虎　力¹，温佩彤¹，张伟波¹，高　原¹，寿　崟¹，王晓南²，徐　平¹

（1上海中医药大学针灸推拿学院，上海2012032；2美国埃默里大学医学院,美国，亚特兰大30322）

摘要：研究背景：目前世界各国均面临着严重的人口老龄化问题。衰老过程中肌肉萎缩、纤维化、脂肪沉积是造成肌肉功能降低的主要原因，肌肉萎缩程度已经成为死亡率的一个重要预测指标。骨骼肌属于中医学“经筋”的范畴，增龄性骨骼肌萎缩过程中表现的“筋肉迟缓、肢体不用”等符合中医学“痿证”的范畴。《素问•痿论》有：“治痿独取阳明”，同时，中医认为“肾主骨生髓”，故选足三里和肾俞对增龄性骨骼肌萎缩具有一定的延缓作用。研究目的：基于TGF-β/Smad信号通路，探讨低频电针减缓增龄性大鼠骨骼肌萎缩的效应及其机制，为延缓增龄所致的骨骼肌萎缩提供依据。研究方法：采用2Hz低频电针，分别对18月龄(YA)、20月龄(EA)和22月龄(JA)自然衰老模型大鼠肾俞和足三里穴进行针刺，得气后连接G6805-2型电针仪，连续波，电压2～4V，电流1～3mA，刺激强度以大鼠四肢轻微抖动为宜，每次15分钟，隔日1次，3周后休息1周。各组的干预周期为YA(12月-18月) ，EA(12月-20月)，JA(12月-22月)。观测指标为腓肠肌、比目鱼肌的肌肉湿重，血清SOD、MDA含量，比目鱼肌组织HE染色，Western Blot检测骨骼肌蛋白表达，包括：(1)与骨骼肌纤维化相关的信号蛋白TGF-β/P-smad2/P-smad3/Smad2-3/Myostatin；(2)与骨骼肌生成相关的信号蛋白MyoD/Myogenin/eMyHC。研究结果：(1)与空白组相比，18月龄、20月龄和22月龄针刺组大鼠体重均下降，但比目鱼肌、腓肠肌的湿重均有所增加；(2)不同月龄空白组大鼠血清SOD显著降低，MDA显著增加，而针刺干预能够有效逆转这一趋势(P<0.05)；(3)不同月龄对照组大鼠骨骼肌均出现不同程度的衰老迹象，表现为肌纤维排列较为松散，出现圆形化的肌纤维，细胞核中心化现象多见；针刺干预后，肌纤维排列较为紧密，圆形化肌纤维及细胞核中心化现象减少；(4)与18月龄(YC)、20月龄空白组(EC)相比，针刺组TGF-β/Smad通路相关的信号蛋白(TGF-β,Smad2,P-smad2,Smad3,P-smad3, myostatin)表达均增加；与22月龄对照组(JC)相比，22月龄针刺组(JA)上述信号蛋白表达显著下降(P<0.05)。研究结论：低频电针干预能够减缓增龄性骨骼肌萎缩的进程，其机制可能与电针干预促进骨骼肌再生和/或后期降低骨骼肌的纤维化有关。

关键词:骨骼肌萎缩；增龄；骨骼肌卫星细胞；TGF-β/Smad

Exploring the Mechanism of Low Frequency Electroacupuncture on Aging Rats Skeletal Muscle Atrophy by TGF-β/Smad Signaling Pathway

HU Li 1, WEN Peitong1, ZHANGWeibo 1, GAO Yuan 1, SHOUYin 1, WANG Xiaonan2, and XU Ping 1

(1 Acumox&Tuina College of Shanghai University of Traditional Chinese Medicine, Shanghai 201203; 2 Department of Medicine of Emory University, Atlanta, GA 30322)

Abstract: Background:At present,all countries in the world are facing a serious ageing of population. In the course of aging, muscle atrophy, fibrosis and fat deposition are the main causes of the decrease of muscle function, and the degree of muscular atrophy has become an important predictor of mortality rate. In Traditional Chinese Medicine(TCM), skeletal muscle belongs to "the muscle meridian" (Jing Jin).The symptoms of muscle movement and physical activity dysfunction, during the age-related skeletal muscle atrophy, accord with "Wei Syndrome" in TCM.Based on TCM theory of “treatment for flaccidity aiming at Yang Ming meridian” and"the kidney dominating bone ", we hypothesized that combined togetherZusanli and Shenshucould attenuate aging-induced skeletal muscle atrophy.Objective: Based on TGF-β/Smad signaling pathway to explore the mechanism of low frequency electroacupuncture on aging rats skeletal muscle atrophy, to provide the basis for attenuating age-related skeletal muscle atrophy. Methods:The aging rats of 18 months(YA), 20 months (EA) and 22 months(JA) were used in this study. Two acupoints, Shenshu(BL23) and Zusanli(ST36), were selected. The needles were connected into G6805-2 electronic acupuncture instrument using consistent pulse,electric frequency 2Hz,voltage 2～4V, electric current 1mA～3mA. The stimulation intensity was suitable for rats with slight limb jitter. The treatment was 15 minutes each time on alternate days with one week of rest after three weeks treatment continually. Treatment durations for each group were YA(12 -18 months)，EA(12 -20 months)，JA(12-22 months). Skeletal muscles of gastrocnemius and soleus,SOD and MDA in serum and HE staining for soleus were measured respectively. Protein expressions, including TGF-β/P-smad2/P-smad3/Smad2-3/Myostatin, which related to skeletal muscle fibrosis signaling proteins and MyoD /Myogenin/eMyHC, which related to muscle myogenesis were measured by Western blot. T-test and ANOVA were used for results statistics.Results: (1)Compared with the controlgroup, the weight of the rats for the 18 months, 20 months and 22 months decreased after the acupuncture treatment but the wet weight of the soleus and gastrocnemius increased. (2)The serum expression of SOD decreased significantly and the MDA expression increased significantlyfor different months, while acupuncture treatment could effectively reverse this tendency (P< 0.05). (3)In control groupof different months, there were signs of aging, which showed that the muscle fibers were more loosely arranged, and the fibroid was round, and the nuclei were more central. After the acupuncture treatment, the muscle fibers were more closely aligned, and the circular fibrosis and nucleus centralization decreased. (4) Compared with 18 months(YC) and 20 months of control groups(EC), acupuncture treatment increased the protein expressionsof TGF-β/Smadsignaling pathway(TGF-β, Smad2, p-smad2, Smad3, p-smad3, myostatin), while compared with 22 months control group(JA), acupuncture treatment decreasedthe protein expression significantly (P< 0.05).Conclusions: We hold that electric acupuncture could attenuate aging-related rat skeletal muscle atrophy by promoting muscle regeneration and/or reducing skeletal muscle fibrosis in the later stage.

Key words:Skeletal muscle atrophy, Age-related, Skeletal muscle satellite cell, TGF-β/Smad、