神经肌肉电刺激对肌少-骨质疏松大鼠骨血管及循环外泌体miRNA表达的影响

The Effect of Neuromuscular Electrical Stimulation on the Expression of miRNA in Bone Vessels and Circulating Extracellular Vesicles of Rats with Muscle Deficiency Osteoporosis

目的:探讨神经肌肉电刺激对失神经诱导的肌少-骨质疏松大鼠骨血管生成及骨质疏松的影响;检测循环外泌体浓度及其miRNA的表达改变,探究循环外泌体中肌源性miRNA的表达水平与骨血管生成及骨质疏松的关系。方法:10周龄雄性SD大鼠随机分为假手术对照组(CON组,n=8)、双腿失神经组(DD组,n=8)和双腿失神经+右腿电刺激组(D+ES组,n=8)。DD组及D+ES组进行双腿胫神经去除手术,D+ES组在胫神经去除术后恢复1周,进行8周的神经肌肉电刺激干预。末次干预24小时后取材,使用micro-CT检测胫骨骨小梁参数;HE染色观察胫骨的骨小梁形态;免疫组化染色鉴定血管标志物血小板–内皮细胞粘附分子(PECAM-1/CD31)和促血管生成因子–血管内皮生长因子(VEGFA)。提取并纯化血浆中的外泌体,使用透射电镜、纳米颗粒跟踪分析和Western Blot对外泌体进行鉴定;利用Real-time PCR测定外泌体中肌源性miR-1、miR-133a、miR-133b以及miR-206的表达。结果:(1)与CON组相比,DD组大鼠的腓肠肌、比目鱼肌的体积和重量明显降低(P<0.001),胫骨骨小梁参数发生病理性改变;经8周神经肌肉电刺激干预后,与DD组相比,D+ES组腓肠肌和比目鱼肌的体积和重量显著升高(P<0.05),胫骨骨小梁参数显著改善。(2)免疫组化染色结果表明,与CON组相比,DD组大鼠胫骨CD31和VEGFA阳性染色面积百分比显著减小;与DD组大鼠相比,D+ES组大鼠的CD31和VEGFA阳性染色面积百分比显著增大。(3)纳米颗粒跟踪分析(NTA)结果表明,与CON组相比,DD组循环外泌体浓度显著降低(P<0.001),PCR结果表明外泌体中miR-1、miR-133a、miR-133b和miR-206表达显著升高(分别为P<0.05,P<0.01,P<0.05,P<0.05);与DD组相比,D+ES组循环外泌体浓度升高(P<0.05),且上述四种miRNA的表达都显著回落(均为P<0.05)。结论:胫骨神经去除术成功建立了肌少-骨质疏松模型;神经肌肉电刺激干预明显改善了失神经诱导的骨质疏松,并促进了胫骨内的血管生成;电刺激干预使循环外泌体浓度显著升高,其携带的肌源性miRNA的表达也发生显著改变,提示电刺激诱导的循环外泌体miRNA可能与骨血管生成及骨质疏松进展有关。

Objective To observe the effect of neuromuscular electrical stimulation on bone angiogenesis and osteoporosis induced by denervation in sarcopenia osteoporosis rats,and explore the correlation of the miRNA expression level in circulating extracellular vesicles to bone angiogenesis and osteoporosis.Methods Ten-week-old male Sprague-Dawley rats were randomly divided into a sham operation control group(con,n=8),a double leg tibial nerve loss group(DD,n=8)and a double leg tibial nerve loss+right leg electrical stimulation group(D+ES,n=8).The tibial nerve of both legs was removed in DD and D+ES groups,and one week later the D+ES group received 8-week neuromuscular electrical stimulation.Samples were taken 24 hours after the last stimulation.Trabecular bone parameters were detected using micro-CT and its morphology was observed using hematoxylin and eosin(HE)staining.Immunohistochemical staining was used to identify the vascular markers platelet endothelial cell adhesion molecule-1(PECAM-1/CD31)and vascular endothelial growth factor A(VEGFA).Moreover,plasma exosomes were extracted and purified,and the vesicles were identified by electron microscopy,nanoparticle tracking analysis(NTA)and Western blotting.Meanwhile,the real time PCR was used to determine the expression of myogenic miR-1,miR-133a,miR-133b and miR-206 in exosomes.Results(1)Compared with the CON group,the volume and weight of gastrocnemius and soleus muscles in the DD group reduced significantly(P<0.001),and the tibial trabecular bone parameters changed pathologically.After 8-week stimulation,compared with the DD group,the volume and weight of gastrocnemius and soleus muscles in D+ES group increased significantly(P<0.05),and tibial trabecular bone parameters improved significantly.(2)The immunohistochemical staining results indicated that,compared with CON group,the expression of CD31 and VEGFA in the tibia of DD group reduced significantly,while that of the D+ES group increased significantly compared with DD group.(3)The results of NTA showed that,compared with CON group,the concentration of circulating extracellular vesicles in the DD group decreased significantly(P<0.001),and the expression of miR-1,miR-133a,miR-133b and miR-206 in exosomes increased significantly(P<0.05,P<0.01,P<0.05,P<0.05 respectively).Compared with the DD group,the concentration of circulating exosomes in D+ES group increased significantly(P<0.05),and the expression of the above four miRNAs decreased significantly(P<0.05 for all).Conclusion Tibial nerve denervation can successfully establish the model of sarcopenia osteoporosis and neuromuscular electrical stimulation significantly relieves such denervation induced changes.Moreover,electrical stimulation also significantly increases the concentration of circulating exosomes,and the expression of myogenic miRNAs carried by them,which suggests that the latter may be related to bone angiogenesis and the progression of osteoporosis.