and post flight. Therefore, it is important to find effective drugs to prevent or treat muscle atrophy caused by weightlessness in the fields of clinical, aerospace and rehabilitation medicine. Under weightlessness or stimulated weightlessness conditions, postural muscle spindle afferent discharge declines, which may contribute to the develop of muscle atrophy. Countermeasures, which can excite muscle spindles such as intermittent stretch, have been shown to alleviate disuse atrophy. On the other hand, real or simulated microgravity may evoke cardiac and hemostatic problems, several reports have documented the risk of deep venous thrombosis or capillary thromboembolism increased. Accordingly, inactivity-induced thrombosis may form a vicious cycle in combination with muscle atrophy. Therefore, drugs that enhance the afferent muscle spindle discharge and at the same time improve muscular perfusion would therefore be ideal to fight weightlessness induced muscle atrophy. Therefore, we choose Angelica sinensis that activate the blood circulation, which can distinctively tingle the spontaneous electrical activity of muscle spindle. Another classical TCM we are interested in is Ligusticum whose main effective ingredient to activate blood circulation is Ligustrazine. Ligustrazine may also be useful to fight weightlessness induced muscle atrophy. If the Ligustrazine is the main component in Ligusticum to fight weightlessness induced muscle atrophy? We will investigate the question in this study. The tail-suspended rat was taken as model to research the morphological structure, biochemical metabolism, hemorrheology of skeletal muscle, so as to provide experimental data for drug development of muscle atrophy.
Objective: This research attempts to observe the effects of Angelica sinensis and Ligusticum on muscle atrophy caused by microgravity and probes into their mechanism. Investigate if the Ligustrazine is the main component in Ligusticum to fight weightlessness induced muscle atrophy?
Methods: Tail-suspended rat was taken as the disuse atrophy model. (1) The ratwas treated with Angelica sinensis and Ligusticum. The effects of Angelica sinensis and Ligusticum on the structure and CSA of soleus muscle in tail-suspended rat were studied and compared respectively. (2) Immunohistochemical method was used to study the effects of expression of MHC on Angelica sinensis and Ligusticum in tail-suspended rat soleus muscle. (3) We observed the effect of Angelica sinensis and Ligusticum on hemorrheology by whole blood viscocity, blood plasm viscocity, , hematocrit value and erythrocyte deformability. (4) The effects of Angelica sinensis and Ligusticum on the ultrastructure of soleus muscle spindle in tail-suspended rat were studied and compared respectively, by electron microscope.
Results:
(1) Effects of Angelica sinensis and Ligusticum on the structure and CSA ofsoleus muscle in tail-suspended rat
1)Compared to TS+W group, muscle fiber of treatment was compacter, deteriorated shape was improved。2) Angelica sinensis could effectively prevent the decreasing of CSA caused by microgravity. 25% type I fiber CSA increase in TS+Lig above TS+W just failed to reach the generally accepted level of statistical significance. Compared to data from our previous work, it is obvious that the atrophy- alleviating effect of a similar dose of Ligusticum is weaker than that of Ligustrazine. Accordingly, we surmise that Ligustrazine is the main ingredient in Ligusticum to alleviate muscle atrophy in simulated weightlessness.
(2) Effects of Angelica sinensis and Ligusticum on tail-suspended rat soleus muscle expression of MHC and fiber percentage
1) After 14 days hindlimb-unloading plus intragastric water instillation (TS+W), the expression of fast muscle MHC increased, slow muscle MHC reduced. Treated with Angelica sinensis and Ligusticum, the expression of fast muscle MHC reduced, and slow muscle MHC increased. 2) Compared to TS+W, type I muscle of treated increased significantly, and type II muscle decreased.
(3) Effects of Angelica sinensis and Ligusticum on hemorrheology of tail-suspended rat
After 14d tail-suspension, the whole blood viscocity, blood plasm viscocity, and erythrocyte deformability of rat increased significantly and hematocrit value reduces. After treatment with Angelica sinensis and Ligusticum for 14 days, the change of them was obviously under control. The results showed that one of the mechanisms of anti- muscle atrophy of Angelica sinensis and Ligusticum was possibly realized by improving the red blood cell mass and promoting blood circulation.
(4) Effects of Angelica sinensis and Ligusticum on the ultrastructure of soleusmuscle spindle in tail-suspended rat
Compared to TS+W group, ultrastructure of soleus muscle spindle was compacter, deteriorated shape was improved。Another mechanisms of anti- muscle atrophy of Angelica sinensis and Ligusticum was possibly realized by exciting muscle spindles.
Conclusion:
(1) Angelica sinensis and Ligusticum could prevent the change of microstructure in muscle fibres caused by simulated weightlessness, and Angelica sinensis could prevent the decrease of CSA. The effect of Ligusticum is weaker than Ligustrazine, so Ligustrazine is the main ingredient to alleviate muscle atrophy in simulated weightlessness in Ligusticum.
(2) Angelica sinensis and Ligusticum could prevent the increase of MHC-II caused by simulated weightlessness.
(3) Angelica sinensis and Ligusticum could drastically inhibit the change ofhemorrheology caused by simulated weightlessness.
(4) Angelica sinensis and Ligusticum could prevent the changes ofultrastructure in muscle fibres caused by simulated weightlessness.
在失重或模拟失重状态下,由于重力作用的减少或消失,肌梭的传入放电减少,肌梭出现明显的退行性改变,这可能是失重性肌萎缩发生的重要原因之一。增加肌梭传入冲动的措施,则可对抗或减缓骨骼肌萎缩。另一方面,失重或模拟失重造成的肌肉萎缩会诱导骨骼肌产生“血瘀样”症状,并且两者互相影响,形成恶性循环。由此,我们认为那些能兴奋肌梭的活血类药物很有可能成为预防和治疗失重性肌萎缩的理想药物。本研究选用前期实验筛选出的可兴奋肌梭的经典活血类药物——当归,以大鼠尾部悬吊建立模拟失重动物模型,观察其对比目鱼肌的形态结构、肌纤维类型的影响,并从血液流变性以及肌梭结构等方面探讨其萎缩机制。此外,本课题组前期已经证实川芎嗪有抗失重性肌萎缩的作用,但是它是否是川芎中抗失重性肌萎缩的主要成分?本次研究将对川芎抗失重性肌萎缩的药效作用进行研究,探讨其主要有效成分是否为川芎嗪。以期为失重性肌萎缩的药物防护提供基础实验资料。
目的:观察当归和川芎对模拟失重大鼠比目鱼肌萎缩的影响,并对其影响机制进行初步探讨。检验川芎中抗肌萎缩的主要成份是否为川芎嗪。
方法:以大鼠尾部悬吊法建立模拟失重模型。(1)在大鼠尾部悬吊的同时,给予大鼠当归或川芎灌胃,利用光镜及免疫组化等方法分别观察并比较当归和川芎对尾部悬吊大鼠比目鱼肌(soleus,SOL)一般形态结构和肌纤维横截面积(crosssectional area,CSA)的影响;(2)应用免疫组织化学方法,观察当归和川芎对模拟失重大鼠比目鱼肌快肌肌球蛋白重链(myosin heavy chain,MHC)表达水平的影响;(3)通过测定全血粘度、血浆粘度、红细胞压积、红细胞变形能力等血液流变学指标,观察当归和川芎对尾部悬吊大鼠血液流变性的影响;(4)利用透射电镜观察并比较当归和川芎对尾部悬吊大鼠比目鱼肌肌梭超微结构的影响。
结果:
1.当归和川芎对尾部悬吊大鼠比目鱼肌形态结构的影响:
1)与吊尾灌溶媒组(TS+W)相比,当归或川芎治疗后,大鼠比目鱼肌肌纤维显微结构紧凑,形态恶化得到改善;2)吊尾同期给予当归治疗,可明显缓解尾部悬吊大鼠比目鱼肌肌纤维横截面积的下降。而川芎的缓解作用有限,提示川芎中对抗失重性肌萎缩的主要有效成分可能是川芎嗪。
2.当归和川芎对大鼠比目鱼肌肌球蛋白重链的表达水平和肌纤维类型的影响:
1)吊尾灌溶媒组(TS+W)的比目鱼肌中快肌肌球蛋白增加显著,而慢肌肌球蛋白亦明显减少;给予当归和川芎治疗后,与溶媒组相比大鼠比目鱼肌中慢肌肌球蛋白重链表达水平显著增加,而快肌肌球蛋白重链表达水平显著减少。2)与吊尾灌溶媒组相比,给药组比目鱼肌中Ⅰ型肌纤维比例均显著升高,Ⅱ型肌纤维比例均显著降低。
3.当归和川芎对尾部悬吊大鼠血液流变性的影响:
尾部悬吊14d后,大鼠的全血粘度、血浆粘度、红细胞刚性指数均明显增高,红细胞压积明显下降,提示模拟失重使大鼠的红细胞质量下降,血粘度提高,出现明显的“血淤症”。给予当归或川芎治疗后,全血粘度、血浆粘度、红细胞刚性指数的增高和红细胞压积的下降均得到明显抑制。提示,当归和川芎抗失重性肌萎缩作用的机制之一可能是通过提高红细胞的质量和改善血粘度而实现。
4.当归和川芎对尾部悬吊大鼠比目鱼肌肌梭超微结构的影响:
与正常组相比,吊尾灌溶媒组大鼠比目鱼肌肌梭超微结构松散,发生退行性变化。与吊尾灌溶媒组(TS+W)相比,当归或川芎治疗后大鼠比目鱼肌肌梭超微结构紧凑,形态恶化得到改善。提示,当归和川芎抗失重性肌萎缩作用的另一机制可能是通过改善肌梭结构而实现的。
结论:
(1)当归和川芎对模拟失重所致的大鼠比目鱼肌显微结构的改变均有明显的对抗作用,且当归对尾部悬吊大鼠肌纤维横截面积的减小有明显对抗作用。川芎对尾部悬吊大鼠肌纤维横截面积的减小仅有对抗趋势。因此,川芎嗪可能是川芎中对抗肌萎缩的主要有效成分。
(2)当归和川芎对模拟失重所致大鼠比目鱼肌快肌肌球蛋白重链表达水平的升高均有明显的抑制作用。
(3)当归和川芎对模拟失重所致大鼠血液粘度、血浆粘度、红细胞压积和红细胞刚性指数等血液流变性的改变有明显的抑制作用。
(4)当归和川芎对模拟失重所致大鼠比目鱼肌肌梭超微结构的退行性改变有抑制作用。
Background
A great deal of scientific data and experiments have indicated weightlessness andsimulated microgravity can lead to apparent muscle atrophy. Muscle atrophy affects astronaut performance both in- and post flight. Therefore, it is important to find effective drugs to prevent or treat muscle atrophy caused by weightlessness in the fields of clinical, aerospace and rehabilitation medicine. Under weightlessness or stimulated weightlessness conditions, postural muscle spindle afferent discharge declines, which may contribute to the develop of muscle atrophy. Countermeasures, which can excite muscle spindles such as intermittent stretch, have been shown to alleviate disuse atrophy. On the other hand, real or simulated microgravity may evoke cardiac and hemostatic problems, several reports have documented the risk of deep venous thrombosis or capillary thromboembolism increased. Accordingly, inactivity-induced thrombosis may form a vicious cycle in combination with muscle atrophy. Therefore, drugs that enhance the afferent muscle spindle discharge and at the same time improve muscular perfusion would therefore be ideal to fight weightlessness induced muscle atrophy. Therefore, we choose Angelica sinensis that activate the blood circulation, which can distinctively tingle the spontaneous electrical activity of muscle spindle. Another classical TCM we are interested in is Ligusticum whose main effective ingredient to activate blood circulation is Ligustrazine. Ligustrazine may also be useful to fight weightlessness induced muscle atrophy. If the Ligustrazine is the main component in Ligusticum to fight weightlessness induced muscle atrophy? We will investigate the question in this study. The tail-suspended rat was taken as model to research the morphological structure, biochemical metabolism, hemorrheology of skeletal muscle, so as to provide experimental data for drug development of muscle atrophy.
Objective: This research attempts to observe the effects of Angelica sinensis and Ligusticum on muscle atrophy caused by microgravity and probes into their mechanism. Investigate if the Ligustrazine is the main component in Ligusticum to fight weightlessness induced muscle atrophy?
Methods: Tail-suspended rat was taken as the disuse atrophy model. (1) The ratwas treated with Angelica sinensis and Ligusticum. The effects of Angelica sinensis and Ligusticum on the structure and CSA of soleus muscle in tail-suspended rat were studied and compared respectively. (2) Immunohistochemical method was used to study the effects of expression of MHC on Angelica sinensis and Ligusticum in tail-suspended rat soleus muscle. (3) We observed the effect of Angelica sinensis and Ligusticum on hemorrheology by whole blood viscocity, blood plasm viscocity, , hematocrit value and erythrocyte deformability. (4) The effects of Angelica sinensis and Ligusticum on the ultrastructure of soleus muscle spindle in tail-suspended rat were studied and compared respectively, by electron microscope.
Results:
(1) Effects of Angelica sinensis and Ligusticum on the structure and CSA ofsoleus muscle in tail-suspended rat
1)Compared to TS+W group, muscle fiber of treatment was compacter, deteriorated shape was improved。2) Angelica sinensis could effectively prevent the decreasing of CSA caused by microgravity. 25% type I fiber CSA increase in TS+Lig above TS+W just failed to reach the generally accepted level of statistical significance. Compared to data from our previous work, it is obvious that the atrophy- alleviating effect of a similar dose of Ligusticum is weaker than that of Ligustrazine. Accordingly, we surmise that Ligustrazine is the main ingredient in Ligusticum to alleviate muscle atrophy in simulated weightlessness.
(2) Effects of Angelica sinensis and Ligusticum on tail-suspended rat soleus muscle expression of MHC and fiber percentage
1) After 14 days hindlimb-unloading plus intragastric water instillation (TS+W), the expression of fast muscle MHC increased, slow muscle MHC reduced. Treated with Angelica sinensis and Ligusticum, the expression of fast muscle MHC reduced, and slow muscle MHC increased. 2) Compared to TS+W, type I muscle of treated increased significantly, and type II muscle decreased.
(3) Effects of Angelica sinensis and Ligusticum on hemorrheology of tail-suspended rat
After 14d tail-suspension, the whole blood viscocity, blood plasm viscocity, and erythrocyte deformability of rat increased significantly and hematocrit value reduces. After treatment with Angelica sinensis and Ligusticum for 14 days, the change of them was obviously under control. The results showed that one of the mechanisms of anti- muscle atrophy of Angelica sinensis and Ligusticum was possibly realized by improving the red blood cell mass and promoting blood circulation.
(4) Effects of Angelica sinensis and Ligusticum on the ultrastructure of soleusmuscle spindle in tail-suspended rat
Compared to TS+W group, ultrastructure of soleus muscle spindle was compacter, deteriorated shape was improved。Another mechanisms of anti- muscle atrophy of Angelica sinensis and Ligusticum was possibly realized by exciting muscle spindles.
Conclusion:
(1) Angelica sinensis and Ligusticum could prevent the change of microstructure in muscle fibres caused by simulated weightlessness, and Angelica sinensis could prevent the decrease of CSA. The effect of Ligusticum is weaker than Ligustrazine, so Ligustrazine is the main ingredient to alleviate muscle atrophy in simulated weightlessness in Ligusticum.
(2) Angelica sinensis and Ligusticum could prevent the increase of MHC-II caused by simulated weightlessness.
(3) Angelica sinensis and Ligusticum could drastically inhibit the change ofhemorrheology caused by simulated weightlessness.
(4) Angelica sinensis and Ligusticum could prevent the changes ofultrastructure in muscle fibres caused by simulated weightlessness.
引文
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