电针经微小核糖核酸-133a和沉默交配型信息调节2同源基因1促进废用性肌萎缩恢复的实验研究

Electroacupuncture promotes recovery from disuse muscular atrophy through the miR-133a/SIRT1 pathway

目的探讨微小核糖核酸-133a(miR-133a)和沉默交配型信息调节2同源基因1(SIRT1)在电针促进废用性肌萎缩恢复中的作用。方法将C57BL/6小鼠30只按随机数字表法随机分为正常组、实验对照组、实验组,每组10只小鼠,将实验对照组和实验组小鼠进行尾悬吊构建废用性肌萎缩模型,实验组在尾悬吊的同时进行电针刺激,刺激穴位为阳陵泉和足三里,每日刺激1次,每次15 min,连续治疗14 d。正常组和实验对照组则常规饲养,不进行任何干预。3组大鼠均于实验组干预14 d后统一取材,测定比目鱼肌、腓肠肌的湿重比和横截面积,采用透射电镜观察其骨骼肌和线粒体结构,Western Blot检测沉默交配型信息调节2同源基因1(SIRT1)、过氧化物酶体增殖物激活受体C辅激活因子1a(PGC-1a)、尼克酰胺磷酸核糖转移酶(NAMPT)、腺苷酸活化蛋白激酶α(AMPK-a)以及磷酸化-AMPK-a(P-AMPK-a)蛋白的表达,实时荧光聚合酶链式反应(RT-PCR)检测肌肉萎缩F盒蛋白(Atrogin-1)、肌肉特异性环指蛋白1(MuRF1)、微小核糖核酸-133a(miR-133a)、SIRT1、配对盒基因(Pax7)、生肌调节因子(MyoD)和肌细胞生成素(MyoG)基因的表达,试剂盒检测尼克酰胺腺嘌呤二核苷酸(NAD+)的浓度和NAD+/还原型烟酰胺腺嘌呤二核苷酸(NADH)。结果干预14 d后,与实验对照组比较,实验组比目鱼肌的湿重和横截面积分别增加了21.03%、30.25%(P<0.05),腓肠肌的湿重和横截面积与实验对照组比较,分别增加了5.24%、16.96%(P<0.05)。实验组Atrogin-1、MuRF1、SIRT1、PGC-1a、NAMPT、P-AMPK-a/AMPK-a的表达和NAD+浓度、NAD+/NADH均显著低于实验对照组(P<0.05),而实验组miR-133a的表达则较实验对照组干预14 d后增加了163.3%(P<0.05)。干预14 d后,与细胞增殖相关的Pax7、MyoD基因在实验对照组中表达的显著上调,与正常组和实验组比较,差异均有统计学意义(P<0.05);实验组与细胞分化相关的MyoG基因则呈高表达状态,与正常组和实验对照组比较,差异均有统计学意义(P<0.05)。结论SIRT1相关通路属于机体反射性保护机制之一,参与介导骨骼肌的自然恢复;电针刺激经miR-133a/SIRT1增强成肌细胞分化,改善线粒体能量代谢,从而促进废用性肌萎缩的恢复。

Objective To explore the role of microRNA-133a(miR-133a)and silent mating information regulation 2 homolog 1(SIRT1)in the effects of electroacupuncture on persons with disuse muscular atrophy.Methods Thirty C57BL/6 mice were randomly divided into a control group,an experimental control group and an experimental group,each of 10.Disuse muscular atrophy was induced in the mice of the experimental and experimental control groups using tail suspension.The mice in the electroacupuncture group were given 15 minutes of electroacupuncture over the Yanglingquan and Zusanli points every day for 14 days.Wet weight ratio and the cross-sectional area of the gastrocnemius and soleus were tracked,and the structure of the mitochondria in the skeletal muscles was observed using a transmission electron microscope.The protein expressions of SIRT1,peroxisome proliferator-activated receptorγcoactivator-1a(PGC-1a),nicotinamide phosphoribosyl transferase(NAMPT),adenosine 5-monophosphate-activated protein kinase-a(AMPK-a)and phospho-AMPK-a(P-AMPK-a)were detected using western blotting.The expressions of the muscle atrophy F-box(Atrogin-1),muscle ring finger1(MuRF1),miR-133a,SIRT1,paired box gene 7(Pax7),myogenic determination(MyoD)and myogenin(MyoG)genes were detected through polymerase chain reactions.The concentration of Niacylamide adenine dinucleotide(NAD+)and the ratio of NAD+to reduced nicotinamide adenine dinucleotide were also measured.Results Compared with the experimental control group,the average wet weight increased by 21%and the cross-sectional area of the soleus increased by 30%in the experimental group.The average wet weight of the gastrocnemius increased by 5%and the area by 17%.The average expressions of Atrogin-1,MuRF1,SIRT1,PGC-1a and NAMPT,the concentration of NAD+,as well as the average value of P-AMPK-a/AMPK-a and NAD+/NADH were significantly lower in the experimental group than in the experimental control group,while the average expression of miR-133a in the experimental group was significantly(163%)higher.The average expressions of Pax7 and MyoD were significantly up-regulated in the experimental control group compared with the other two groups,while MyoG was highly expressed in the experimental group compared with the other 2 groups.Conclusions The SIRT1 pathway is one of the reflexive protective mechanisms that mediate in the natural recovery of skeletal muscles.Electroacupuncture enhances myoblast differentiation,improves energy metabolism in the mitochondria,and thus promotes recovery from disuse muscular atrophy.