长白山黄芪对骨骼肌的抗疲劳作用及其机制初探
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摘要
[目的]研究长白山黄芪5种提取液对骨骼肌的抗疲劳作用效果及其剂量效应关系,筛选出最佳提取液,结合应用生物化学和RT-PCR方法深入探讨长白山黄芪抗疲劳作用的可能的生物化学和分子机制。
[方法]本实验采用长白山黄芪,严格按照中药提取方法制备5种提取液(黄芪水、85%酒精、50%酒精、正丁醇和乙酸乙酯)。实验动物选用昆明种雄性小鼠和Wistar系雄性大鼠,小鼠用于测定负重游泳力竭时间和血清超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,大鼠用于骨骼肌收缩力学实验和骨骼肌SOD活性及MDA含量等生化指标的测定。骨骼肌组织测定α-actin的表达。
[结果]1.长白山黄芪不同提取液对小鼠负重游泳时间和体重的影响:黄芪的五种提取液均在不同程度上延长小鼠负重游泳时间;2.长白山黄芪水提取液对大鼠骨骼肌收缩力的影响:①黄芪水提取液中、大剂量组明显增强腓肠肌和比目鱼肌单收缩的最大收缩幅度(Pt),但是腓肠肌潜伏期(CT)和1/2舒张时间(HRT)延长,而比目鱼肌单收缩的CT和HRT缩短;②黄芪水提取液中、大剂量提高腓肠肌和比目鱼肌强直收缩的Pt、减小疲劳指数(FI);3.长白山黄芪85%酒精提取液对大鼠骨骼肌收缩力的影响:①黄芪85%酒精提取液中剂量组使腓肠肌单收缩的CT和HRT明显延长、Pt增强,在中、大剂量组使目鱼肌的单收缩CT和HRT缩短、Pt增强;②黄芪85%酒精提取液中、大剂量提高腓肠肌和比目鱼肌强直收缩的Pt、减小FI;4.长白山黄芪水提取液对小鼠血清和大鼠骨骼肌中SOD、MDA含量的影响:①黄芪水提取液中、大剂量组使小鼠血清中的SOD活性增强,MDA含量显著降低;②使大鼠腓肠肌和比目鱼肌中的SOD活性均明显增强、MDA含量均显著降低;5.长白山黄芪85%酒精提取液对小鼠血清和大鼠骨骼肌中SOD、MDA含量的影响:①黄芪85%酒精提取液仅大剂量组增加了小鼠血清中SOD活性,其余各剂量组无统计学差异;MDA含量变化无统计学差异;②黄芪酒精提取液对大鼠骨骼肌中SOD、MDA含量的变化也无统计学差异;6.长白山黄芪水提取液对大鼠骨骼肌内α-actin表达的影响:同样长白山黄芪水提液灌胃后发现其中、大剂量组与对照组比较,大鼠骨骼肌中的α-actin的表达明显增加。
[结论]1.长白山黄芪的5种提取液(水、85%酒精、50%酒精、正丁醇、乙酸乙酯)在不同程度上均具有一定的抗疲劳作用,并且具有一定的剂量依赖关系,而且以水提液的作用最明显;2.长白山黄芪能够提高骨骼肌的收缩能力;3.长白山黄芪可以增强血液和骨骼肌中抗氧化酶活性,抑制脂质过氧化反应;4.长白山黄芪可以增加大鼠骨骼肌中α-actin的表达;5.提示,长白山黄芪可能通过加强血液和骨骼肌的抗自由基能力和提高骨骼肌内α-actin的表达起到抗疲劳作用。
[Objective] To evaluate the antifatigue effects of Changbai Mountain Radix Astragali (CMRA) extracts (aqueous extract, 85% ethanol extract, 50% ethanol extract, n-butanol extract, and ethyl acetate extract) on skeletal muscle and investigate their dose-effect relationship. To study the biochemical and molecular mechanisms of action for the extract possesing strongest activity by the method of biochemical and RT-PCR.
[Method] Five kinds of CMRA extracts (aqueous extract, 85% ethanol extract, 50% ethanol extract, n-butanol extract, and ethyl acetate extract) were prepared according to the traditional Chinese herbal extraction procedure. Kunming mouse (male, weight 20-22g) and Wistar rat (male, weight 180-200g) were adopted to the tests. Mice were used to determine the swimming time, the superoxide dismutase (SOD) activity, and the content of malondialdehyde (MDA). Wistar rats were used to measure the skeletal muscle myotility, the superoxide dismutase (SOD) activity and the content of malondialdehyde (MDA) of the skeletal muscle, and the expression ofα-actin of the skeletal muscle in rat was also measured.
[Results] 1. Effects of CMRA extracts on loading swimming time and weight of mice: All five kinds of extracts showed the prolonged loading swimming time in different extent. 2. Effects of CMRA aqueous extract on skeletal muscle myotility: (1) A significant enhancement of Pt for the single contraction of the gastrocnemius muscle and soleus muscle was found for the middle dose group and large dose group of the aqueous extract, but the incubation period and half relaxation time for the gastrocnemius muscle were prolonged, while a decurtated incubation period and half relaxation time were found for the single contraction of the soleus muscle; (2) An increased tetanus Pt and a decreased fatigue index (FI) of the the gastrocnemius muscle and soleus muscle were found for the aqueous extract. 3. Effects of CMRA 85% ethanol extract on the skeletal muscle myotility: (1) A prolongation of the decurtated incubation period and half relaxation time of the single contraction, and an increased Pt for the gastrocnemius muscle were found in the large dose group. A decurtated incubation period and half relaxation time, and an increased Pt for the soleus muscle were foud in the middle and large dose groups of the 85% ethanol extract. (2) An increased tetanus Pt and a decreased fatigue index (FI) of the the gastrocnemius muscle and soleus muscle were found for the 85% ethanol extract. 4. Effects of CMRA aqueous extract on the contents of SOD and MDA in the blood serum of mice and the skeletal muscle of rats: (1) An increased activity of SOD in the blood serum of mouse and a significant decrease of the MDA content were found in the middle and large groups of the aqueous extrat. (2) An obvious enhancement of SOD activity and a dignificant decrease of the MDA content were found in the gastrocnemius muscle and soleus muscle in rats. 5. Effects of CMRA 85% ethanol extract on the contents of SOD and MDA in blood serum of mice and skeletal muscle of rats: (1) Only the large dose group of 85% ethanol extract enhanced the SOD activity, while the other dosage groups showed no significant differences and the changes of the contents showed no significant. (2) The changes of the content of SOD and MDA in rat skeletal muscle also showed no significant. 6. Effects of CMRA aqueous extract on theα-actin 's expression_ in rat skeletal muscle: The middle dose and large dose groups showed a significant enhancement for theα-actin expression_ in rat skeletal muscle after intragastric administration of CMRA aqueous extract compaired with control group.
[Conclusion] (1) Aqueous extract, 85% ethanol extract, 50% ethanol extract, n-butanol extract and ethyl acetate extract of CMRA showed antifatigue activities to a certen exent and a dose depended propertis, among which the aqueous extrat showed the most significant activity. (2) CMRA could raise the skeletal muscle's contractibility. (3) CMRA increase the activity of SOD, and inhibit the lipid peroxidation in blood and skeletal muscle tissue. (4) CMRA enhance the expression_ ofα-actin mRNA in rat skeletal muscle. (5) It is possible that CMRA exhibit its antifatigue activity via free radical inhibiting effect in blood and skeletal muscle and via enhancing the expression ofα-actin mRNA in skeletal muscle.
[方法]本实验采用长白山黄芪,严格按照中药提取方法制备5种提取液(黄芪水、85%酒精、50%酒精、正丁醇和乙酸乙酯)。实验动物选用昆明种雄性小鼠和Wistar系雄性大鼠,小鼠用于测定负重游泳力竭时间和血清超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,大鼠用于骨骼肌收缩力学实验和骨骼肌SOD活性及MDA含量等生化指标的测定。骨骼肌组织测定α-actin的表达。
[结果]1.长白山黄芪不同提取液对小鼠负重游泳时间和体重的影响:黄芪的五种提取液均在不同程度上延长小鼠负重游泳时间;2.长白山黄芪水提取液对大鼠骨骼肌收缩力的影响:①黄芪水提取液中、大剂量组明显增强腓肠肌和比目鱼肌单收缩的最大收缩幅度(Pt),但是腓肠肌潜伏期(CT)和1/2舒张时间(HRT)延长,而比目鱼肌单收缩的CT和HRT缩短;②黄芪水提取液中、大剂量提高腓肠肌和比目鱼肌强直收缩的Pt、减小疲劳指数(FI);3.长白山黄芪85%酒精提取液对大鼠骨骼肌收缩力的影响:①黄芪85%酒精提取液中剂量组使腓肠肌单收缩的CT和HRT明显延长、Pt增强,在中、大剂量组使目鱼肌的单收缩CT和HRT缩短、Pt增强;②黄芪85%酒精提取液中、大剂量提高腓肠肌和比目鱼肌强直收缩的Pt、减小FI;4.长白山黄芪水提取液对小鼠血清和大鼠骨骼肌中SOD、MDA含量的影响:①黄芪水提取液中、大剂量组使小鼠血清中的SOD活性增强,MDA含量显著降低;②使大鼠腓肠肌和比目鱼肌中的SOD活性均明显增强、MDA含量均显著降低;5.长白山黄芪85%酒精提取液对小鼠血清和大鼠骨骼肌中SOD、MDA含量的影响:①黄芪85%酒精提取液仅大剂量组增加了小鼠血清中SOD活性,其余各剂量组无统计学差异;MDA含量变化无统计学差异;②黄芪酒精提取液对大鼠骨骼肌中SOD、MDA含量的变化也无统计学差异;6.长白山黄芪水提取液对大鼠骨骼肌内α-actin表达的影响:同样长白山黄芪水提液灌胃后发现其中、大剂量组与对照组比较,大鼠骨骼肌中的α-actin的表达明显增加。
[结论]1.长白山黄芪的5种提取液(水、85%酒精、50%酒精、正丁醇、乙酸乙酯)在不同程度上均具有一定的抗疲劳作用,并且具有一定的剂量依赖关系,而且以水提液的作用最明显;2.长白山黄芪能够提高骨骼肌的收缩能力;3.长白山黄芪可以增强血液和骨骼肌中抗氧化酶活性,抑制脂质过氧化反应;4.长白山黄芪可以增加大鼠骨骼肌中α-actin的表达;5.提示,长白山黄芪可能通过加强血液和骨骼肌的抗自由基能力和提高骨骼肌内α-actin的表达起到抗疲劳作用。
[Objective] To evaluate the antifatigue effects of Changbai Mountain Radix Astragali (CMRA) extracts (aqueous extract, 85% ethanol extract, 50% ethanol extract, n-butanol extract, and ethyl acetate extract) on skeletal muscle and investigate their dose-effect relationship. To study the biochemical and molecular mechanisms of action for the extract possesing strongest activity by the method of biochemical and RT-PCR.
[Method] Five kinds of CMRA extracts (aqueous extract, 85% ethanol extract, 50% ethanol extract, n-butanol extract, and ethyl acetate extract) were prepared according to the traditional Chinese herbal extraction procedure. Kunming mouse (male, weight 20-22g) and Wistar rat (male, weight 180-200g) were adopted to the tests. Mice were used to determine the swimming time, the superoxide dismutase (SOD) activity, and the content of malondialdehyde (MDA). Wistar rats were used to measure the skeletal muscle myotility, the superoxide dismutase (SOD) activity and the content of malondialdehyde (MDA) of the skeletal muscle, and the expression ofα-actin of the skeletal muscle in rat was also measured.
[Results] 1. Effects of CMRA extracts on loading swimming time and weight of mice: All five kinds of extracts showed the prolonged loading swimming time in different extent. 2. Effects of CMRA aqueous extract on skeletal muscle myotility: (1) A significant enhancement of Pt for the single contraction of the gastrocnemius muscle and soleus muscle was found for the middle dose group and large dose group of the aqueous extract, but the incubation period and half relaxation time for the gastrocnemius muscle were prolonged, while a decurtated incubation period and half relaxation time were found for the single contraction of the soleus muscle; (2) An increased tetanus Pt and a decreased fatigue index (FI) of the the gastrocnemius muscle and soleus muscle were found for the aqueous extract. 3. Effects of CMRA 85% ethanol extract on the skeletal muscle myotility: (1) A prolongation of the decurtated incubation period and half relaxation time of the single contraction, and an increased Pt for the gastrocnemius muscle were found in the large dose group. A decurtated incubation period and half relaxation time, and an increased Pt for the soleus muscle were foud in the middle and large dose groups of the 85% ethanol extract. (2) An increased tetanus Pt and a decreased fatigue index (FI) of the the gastrocnemius muscle and soleus muscle were found for the 85% ethanol extract. 4. Effects of CMRA aqueous extract on the contents of SOD and MDA in the blood serum of mice and the skeletal muscle of rats: (1) An increased activity of SOD in the blood serum of mouse and a significant decrease of the MDA content were found in the middle and large groups of the aqueous extrat. (2) An obvious enhancement of SOD activity and a dignificant decrease of the MDA content were found in the gastrocnemius muscle and soleus muscle in rats. 5. Effects of CMRA 85% ethanol extract on the contents of SOD and MDA in blood serum of mice and skeletal muscle of rats: (1) Only the large dose group of 85% ethanol extract enhanced the SOD activity, while the other dosage groups showed no significant differences and the changes of the contents showed no significant. (2) The changes of the content of SOD and MDA in rat skeletal muscle also showed no significant. 6. Effects of CMRA aqueous extract on theα-actin 's expression_ in rat skeletal muscle: The middle dose and large dose groups showed a significant enhancement for theα-actin expression_ in rat skeletal muscle after intragastric administration of CMRA aqueous extract compaired with control group.
[Conclusion] (1) Aqueous extract, 85% ethanol extract, 50% ethanol extract, n-butanol extract and ethyl acetate extract of CMRA showed antifatigue activities to a certen exent and a dose depended propertis, among which the aqueous extrat showed the most significant activity. (2) CMRA could raise the skeletal muscle's contractibility. (3) CMRA increase the activity of SOD, and inhibit the lipid peroxidation in blood and skeletal muscle tissue. (4) CMRA enhance the expression_ ofα-actin mRNA in rat skeletal muscle. (5) It is possible that CMRA exhibit its antifatigue activity via free radical inhibiting effect in blood and skeletal muscle and via enhancing the expression ofα-actin mRNA in skeletal muscle.
引文
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