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目的通过蛋白质组学方法分析失神经支配后胫前肌与比目鱼肌的蛋白表达变化,探讨肿瘤坏死因子受体相关因子6(TRAF6)在失神经支配后胫前肌与比目鱼肌中的表达变化及其意义。方法制备大鼠坐骨神经离断模型,通过同位素相对标记与绝对定量(i TRAQ)技术方法分析胫前肌与比目鱼肌在失神经支配后的蛋白表达变化,比较差异表达的蛋白在胫前肌与比目鱼肌中的差异,筛选出在胫前肌与比目鱼肌中的表达变化差异显著的关键蛋白,并通过免疫印迹法验证其表达,进一步通过体外方法验证关键蛋白的生物学作用。结果失神经支配后比目鱼肌萎缩速度和程度明显大于胫前肌(P<0.05,n=20),蛋白质组学研究发现有30个蛋白在胫前肌与比目鱼肌中表达变化差异较为明显,这些蛋白主要包括代谢相关蛋白、伴侣蛋白、收缩蛋白以及信号分子等,其中TRAF6在失神经支配后比目鱼肌中表达显著上升,蛋白免疫印迹法检测结果也证实了蛋白质组学的结果,TRAF6的靶基因肌肉环状指蛋白1(MuRF1)和肌肉萎缩盒F基因(MAFbx)在失神经支配后比目鱼肌中的表达也高于其在胫前肌中的表达(P<0.05,n=6);为了进一步探讨TRAF6对肌萎缩的影响,在肌管中转染TRAF6 siRNA和对照siRNA,然后用地塞米松诱导C2C12肌管萎缩,发现TRAF6 siRNA转染组肌管的直径明显大于转染对照siRNA组(P<0.05,n=6);TRAF6、MuRF1和MAFBx在TRAF6 siRNA转染组肌管中的表达显著低于转染对照siRNA组(P<0.05,n=6),结果提示TRAF6 siRNA有效抑制了靶基因TRAF6的表达,也同时抑制了其下游靶基因MuRF1和MAFBx的表达。结论 TRAF6在失神经支配后比目鱼肌中的表达量显著高于其在胫前肌中的表达,抑制TRAF6的表达可以减轻地塞米松引起的肌管萎缩,由此推测失神经支配后比目鱼肌萎缩较胫前肌严重可能与失神经支配后比目鱼肌中TRAF6的升高更显著有关。
Objective To analyze the protein expression changes of anterior tibial and soleus muscles after denervation by proteomics and to explore the expression changes of TRAF6 in the anterior tibial and soleus muscles after denervation Its meaning. Methods The model of sciatic nerve in rats was prepared and the changes of protein expression of the anterior tibialis and soleus muscle after denervation were analyzed by isotope relative labeling and i TRAQ method. The differences between the anterior tibialis muscle and the soleus The differences between the anterior tibialis and the soleus muscle were observed by immunohistochemistry. The key proteins that were significantly different from those in the anterior tibial and soleus muscles were screened out and their expression was verified by immunoblotting. The biological effects of key proteins were further verified by in vitro methods. Results The speed and extent of soleus muscle atrophy after denervation were significantly higher than those of the anterior tibialis muscle (P <0.05, n = 20). The proteomic analysis showed that there were 30 proteins which showed obvious differences in the anterior tibial and soleus The proteins mainly include metabolism-related proteins, chaperones, contractile proteins and signal molecules. The expression of TRAF6 in the soleus muscle after denervation was significantly increased. The result of Western blot also confirmed the results of proteomics. The target gene of TRAF6 The expression of MuRF1 and MAFbx in the soleus muscle after denervation was also higher than that in the anterior tibialis muscle (P <0.05, n = 6) To further investigate the effect of TRAF6 on muscle atrophy, TRAF6 siRNA and control siRNA were transfected into myotubes, and then C2C12 myotube was induced by dexamethasone. The diameter of myotubes in TRAF6 siRNA transfected group was significantly larger than that in transfected siRNA group (P <0.05, n = 6). The expression of TRAF6, MuRF1 and MAFBx in myotube of TRAF6 siRNA transfected group was significantly lower than that in transfected siRNA group (P <0.05, n = 6) The expression of the gene TRAF6 is also suppressed The downstream target genes MuRF1 and MAFBx were expressed. Conclusion The expression of TRAF6 in the soleus muscle after denervation is significantly higher than that in the anterior tibial muscle. Inhibition of TRAF6 expression can reduce the atrophy of myotubes induced by dexamethasone. Severe than the anterior tibialis may be denervated innervated greater than a significant increase in TRAF6 soleus muscle.