基于bFGF/ERK信号通路探讨电针对骨骼肌损伤修复的影响
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摘要
目的：基于bFGF/ERK信号通路探讨电针对骨骼肌损伤后肌肉修复再生的作用与机制方法：
第一部分：电针阿是穴对大鼠腓肠肌损伤早期肌肉修复再生的影响。通过积累性钝挫伤结合动物跑台离心运动的方法建立SD大鼠腓肠肌损伤模型,观察电针阿是穴对大鼠腓肠肌损伤后1d、4d、7d的组织形态学改变以及PCNA、Desmin、bFGF表达的影响,探讨电针阿是穴对大鼠腓肠肌损伤后肌卫星细胞增殖分化的作用与机制；
第二部分：电针委中穴与阿是穴对兔急性腰肌钝挫伤修复期肌肉修复再生及bFGF/ERK信号通路的影响。通过钝挫伤的方法建立新西兰大耳兔急性腰肌损伤模型,观察电针委中穴与阿是穴对损伤后1d、4d、7d、14d兔血清CK活性的动态调节,结合损伤后14d兔腰肌肌细胞数量、肌纤维平均横截面积、每肌纤维细胞核数、Ⅰ级肌束膜厚度、组织形态改变等组织学指标的变化,分析比较电针委中穴与阿是穴的作用差异,并且通过观察损伤后14d兔腰肌PCNA、Desmin、ERK1/2的表达变化,结合血清、腰肌以及脊髓中bFGF含量的改变,探讨电针委中穴与阿是穴对兔急性腰肌钝挫伤肌肉修复再生的作用机制；
第三部分：电针阿是穴对应用U0126抑制剂的兔急性腰肌钝挫伤修复期肌肉修复再生及bFGF/ERK信号通路的影响。通过钝挫伤的方法建立新西兰大耳兔急性腰肌损伤模型,应用ERK抑制剂U0126观察电针阿是穴对损伤后1d、14d兔血清CK活性的变化,以及兔腰肌肌细胞数量、肌纤维平均横截面积、每肌纤维细胞核数、Ⅰ级肌束膜厚度、组织形态改变等组织学指标的变化,结合兔腰肌PCNA、Desmin、ERK1/2与bFGF的表达变化,进一步探讨bFGF/ERK信号通路对电针阿是穴促兔腰肌钝挫伤后肌肉修复再生的介导作用。结果：
1.电针阿是穴对大鼠腓肠肌损伤早期肌肉修复再生的影响：组织形态学结果显示,损伤后4d电针组腓肠肌炎症细胞浸润有所减轻,肌细胞再生较丰富；损伤后7d电针组结缔组织增生程度低于模型组,且组织形态学损伤评分明显低于模型组(P<0.01)；电针组腓肠肌Desmin与bFGF在损伤后1d均高于模型组(P<0.01,P<0.05),在损伤后4d腓肠肌PCNA、Desmin与bFGF均高于模型组(P<0.05,P<0.01,P<0.01)。
2.电针委中穴与阿是穴对兔急性腰肌钝挫伤修复期肌肉修复再生及bFGF/ERK信号通路的影响：从组织病理切片观察,损伤后14d,模型组肌纤维排列不齐,结缔组织增生明显,再生肌细胞较少,电针委中组肌细胞体积较大,可见肌细胞再生并逐渐融合,结缔组织增生程度较轻,而电针阿是穴组可见较多新生的肌细胞,结缔组织增生程度同样较轻；模型组肌细胞数量MM、肌纤维平均横截面积CSA、每肌纤维细胞核数NPF均明显低于空白组(P<0.05,P<0.01,P<0.01),组织形态学损伤评分以及Ⅰ级肌束膜厚度均明显高于空白组(P<0.01),其中肌纤维CSA、NPF均低于电针委中组(P<0.05,P<0.05)与电针阿是穴组(P<0.05,P<0.05),组织形态学损伤评分以及Ⅰ级肌束膜厚度均明显高于电针委中组(P<0.01,P<0.05)与电针阿是穴组(P<0.01,P<0.05),而电针委中组组织形态学损伤评分低于电针阿是穴组(P<0.05);
损伤后1d,模型组、电针委中组与电针阿是穴组血清CK含量均明显高于空白组(P<0.01),在损伤后4d电针委中组与电针阿是穴组均高于模型组(P<0.01,P<0.05),在损伤后7d模型组再次回升明显高于空白组(P<0.01),电针委中组明显低于模型组(P<0.01)与电针阿是穴组(P<0.05),电针阿是穴组低于模型组(P<0.05),在损伤后14d模型组仍明显高于空白组(P<0.05),而电针委中组与电针阿是穴组均低于模型组(P<0.01,P<0.01);
损伤后14d,模型组兔腰肌PCNA、ERK1/2以及bFGF的表达均高于空白组(P<0.01,P<0.05,P<0.01),其PCNA、Desmin、ERK1/2以及bFGF的表达均低于电针委中组(P<0.05,P<0.05,P<0.05,P<0.01)与电针阿是穴组(P<0.01,P<0.05,P<0.05,P<0.01)；电针委中组兔腰肌PCNA与bFGF的表达低于电针阿是穴组(P<0.05,P<0.01)；模型组兔腰段脊髓bFGF含量高于空白组(P<0.05),并且低于电针委中组(P<0.01);
损伤后4d,模型组血清bFGF均高于空白组(P<0.01)、电针委中组(P<0.05)与阿是穴组(P<0.05)；损伤后7d,各组均无统计学差异；损伤后14d,模型组低于空白组(P<0.01),而电针阿是穴组低于电针委中组(P<0.05)。
3.电针阿是穴对应用U0126抑制剂的兔急性腰肌钝挫伤修复期肌肉修复再生及bFGF/ERK信号通路的影响：从组织病理切片观察,损伤后14d,模型组肌纤维排列不齐,结缔组织增生明显,再生肌细胞较少。模型抑制组肌细胞缺失明显,肌细胞体积明显减小,仅有少量结缔组织增生。电针组肌纤维排列较整齐,可见较多新生的肌细胞,轻度结缔组织增生。电针抑制组有少量肌细胞再生,较严重的结缔组织增生；模型组组织形态学损伤评分明显高于空白组(P<0.01,P<0.01),低于模型抑制组(P<0.01),电针组均明显低于模型组(P<0.01)与电针抑制组(P<0.01)；模型组肌细胞数量MM、肌纤维平均横截面积CSA、每肌纤维细胞核数NPF明显低于空白组(P<0.05,P<0.01,P<0.01),高于模型抑制组(P>0.05,P<0.01,P<0.05),电针组高于模型组(P>0.05,P<0.05,P<0.05)与电针抑制剂组(P>0.05,P<0.05,P<0.01)；模型组Ⅰ级肌束膜厚度均明显高于空白组(P<0.01)与模型抑制剂组(P<0.05),电针组低于模型组(P<0.05),与电针抑制剂组无显著差异(P>0.05)；模型组血清CK含量高于空白组(P<0.01),与模型抑制剂组无显著差异(P>0.05),电针组低于模型组(P<0.05)与电针抑制剂组(P<0.05)。
损伤后14d,模型组兔腰肌PCNA、ERK1/2以及bFGF的表达均高于空白组(P<0.01,P<0.01,P<0.05),模型抑制剂组PCNA、Desmin、ERK1/2的表达均低于模型组(P<0.01,P<0.01,P<0.01),电针组PCNA、Desmin、ERK1/2以及bFGF的表达均高于模型组(P<0.01,P<0.05,P<0.05,P<0.01)；电针抑制剂组PCNA、Desmin、 ERK1/2的表达均低于电针组(P<0.05,P<0.01,P<0.01)。结论：
1.电针阿是穴能促进大鼠腓肠肌损伤早期肌卫星细胞的增殖与分化,并且可能与其上调bFGF的表达有关；
2.电针委中穴与阿是穴均能够促进兔腰肌急性钝挫伤修复期肌卫星细胞的增殖、分化以及组织再生修复的成熟度,其机制可能与调节局部组织、血清以及脊髓中bFGF的含量变化有关,ERK1/2信号通路可能是介导电针发挥作用的主要通路之一；
3.电针委中穴对兔腰肌损伤的促修复再生作用有优于电针阿是穴的趋势,两者的作用途径可能存在差异：电针委中穴可能主要通过神经-内分泌的整体调节发挥作用,因此能更明显的促进脊髓bFGF的释放,两者的明确机制有待于进一步研究,而电针阿是穴可能主要通过局部机械牵拉刺激发挥作用,因此对局部组织中bFGF的促进调节更明显；
4.应用ERK抑制剂U0126可部分阻断电针阿是穴对兔腰肌损伤后肌卫星细胞增殖分化以及肌肉的再生成熟度的促进作用,bFGF/ERK信号通路可能是电针阿是穴促肌肉修复再生的主要途径之一。
Objective:To observe the effects and discuss the mechanism of electro-acupuncture's function of repairing skeletal muscle injury based on bFGF/ERK signal passway. Methods:
Part Ⅰ:The observation of a-shi point electro-acupuncture's early recovery and regeneration effects on rats'gastrocnemius muscle damage and bFGF expression. Rat model of gastrocnemius muscle damage was set up by cumulative contusion combined with treadmill exercise. The morphological changes and PCNA, Desmin, bFGF expressions of the gastroncnemius muscle were observed at the first,4th, and7th day after the application of electro-acupuncture on a-shi point for the evaluation and mechanism study of a-shi point electro-acupuncture's effects on the proliferation and differentiation rate of rats'muscular satellite cells after gastrocnemius muscle injury.
Part Ⅱ:Effects ot weizhong (BL40) and a-shi point electro-acupuncture on rabbits'acute lumbar muscle contusion and bFGF/ERK pass-way. Experiment in part Ⅱ applied dynamic observation of weizhong (BL40) and a-shi point electro-acupuncture's regulatory effects on rabbits' serum CK activity at the first,4th,7th, and14th day. Taking rabbits' average lumbar muscle cell number, cross section area of the muscle fiber, nuclei number per fiber, thickness of grade I perimysium, and histomorphological changes at the14th day into consideration, the study compared and analyzed the differences between electro-acupuncture on weizhong (BL40) and a-shi point. It also discussed the mechanism of the regenerative effects of weizhong (BL40) and a-shi point electro-acupuncture on rabbit's acute lumbar muscle contusion by observing changes of lumbar muscle PCNA, Desmin, ERK1/2, and bFGF in blood serum and spinal cord at the14th day.
Part Ⅲ:A-shi point electro-acupuncture's effects on muscle recovery and regeneration of acute lumbar muscle contusion rabbits and bFGF/ERK pass-way of rabbits administrated with U0126. The study applied dynamic observation of weizhong (BL40) and a-shi point electro-acupuncture's regulatory effects on rabbits' serum CK activity at the1th,14th day, after ERK inhibitor, U0126, had been administrated. It also combined rabbits'average lumbar muscle cell number, cross section area of the muscle fiber, nuclei number per fiber, thickness of grade I perimysium, and histomorphological changes with expression changes of PCNA, Desmin, ERK1/2, and bFGF in rabbits'lumbar muscle for the further discovery of bFGF/ERK pass-way's mediation of a-shi point electro-acupuncture's regenerative effects on rabbits'lumbar muscle contusion.
Results:
1. A-shi point electro-acupuncture's early regenerative effects on rats' gastrocnemius muscle injury. Results of histomorphological test showed that, at the4th day after injury, rats in the electro-acupuncture group were of relieved inflammatory cell infiltration of the gastrocnemius muscle and comparatively massive muscular cell regeneration. At the7th day, rats in the electro-acupuncture group were of lower connective tissue proliferation rate and histomorphological score, compared with that of the model group (p<0.01). Rats in the electro-acupuncture group were of significantly higher gastrocnemius Desmin and bFGF one day after the injury, compared with that of the model group (p<0.01, p<0.05). At the4th day, gastrocnemius PCNA, Desmin, and bFGF of the electro-acupuncture group were remarkably higher than that of the model group (p<0.05, p<0.01, p<0.01).
2. Effects of Weizhong (BL40) and a-shi point electro-acupuncture on acute lumbar muscle contusion rabbits'muscle regeneration and bFGF/ERK pass-way in recovery phase. Histopathological sections showed that,14days after the injury, rabbits in the model group were of disarranged muscle fiber, remarkably increased proliferation of connective tissue, and decreased muscular cell regeneration. Rats in we thong (BL40) electro-acupuncture group were of comparatively bigger muscular cells, low proliferation of connective tissue, with regeneration and fusion of muscular cells observed. In a-shi point electro-acupuncture group, more regenerated muscular cells can be observed, with low proliferation of connective tissue. Rabbits in the model group were of drastically decreased muscular cell number, average cross sectional area of muscle fiber (CSA), nuclei number per fiber(NPF)(p<0.05, p<0.01, p<0.01), and significantly higher histomorphological score and thickness of grade I perimysium (p<0.01, p<0.05), compared with that of the blank control group. Among the indices, CSA and NPF were also significantly lower than that in weizhong (BL40) electro-acupuncture group (p<0.05, p<0.05) and a-shi point electro-acupuncture group (p<0.05, p<0.05). The histomorphological score and thickness of grade I perimysium were remarkably higher than that in the weizhong (BL40) electro-acupuncture group (p<0.01, p<0.05) and a-shi point electro-acupuncture group (p<0.01, p<0.05). Histomorphological score of weizhong (BL40) electro-acupuncture group is remarkably lower than that of a-shi point electro-acupuncture group (p<0.05). One day after the injury, serum CK concentrations of the model group, weizhong (BL40) electro-acupuncture group, and a-shi point electro-acupuncture group were significantly higher than that of the blank control group (p<0.01). The index of the model group was significantly lower than that of the weizhong (BL40) electro-acupuncture group and a-shi point electro-acupuncture group (p<0.01, p<0.05) at the4th day after the injury, and rose again to a level remarkably higher than the blank control group (p<0.01) at the7th day. While serum CK contents of weizhong (BL40) electro-acupuncture group was significantly lower than that of the model group (p<0.01) and a-shi point electro-acupuncture group (p<0.05), with that of the a-shi point electro-acupuncture group greatly lower than the model group (p<0.05). At the14th day after the injury, serum CK of the model group was significantly higher than that of the blank control group (p<0.05), while that of the weizhong (BL40) electro-acupuncture group and a-shi point electro-acupuncture group reduced to a level remarkably lower than the model group (p<0.01, p<0.01). At the14th day after the injury, lumbar muscle PCNA, ERK1/2, and bFGF expressions of rabbits in the model group were significantly higher than that in the blank control group (p<0.01, p<0.05, p<0.01). And its PCNA, Desmin, ERK1/2, and bFGF expressions were remarkably lower than that of weizhong (BL40) electro-acupuncture group (p<0.05, p<0.05, p<0.05, p<0.01) and a-shi point electro-acupuncture group (p<0.01, p<0.05, p<0.05, p<0.01). Lumbar muscle PCNA and bFGF expressions of weizhong (BL40) electro-acupuncture group were remarkably lower than a-shi point electro-acupuncture group (p<0.05, p<0.01). And bFGF in lumbar spinal cord of rabbits in the model group was greatly higher than that of the blank control group (p<0.01), but significantly lower than weizhong (BL40) electro-acupuncture group (p<0.01). At the4th day after the injury, serum bFGF of the model group was remarkably higher than that of the blank control group (p<0.01), weizhong (BL40) electro-acupuncture group (p<0.05), and a-shi point electro-acupuncture group (p<0.05). But there's no significant difference of the index between all the groups at the7th day. At the14th day, serum bFGF of the model group was significantly lower than that of the blank control group. The same index of a-shi point electro-acupuncture group was greatly lower than that of the model group (p<0.01) and weizhong (BL40) electro-acupuncture group.
3. Effects of a-shi point electro-acupuncture on U0126-intervened acute lumbar muscle contusion rabbits'muscle regeneration and bFGF/ERK signal pass-way. At the14th day after the injury, histopathological sections of the model group showed disarranged muscle fiber, obviously proliferated connective tissue, and less regenerated muscle cells. U0126-intervened model group were of obviously decreased and shrunk muscular cells, and less regenerated connective tissue. Rabbits in the electro-acupuncture group were of orderly arranged muscle fiber, more regenerated muscular cells, and slightly proliferation of connective tissue. Rabbits of the U0126-intervened electro-acupuncture group had a small amount of connective tissue proliferation and comparatively severe proliferation of connective tissue. The histomorphological score of the model group was significantly higher than that of the blank control group (p<0.01, p<0.01), and greatly lower than that of the U0126-intervened model group (p<0.01). The score of electro-acupuncture group was remarkably lower than that of the model group (p<0.01) and U0126-intervened electro-acupuncture group (p<0.01). The number of muscular cells, CSA, and NPF of the model group were greatly lower than that of the blank control group (p<0.05, p<0.01, p<0.01), but higher than U0126-intervened model group (p>0.05, p<0.01, p<0.05). The indices of electro-acupuncture groups were higher than that of the model group (p>0.05, p<0.05, p<0.05) and U0126-intervened electro-acupuncture group (p>0.05, p<0.05, p<0.01). Thickness of grade I perimysium of the model group was significantly higher than that of the blank control group (p<0.01) and U0126-intervened model group (p<0.05). The same index of the electro-acupuncture group was lower than that of the model group (p<0.05), but had no significant difference with U0126-intervened electro-acupuncture group (p>0.05). Serum CK content of the model group was remarkably higher than that of the blank control group (p<0.01), and had no significant difference with U0126-intervened model group (p>0.05). The index of the electro-acupuncture group was greatly lower than that of the model group (p<0.05) and U0126-intervened electro-acupuncture group (p<0.05).14days after the injury, lumbar muscle PCNA, ERK1/2, and bFGF of rabbits in the model group were significantly higher than that of the blank control group (p<0.01, p<0.01, p<0.05). Expressions of PCNA, Desmin, and ERK1/2of rabbits in the U0126-intervened model group was remarkably lower than that of the model group (p<0.01, p<0.01, p<0.01). PCNA, Desmin, ERK1/2, and bFGF expressions of rabbits in the electro-acupuncture group were greatly higher than the model group (p<0.01, p<0.05, p<0.05, p<0.01). PCNA, Desmin, and ERK1/2expressions of rabbits in U0126-intervened electro-acupuncture group were lower than that of the electro-acupuncture group (p<0.05, p<0.01, p<0.01).
Conclusion:
1.A-shi point electro-acupuncture can boost the proliferation and differentiation of satellite cells of gastrocnemius muscle damaged rats. The mechanism may lies in the up-regulation of bFGF expression.
2. Electro-acupuncture on weizhong (BL40) point and a-shi can both boost proliferation and differentiation of satellite cells, and regenerative repair of the tissue in recovery stage of acute lumbar muscle contusion rabbits. The mechanism may concerns regulation of bFGF contents in local tissue, blood serum, and spinal cord. ERK1/2pass-way may be one of the important pass-ways mediating curative the effects of electro-acupuncture.
3. Effect on weizhong (BL40) of promoting repairmen is batter than a-shi point, and there may be differences between the action pass-ways of a-shi point electro-acupuncture and weizhong (BL40) electro-acupuncture. Weizhong (BL40) electro-acupuncture works mainly by general regulation of the nerve-endocrine network. It is thereby of morc obvious effect on bFGF release in the spinal cord. The details of the two mechanisms are to be further studied. On the contrary, A-shi point electro-acupuncture works mainly by pulling stimulation, which had more obviously regulatory effects on bFGF in local tissue.
4. The application of ERK inhibitor, U0126, can partly block a-shi point electro-acupuncture's effects of stimulating proliferation and differentiation of satellite cells and regenerating muscles on acute lumbar muscle contusion rabbits. bFGF/ERK signal pass-way may be one of the important ways for muscle recovery and regeneration of a-shi point electro-acupuncture.
第一部分：电针阿是穴对大鼠腓肠肌损伤早期肌肉修复再生的影响。通过积累性钝挫伤结合动物跑台离心运动的方法建立SD大鼠腓肠肌损伤模型,观察电针阿是穴对大鼠腓肠肌损伤后1d、4d、7d的组织形态学改变以及PCNA、Desmin、bFGF表达的影响,探讨电针阿是穴对大鼠腓肠肌损伤后肌卫星细胞增殖分化的作用与机制；
第二部分：电针委中穴与阿是穴对兔急性腰肌钝挫伤修复期肌肉修复再生及bFGF/ERK信号通路的影响。通过钝挫伤的方法建立新西兰大耳兔急性腰肌损伤模型,观察电针委中穴与阿是穴对损伤后1d、4d、7d、14d兔血清CK活性的动态调节,结合损伤后14d兔腰肌肌细胞数量、肌纤维平均横截面积、每肌纤维细胞核数、Ⅰ级肌束膜厚度、组织形态改变等组织学指标的变化,分析比较电针委中穴与阿是穴的作用差异,并且通过观察损伤后14d兔腰肌PCNA、Desmin、ERK1/2的表达变化,结合血清、腰肌以及脊髓中bFGF含量的改变,探讨电针委中穴与阿是穴对兔急性腰肌钝挫伤肌肉修复再生的作用机制；
第三部分：电针阿是穴对应用U0126抑制剂的兔急性腰肌钝挫伤修复期肌肉修复再生及bFGF/ERK信号通路的影响。通过钝挫伤的方法建立新西兰大耳兔急性腰肌损伤模型,应用ERK抑制剂U0126观察电针阿是穴对损伤后1d、14d兔血清CK活性的变化,以及兔腰肌肌细胞数量、肌纤维平均横截面积、每肌纤维细胞核数、Ⅰ级肌束膜厚度、组织形态改变等组织学指标的变化,结合兔腰肌PCNA、Desmin、ERK1/2与bFGF的表达变化,进一步探讨bFGF/ERK信号通路对电针阿是穴促兔腰肌钝挫伤后肌肉修复再生的介导作用。结果：
1.电针阿是穴对大鼠腓肠肌损伤早期肌肉修复再生的影响：组织形态学结果显示,损伤后4d电针组腓肠肌炎症细胞浸润有所减轻,肌细胞再生较丰富；损伤后7d电针组结缔组织增生程度低于模型组,且组织形态学损伤评分明显低于模型组(P<0.01)；电针组腓肠肌Desmin与bFGF在损伤后1d均高于模型组(P<0.01,P<0.05),在损伤后4d腓肠肌PCNA、Desmin与bFGF均高于模型组(P<0.05,P<0.01,P<0.01)。
2.电针委中穴与阿是穴对兔急性腰肌钝挫伤修复期肌肉修复再生及bFGF/ERK信号通路的影响：从组织病理切片观察,损伤后14d,模型组肌纤维排列不齐,结缔组织增生明显,再生肌细胞较少,电针委中组肌细胞体积较大,可见肌细胞再生并逐渐融合,结缔组织增生程度较轻,而电针阿是穴组可见较多新生的肌细胞,结缔组织增生程度同样较轻；模型组肌细胞数量MM、肌纤维平均横截面积CSA、每肌纤维细胞核数NPF均明显低于空白组(P<0.05,P<0.01,P<0.01),组织形态学损伤评分以及Ⅰ级肌束膜厚度均明显高于空白组(P<0.01),其中肌纤维CSA、NPF均低于电针委中组(P<0.05,P<0.05)与电针阿是穴组(P<0.05,P<0.05),组织形态学损伤评分以及Ⅰ级肌束膜厚度均明显高于电针委中组(P<0.01,P<0.05)与电针阿是穴组(P<0.01,P<0.05),而电针委中组组织形态学损伤评分低于电针阿是穴组(P<0.05);
损伤后1d,模型组、电针委中组与电针阿是穴组血清CK含量均明显高于空白组(P<0.01),在损伤后4d电针委中组与电针阿是穴组均高于模型组(P<0.01,P<0.05),在损伤后7d模型组再次回升明显高于空白组(P<0.01),电针委中组明显低于模型组(P<0.01)与电针阿是穴组(P<0.05),电针阿是穴组低于模型组(P<0.05),在损伤后14d模型组仍明显高于空白组(P<0.05),而电针委中组与电针阿是穴组均低于模型组(P<0.01,P<0.01);
损伤后14d,模型组兔腰肌PCNA、ERK1/2以及bFGF的表达均高于空白组(P<0.01,P<0.05,P<0.01),其PCNA、Desmin、ERK1/2以及bFGF的表达均低于电针委中组(P<0.05,P<0.05,P<0.05,P<0.01)与电针阿是穴组(P<0.01,P<0.05,P<0.05,P<0.01)；电针委中组兔腰肌PCNA与bFGF的表达低于电针阿是穴组(P<0.05,P<0.01)；模型组兔腰段脊髓bFGF含量高于空白组(P<0.05),并且低于电针委中组(P<0.01);
损伤后4d,模型组血清bFGF均高于空白组(P<0.01)、电针委中组(P<0.05)与阿是穴组(P<0.05)；损伤后7d,各组均无统计学差异；损伤后14d,模型组低于空白组(P<0.01),而电针阿是穴组低于电针委中组(P<0.05)。
3.电针阿是穴对应用U0126抑制剂的兔急性腰肌钝挫伤修复期肌肉修复再生及bFGF/ERK信号通路的影响：从组织病理切片观察,损伤后14d,模型组肌纤维排列不齐,结缔组织增生明显,再生肌细胞较少。模型抑制组肌细胞缺失明显,肌细胞体积明显减小,仅有少量结缔组织增生。电针组肌纤维排列较整齐,可见较多新生的肌细胞,轻度结缔组织增生。电针抑制组有少量肌细胞再生,较严重的结缔组织增生；模型组组织形态学损伤评分明显高于空白组(P<0.01,P<0.01),低于模型抑制组(P<0.01),电针组均明显低于模型组(P<0.01)与电针抑制组(P<0.01)；模型组肌细胞数量MM、肌纤维平均横截面积CSA、每肌纤维细胞核数NPF明显低于空白组(P<0.05,P<0.01,P<0.01),高于模型抑制组(P>0.05,P<0.01,P<0.05),电针组高于模型组(P>0.05,P<0.05,P<0.05)与电针抑制剂组(P>0.05,P<0.05,P<0.01)；模型组Ⅰ级肌束膜厚度均明显高于空白组(P<0.01)与模型抑制剂组(P<0.05),电针组低于模型组(P<0.05),与电针抑制剂组无显著差异(P>0.05)；模型组血清CK含量高于空白组(P<0.01),与模型抑制剂组无显著差异(P>0.05),电针组低于模型组(P<0.05)与电针抑制剂组(P<0.05)。
损伤后14d,模型组兔腰肌PCNA、ERK1/2以及bFGF的表达均高于空白组(P<0.01,P<0.01,P<0.05),模型抑制剂组PCNA、Desmin、ERK1/2的表达均低于模型组(P<0.01,P<0.01,P<0.01),电针组PCNA、Desmin、ERK1/2以及bFGF的表达均高于模型组(P<0.01,P<0.05,P<0.05,P<0.01)；电针抑制剂组PCNA、Desmin、 ERK1/2的表达均低于电针组(P<0.05,P<0.01,P<0.01)。结论：
1.电针阿是穴能促进大鼠腓肠肌损伤早期肌卫星细胞的增殖与分化,并且可能与其上调bFGF的表达有关；
2.电针委中穴与阿是穴均能够促进兔腰肌急性钝挫伤修复期肌卫星细胞的增殖、分化以及组织再生修复的成熟度,其机制可能与调节局部组织、血清以及脊髓中bFGF的含量变化有关,ERK1/2信号通路可能是介导电针发挥作用的主要通路之一；
3.电针委中穴对兔腰肌损伤的促修复再生作用有优于电针阿是穴的趋势,两者的作用途径可能存在差异：电针委中穴可能主要通过神经-内分泌的整体调节发挥作用,因此能更明显的促进脊髓bFGF的释放,两者的明确机制有待于进一步研究,而电针阿是穴可能主要通过局部机械牵拉刺激发挥作用,因此对局部组织中bFGF的促进调节更明显；
4.应用ERK抑制剂U0126可部分阻断电针阿是穴对兔腰肌损伤后肌卫星细胞增殖分化以及肌肉的再生成熟度的促进作用,bFGF/ERK信号通路可能是电针阿是穴促肌肉修复再生的主要途径之一。
Objective:To observe the effects and discuss the mechanism of electro-acupuncture's function of repairing skeletal muscle injury based on bFGF/ERK signal passway. Methods:
Part Ⅰ:The observation of a-shi point electro-acupuncture's early recovery and regeneration effects on rats'gastrocnemius muscle damage and bFGF expression. Rat model of gastrocnemius muscle damage was set up by cumulative contusion combined with treadmill exercise. The morphological changes and PCNA, Desmin, bFGF expressions of the gastroncnemius muscle were observed at the first,4th, and7th day after the application of electro-acupuncture on a-shi point for the evaluation and mechanism study of a-shi point electro-acupuncture's effects on the proliferation and differentiation rate of rats'muscular satellite cells after gastrocnemius muscle injury.
Part Ⅱ:Effects ot weizhong (BL40) and a-shi point electro-acupuncture on rabbits'acute lumbar muscle contusion and bFGF/ERK pass-way. Experiment in part Ⅱ applied dynamic observation of weizhong (BL40) and a-shi point electro-acupuncture's regulatory effects on rabbits' serum CK activity at the first,4th,7th, and14th day. Taking rabbits' average lumbar muscle cell number, cross section area of the muscle fiber, nuclei number per fiber, thickness of grade I perimysium, and histomorphological changes at the14th day into consideration, the study compared and analyzed the differences between electro-acupuncture on weizhong (BL40) and a-shi point. It also discussed the mechanism of the regenerative effects of weizhong (BL40) and a-shi point electro-acupuncture on rabbit's acute lumbar muscle contusion by observing changes of lumbar muscle PCNA, Desmin, ERK1/2, and bFGF in blood serum and spinal cord at the14th day.
Part Ⅲ:A-shi point electro-acupuncture's effects on muscle recovery and regeneration of acute lumbar muscle contusion rabbits and bFGF/ERK pass-way of rabbits administrated with U0126. The study applied dynamic observation of weizhong (BL40) and a-shi point electro-acupuncture's regulatory effects on rabbits' serum CK activity at the1th,14th day, after ERK inhibitor, U0126, had been administrated. It also combined rabbits'average lumbar muscle cell number, cross section area of the muscle fiber, nuclei number per fiber, thickness of grade I perimysium, and histomorphological changes with expression changes of PCNA, Desmin, ERK1/2, and bFGF in rabbits'lumbar muscle for the further discovery of bFGF/ERK pass-way's mediation of a-shi point electro-acupuncture's regenerative effects on rabbits'lumbar muscle contusion.
Results:
1. A-shi point electro-acupuncture's early regenerative effects on rats' gastrocnemius muscle injury. Results of histomorphological test showed that, at the4th day after injury, rats in the electro-acupuncture group were of relieved inflammatory cell infiltration of the gastrocnemius muscle and comparatively massive muscular cell regeneration. At the7th day, rats in the electro-acupuncture group were of lower connective tissue proliferation rate and histomorphological score, compared with that of the model group (p<0.01). Rats in the electro-acupuncture group were of significantly higher gastrocnemius Desmin and bFGF one day after the injury, compared with that of the model group (p<0.01, p<0.05). At the4th day, gastrocnemius PCNA, Desmin, and bFGF of the electro-acupuncture group were remarkably higher than that of the model group (p<0.05, p<0.01, p<0.01).
2. Effects of Weizhong (BL40) and a-shi point electro-acupuncture on acute lumbar muscle contusion rabbits'muscle regeneration and bFGF/ERK pass-way in recovery phase. Histopathological sections showed that,14days after the injury, rabbits in the model group were of disarranged muscle fiber, remarkably increased proliferation of connective tissue, and decreased muscular cell regeneration. Rats in we thong (BL40) electro-acupuncture group were of comparatively bigger muscular cells, low proliferation of connective tissue, with regeneration and fusion of muscular cells observed. In a-shi point electro-acupuncture group, more regenerated muscular cells can be observed, with low proliferation of connective tissue. Rabbits in the model group were of drastically decreased muscular cell number, average cross sectional area of muscle fiber (CSA), nuclei number per fiber(NPF)(p<0.05, p<0.01, p<0.01), and significantly higher histomorphological score and thickness of grade I perimysium (p<0.01, p<0.05), compared with that of the blank control group. Among the indices, CSA and NPF were also significantly lower than that in weizhong (BL40) electro-acupuncture group (p<0.05, p<0.05) and a-shi point electro-acupuncture group (p<0.05, p<0.05). The histomorphological score and thickness of grade I perimysium were remarkably higher than that in the weizhong (BL40) electro-acupuncture group (p<0.01, p<0.05) and a-shi point electro-acupuncture group (p<0.01, p<0.05). Histomorphological score of weizhong (BL40) electro-acupuncture group is remarkably lower than that of a-shi point electro-acupuncture group (p<0.05). One day after the injury, serum CK concentrations of the model group, weizhong (BL40) electro-acupuncture group, and a-shi point electro-acupuncture group were significantly higher than that of the blank control group (p<0.01). The index of the model group was significantly lower than that of the weizhong (BL40) electro-acupuncture group and a-shi point electro-acupuncture group (p<0.01, p<0.05) at the4th day after the injury, and rose again to a level remarkably higher than the blank control group (p<0.01) at the7th day. While serum CK contents of weizhong (BL40) electro-acupuncture group was significantly lower than that of the model group (p<0.01) and a-shi point electro-acupuncture group (p<0.05), with that of the a-shi point electro-acupuncture group greatly lower than the model group (p<0.05). At the14th day after the injury, serum CK of the model group was significantly higher than that of the blank control group (p<0.05), while that of the weizhong (BL40) electro-acupuncture group and a-shi point electro-acupuncture group reduced to a level remarkably lower than the model group (p<0.01, p<0.01). At the14th day after the injury, lumbar muscle PCNA, ERK1/2, and bFGF expressions of rabbits in the model group were significantly higher than that in the blank control group (p<0.01, p<0.05, p<0.01). And its PCNA, Desmin, ERK1/2, and bFGF expressions were remarkably lower than that of weizhong (BL40) electro-acupuncture group (p<0.05, p<0.05, p<0.05, p<0.01) and a-shi point electro-acupuncture group (p<0.01, p<0.05, p<0.05, p<0.01). Lumbar muscle PCNA and bFGF expressions of weizhong (BL40) electro-acupuncture group were remarkably lower than a-shi point electro-acupuncture group (p<0.05, p<0.01). And bFGF in lumbar spinal cord of rabbits in the model group was greatly higher than that of the blank control group (p<0.01), but significantly lower than weizhong (BL40) electro-acupuncture group (p<0.01). At the4th day after the injury, serum bFGF of the model group was remarkably higher than that of the blank control group (p<0.01), weizhong (BL40) electro-acupuncture group (p<0.05), and a-shi point electro-acupuncture group (p<0.05). But there's no significant difference of the index between all the groups at the7th day. At the14th day, serum bFGF of the model group was significantly lower than that of the blank control group. The same index of a-shi point electro-acupuncture group was greatly lower than that of the model group (p<0.01) and weizhong (BL40) electro-acupuncture group.
3. Effects of a-shi point electro-acupuncture on U0126-intervened acute lumbar muscle contusion rabbits'muscle regeneration and bFGF/ERK signal pass-way. At the14th day after the injury, histopathological sections of the model group showed disarranged muscle fiber, obviously proliferated connective tissue, and less regenerated muscle cells. U0126-intervened model group were of obviously decreased and shrunk muscular cells, and less regenerated connective tissue. Rabbits in the electro-acupuncture group were of orderly arranged muscle fiber, more regenerated muscular cells, and slightly proliferation of connective tissue. Rabbits of the U0126-intervened electro-acupuncture group had a small amount of connective tissue proliferation and comparatively severe proliferation of connective tissue. The histomorphological score of the model group was significantly higher than that of the blank control group (p<0.01, p<0.01), and greatly lower than that of the U0126-intervened model group (p<0.01). The score of electro-acupuncture group was remarkably lower than that of the model group (p<0.01) and U0126-intervened electro-acupuncture group (p<0.01). The number of muscular cells, CSA, and NPF of the model group were greatly lower than that of the blank control group (p<0.05, p<0.01, p<0.01), but higher than U0126-intervened model group (p>0.05, p<0.01, p<0.05). The indices of electro-acupuncture groups were higher than that of the model group (p>0.05, p<0.05, p<0.05) and U0126-intervened electro-acupuncture group (p>0.05, p<0.05, p<0.01). Thickness of grade I perimysium of the model group was significantly higher than that of the blank control group (p<0.01) and U0126-intervened model group (p<0.05). The same index of the electro-acupuncture group was lower than that of the model group (p<0.05), but had no significant difference with U0126-intervened electro-acupuncture group (p>0.05). Serum CK content of the model group was remarkably higher than that of the blank control group (p<0.01), and had no significant difference with U0126-intervened model group (p>0.05). The index of the electro-acupuncture group was greatly lower than that of the model group (p<0.05) and U0126-intervened electro-acupuncture group (p<0.05).14days after the injury, lumbar muscle PCNA, ERK1/2, and bFGF of rabbits in the model group were significantly higher than that of the blank control group (p<0.01, p<0.01, p<0.05). Expressions of PCNA, Desmin, and ERK1/2of rabbits in the U0126-intervened model group was remarkably lower than that of the model group (p<0.01, p<0.01, p<0.01). PCNA, Desmin, ERK1/2, and bFGF expressions of rabbits in the electro-acupuncture group were greatly higher than the model group (p<0.01, p<0.05, p<0.05, p<0.01). PCNA, Desmin, and ERK1/2expressions of rabbits in U0126-intervened electro-acupuncture group were lower than that of the electro-acupuncture group (p<0.05, p<0.01, p<0.01).
Conclusion:
1.A-shi point electro-acupuncture can boost the proliferation and differentiation of satellite cells of gastrocnemius muscle damaged rats. The mechanism may lies in the up-regulation of bFGF expression.
2. Electro-acupuncture on weizhong (BL40) point and a-shi can both boost proliferation and differentiation of satellite cells, and regenerative repair of the tissue in recovery stage of acute lumbar muscle contusion rabbits. The mechanism may concerns regulation of bFGF contents in local tissue, blood serum, and spinal cord. ERK1/2pass-way may be one of the important pass-ways mediating curative the effects of electro-acupuncture.
3. Effect on weizhong (BL40) of promoting repairmen is batter than a-shi point, and there may be differences between the action pass-ways of a-shi point electro-acupuncture and weizhong (BL40) electro-acupuncture. Weizhong (BL40) electro-acupuncture works mainly by general regulation of the nerve-endocrine network. It is thereby of morc obvious effect on bFGF release in the spinal cord. The details of the two mechanisms are to be further studied. On the contrary, A-shi point electro-acupuncture works mainly by pulling stimulation, which had more obviously regulatory effects on bFGF in local tissue.
4. The application of ERK inhibitor, U0126, can partly block a-shi point electro-acupuncture's effects of stimulating proliferation and differentiation of satellite cells and regenerating muscles on acute lumbar muscle contusion rabbits. bFGF/ERK signal pass-way may be one of the important ways for muscle recovery and regeneration of a-shi point electro-acupuncture.
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