电针对大鼠失神经支配骨骼肌萎缩及IGF-1/PI3K/AKT表达的影响
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摘要
目的:观察电针对失神经支配骨骼肌萎缩及IGF-1/PI3K/AKT信号通路表达的影响,探讨电针对失神经肌肉萎缩的可能作用机制。方法:36只雄性SD大鼠随机分为对照组、模型组和电针组。模型组和电针组大鼠切断左后肢坐骨神经制备失神经肌萎缩模型。电针组予电针治疗,穴取"足三里"和"上巨虚",疏密波,2 Hz/33 Hz,1.5～2 mA,每次30 min,每日1次,每5次间隔2 d,共20次。对照组与模型组每日同样鼠笼固定,但不电针。疗程结束后观察各组坐骨神经功能指数,称量腓肠肌肌湿重,HE染色测量患/健侧肌纤维横截面积比和肌细胞直径比,TUNEL和免疫荧光双染色的方法评价肌细胞凋亡和增殖分化情况。荧光定量PCR法检测胰岛素样生长因子-1(insulin-like growth factor-1,Igf-1)和蛋白激酶B(protein kinase B,PKB,又被称作Akt)的mRNA表达,免疫蛋白印迹法检测局部肌肉Akt总蛋白及其磷酸化形式p-AKT(Ser473)和p-AKT(Thr308)的表达量。结果:与对照组相比,模型组大鼠的坐骨神经功能指数,患侧/健侧腓肠肌湿重比、肌纤维横截面积比和肌细胞直径比均明显下降,肌细胞凋亡指数明显升高(均P<0.01);与模型组相比,电针组上述指标均得到改善(均P<0.05),与肌卫星细胞增殖分化相关的PAX3和PAX7表达明显增高(均P<0.01),p-AKT(Ser473)蛋白水平增加(P<0.05)。结论:电针可改善失神经肌肉的萎缩情况,减少肌细胞凋亡,促进肌卫星细胞增殖分化,其作用机制可能与提高p-AKT(Ser473)水平、激活AKT信号通路有关。
Objective To observe the effects of electroacupuncture (EA) on denervated skeletal muscle atrophy and expression of IGF-1/PI3 K/AKT signaling pathway in rats, and to explore the possible mechanisms of it. Methods Thirty-six male SD rats were randomly divided into a control group, a model group and an EA group. Denervated muscle atrophy model was made by cutting off the left hindlimb sciatic nerve in the model group and the EA group. In the EA group, EA, dense-disperse wave, 2 Hz/33 Hz and 1.5-2 mA, was connected at "Zusanli" (ST 36) and "Shangjuxu" (ST 37). The treatment was given for 30 min every time, once a day and a total of 20 times were needed, there was 2 days interval every 5 times. The rats in the control group and model group were fixed in the same cage every day but not applied EA. Sciatic nerve function index (SFI) and ratio of muscle wet weight (RWW) were measured after treatment. HE staining was used to determine the ratio of cross-sectional area (RCA) and the ratio of fiber diameter (RFD). TUNEL and double immunofluorescence staining were used to evaluate the myocyte apoptosis, proliferation and differentiation. The mRNA expressions of IGF-1 and AKT were detected by real-time PCR. The total protein of AKT, p-AKT (Ser473) and p-AKT (Thr308) were detected by western blot. Results Compared with the control group, SFI, RWW, RCA and RFD of the model group were significantly decreased and apoptotic index of myocytes were significantly increased (all P<0.01). Compared with the model group, the above indexes of the EA group were improved (all P<0.05), and the expressions of PAX3 and PAX7 related to proliferation and differentiation of muscle satellite cells were significantly increased (both P<0.01), p-AKT (Ser473) protein level was increased (P<0.05). Conclusion EA can improve the atrophy of denervated muscles, reduce myocyte apoptosis, and promote the proliferation and differentiation of muscle satellite cells. The mechanism may be related to the increase of p-AKT (Ser473) level and activation of AKT signaling pathway.
Objective To observe the effects of electroacupuncture (EA) on denervated skeletal muscle atrophy and expression of IGF-1/PI3 K/AKT signaling pathway in rats, and to explore the possible mechanisms of it. Methods Thirty-six male SD rats were randomly divided into a control group, a model group and an EA group. Denervated muscle atrophy model was made by cutting off the left hindlimb sciatic nerve in the model group and the EA group. In the EA group, EA, dense-disperse wave, 2 Hz/33 Hz and 1.5-2 mA, was connected at "Zusanli" (ST 36) and "Shangjuxu" (ST 37). The treatment was given for 30 min every time, once a day and a total of 20 times were needed, there was 2 days interval every 5 times. The rats in the control group and model group were fixed in the same cage every day but not applied EA. Sciatic nerve function index (SFI) and ratio of muscle wet weight (RWW) were measured after treatment. HE staining was used to determine the ratio of cross-sectional area (RCA) and the ratio of fiber diameter (RFD). TUNEL and double immunofluorescence staining were used to evaluate the myocyte apoptosis, proliferation and differentiation. The mRNA expressions of IGF-1 and AKT were detected by real-time PCR. The total protein of AKT, p-AKT (Ser473) and p-AKT (Thr308) were detected by western blot. Results Compared with the control group, SFI, RWW, RCA and RFD of the model group were significantly decreased and apoptotic index of myocytes were significantly increased (all P<0.01). Compared with the model group, the above indexes of the EA group were improved (all P<0.05), and the expressions of PAX3 and PAX7 related to proliferation and differentiation of muscle satellite cells were significantly increased (both P<0.01), p-AKT (Ser473) protein level was increased (P<0.05). Conclusion EA can improve the atrophy of denervated muscles, reduce myocyte apoptosis, and promote the proliferation and differentiation of muscle satellite cells. The mechanism may be related to the increase of p-AKT (Ser473) level and activation of AKT signaling pathway.
引文
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[12]王刚,高谦,杨志丽,等.不同针刺法对大鼠腓肠肌砸伤后微循环血流灌注的影响[J].军医进修学院学报,2011,32(6):651-653.
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[14]Cassano M,Quattrocelli M,Crippa S,et al.Cellular mechanisms and local progenitor activation to regulate skeletal muscle mass[J].J Muscle Res Cell Motil,2009,30(7-8):243-253.
[15]Glass DJ.PI3 kinase regulation of skeletal muscle hypertrophy and atrophy[J].Curr Top Microbiol Immunol,2010,346:267-278.
[16]Latres E,Amini AR,Amini AA,et al.Insulin-like growth factor-1(IGF-1)inversely regulates atrophy-induced genes via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway[J].J Biol Chem,2005,280(4):2737-2744.
[17]华兴邦,李辞蓉,周浩良,等.大鼠穴位图谱的研制[J].实验动物与动物实验,1991,11(1):1-5.
[18]陈家泽.电针对坐骨神经损伤后模型大鼠腓肠肌萎缩及NGFmRNA表达的影响[D].广州:广州中医药大学,2008.
[19]李庆雯,石田寅夫,郭义,等.不同频率电针对大鼠坐骨神经损伤后神经组织形态学与骨骼肌肌电图的影响[J].中国针灸,2005,25(3):73-76.
[20]Su Z,Hu L,Cheng J,et al.Acupuncture plus low-frequency electrical stimulation(Acu-LFES)attenuates denervation-induced muscle atrophy[J].J Appl Physiol(1985),2016,120(4):426-436.
[21]孙迎春.电针不同波形、频率、刺激强度、时间对大鼠坐骨神经损伤修复的实验研究[D].哈尔滨:黑龙江中医药大学,2005.
[22]徐建广,顾玉东,李继峰.大鼠失神经支配骨骼肌退变的实验研究[J].上海医科大学学报,1999,26(4):265-268.
[23]Sakamoto K,Aschenbach WG,Hirshman MF,et al.Akt signaling in skeletal muscle:regulation by exercise and passive stretch[J].Am J Physiol Endocrinol Metab,2003,285(5):E1081-1088.
[24]Latres E,Amini AR,Amini AA,et al.Insulin-like growth factor-1(IGF-1)inversely regulates atrophy-induced genes via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway[J].J Biol Chem,2005,280(4):2737-2744.
[2]Krarup C,Rosen B,Boeckstyns M,et al.Sensation,mechanoreceptor,and nerve fiber function after nerve regeneration[J].Ann Neurol,2017,82(6):940-950.
[3]Al-Temimi MH,Chandrasekaran B,Phelan MJ,et al.Incidence,Risk Factors,and Trends of Motor Peripheral Nerve Injury After Colorectal Surgery:Analysis of the National Surgical Quality Improvement Program Database[J].Dis Colon Rectum,2017,60(3):318-325.
[4]虎力,徐平,王晓南,等.针灸在骨骼肌萎缩中的研究现状与展望[J].中华中医药学刊,2015,33(4):890-893.
[5]Lim HD,Kim MH,Lee CY,et al.Anti-Inflammatory Effects of Acupuncture Stimulation via the Vagus Nerve[J].PLoS One,2016,11(3):e0151882.
[6]李恭,印文考.电针对周围神经损伤的疗效[J].吉林医科大学学报,1962,2(2):62-64.
[7]陈礼娇,田丽.对头针齐刺法与传统针刺疗法治疗肌萎缩178例疗效观察[J].中国针灸,1997,17(8):504.
[8]刘学谦.针刺治疗对注射型坐骨神经损伤肌形态学变化的观察[D].遵义:遵义医学院,2013.
[9]庄伊洢,黄晓卿,叶笑然,等.电针抗下肢肌萎缩的频率组合优化及其机制的初步研究[J].上海针灸杂志,2017,36(4):473-477.
[10]刘学谦,罗怀香,张华,等.针刺对注射型坐骨神经损伤肌萎缩的影响[J].中国医药指南,2012,10(33):65-66.
[11]魏月娥,傅冠英,万选才.针刺后交感神经中枢内神经介质变化的组织化学观察[J].解剖学报,1983,14(2):185-190,235.
[12]王刚,高谦,杨志丽,等.不同针刺法对大鼠腓肠肌砸伤后微循环血流灌注的影响[J].军医进修学院学报,2011,32(6):651-653.
[13]陈家泽,陈传伟,孙锋.针刺对大鼠坐骨神经损伤后腓肠肌萎缩的影响[J].实用医学杂志,2008(8):1313-1315.
[14]Cassano M,Quattrocelli M,Crippa S,et al.Cellular mechanisms and local progenitor activation to regulate skeletal muscle mass[J].J Muscle Res Cell Motil,2009,30(7-8):243-253.
[15]Glass DJ.PI3 kinase regulation of skeletal muscle hypertrophy and atrophy[J].Curr Top Microbiol Immunol,2010,346:267-278.
[16]Latres E,Amini AR,Amini AA,et al.Insulin-like growth factor-1(IGF-1)inversely regulates atrophy-induced genes via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway[J].J Biol Chem,2005,280(4):2737-2744.
[17]华兴邦,李辞蓉,周浩良,等.大鼠穴位图谱的研制[J].实验动物与动物实验,1991,11(1):1-5.
[18]陈家泽.电针对坐骨神经损伤后模型大鼠腓肠肌萎缩及NGFmRNA表达的影响[D].广州:广州中医药大学,2008.
[19]李庆雯,石田寅夫,郭义,等.不同频率电针对大鼠坐骨神经损伤后神经组织形态学与骨骼肌肌电图的影响[J].中国针灸,2005,25(3):73-76.
[20]Su Z,Hu L,Cheng J,et al.Acupuncture plus low-frequency electrical stimulation(Acu-LFES)attenuates denervation-induced muscle atrophy[J].J Appl Physiol(1985),2016,120(4):426-436.
[21]孙迎春.电针不同波形、频率、刺激强度、时间对大鼠坐骨神经损伤修复的实验研究[D].哈尔滨:黑龙江中医药大学,2005.
[22]徐建广,顾玉东,李继峰.大鼠失神经支配骨骼肌退变的实验研究[J].上海医科大学学报,1999,26(4):265-268.
[23]Sakamoto K,Aschenbach WG,Hirshman MF,et al.Akt signaling in skeletal muscle:regulation by exercise and passive stretch[J].Am J Physiol Endocrinol Metab,2003,285(5):E1081-1088.
[24]Latres E,Amini AR,Amini AA,et al.Insulin-like growth factor-1(IGF-1)inversely regulates atrophy-induced genes via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway[J].J Biol Chem,2005,280(4):2737-2744.