TRAF6在蛛网膜下腔出血后早期脑损伤中的作用及机制研究

Early brain injury (EBI) after subarachnoid hemorrhage(SAH) is the main factor that leads to the poor outcome of the patient, however, the exact mechanism still remans unclear. Previous researches of applicant’s suggested that the immune inflammatory, oxidative stress reaction and autophagy play an important role in the occurrence of EBI. TNF receptor-associated factor 6（TRAF6）has wide tissue distribution, with highest levels in brain, lung, liver, skeletal muscle and kidney. TRAF6, which serves as a key regulator, is involved in the signals regulating the adaptive and innate immunity, cell survival, cellular proliferation, cytokine production, inflammatory and oxidative stress reaction, and autophagy. Preliminary studies conducted by the applicant’s group suggested that the enhanced expression of TRAF6 was detected in the cortex after SAH which may play an important role involving in the pathogenesis of EBI after SAH. However, the exact role of TRAF6 remains unclear. Therefore, we hypothesis that TRAF6 plays the role in the pathogenesis of EBI after SAH by involving in the signals of inflammatory and oxidative stress reaction, and autophagy. To testify our hypothesis, we intend to establish SAH models in vitro and in vivo to investigate the exact role and mechanisms of TRAF6 in the pathogenesis of EBI after SAH by means of Western blot, real time PCR, EMSA, plasmid vector transfection, gene over expression and gene silencing, etc. The findings of this study will not only academically clarify the the pathogenesis of EBI caused by SAH based on TRAF6, but also provide a strong academic foundation for making a breakthrough in clinical treatment.

蛛网膜下腔出血（SAH）后早期脑损伤（EBI）是影响患者预后的主要因素，其内在机制尚不够清晰。申请人前期研究提示免疫炎症、氧化应激和自噬等过程在EBI发生中起重要作用。肿瘤坏死因子受体相关因子6（TRAF6）在体内分布广泛，在免疫、细胞因子产生、炎症和氧化应激反应、自噬等过程中起重要作用。我们的预实验提示TRAF6在SAH后过度活化，在EBI的发生中起重要作用，但具体机制有待明确。我们提出假说：TRAF6可能通过影响炎症和氧化应激反应、自噬等信号通路，导致EBI的发生。为验证这一假说，我们将通过大鼠离体和在体SAH模型，采用基因过表达、RNA干扰和抑制剂等手段，多角度验证TRAF6在EBI发生中的作用方式及调控机制。本研究将从TRAF6蛋白这个新视点为揭示SAH后EBI的发生机制奠定基础，为临床治疗SAH后EBI的防治提供新的思路。

肿瘤坏死因子受体相关因子6 (Tumor necrosis factor receptor-associated factor 6, TRAF6)是TRAF家族成员之一，是肿瘤坏死因子(Tumor necrosis factor, TNF)超家族和toll/IL-1受体(toll/IL-1 receptor, TIR)超家族重要的多功能胞内适应因子。TRAF6在几种中枢神经系统疾病(包括缺血性中风、创伤性脑损伤和神经退行性疾病)中被研究，但其在蛛网膜下腔出血(SAH)中的作用尚未被充分阐明。本研究采用Sprague-Dawley大鼠模型，将0.3 ml 非肝素化的自体动脉血注入交叉前池，探讨TRAF6在SAH后早期脑损伤(EBI)中的表达水平变化及可能的作用及机制。首先，与sham组相比，我们发现TRAF6的表达水平在SAH后24小时逐渐升高并达到峰值。其次，应用TRAF6过表达质粒和基因沉默siRNA分别可增加或减少SAH后TRAF6的表达，严重加重或减轻神经元死亡、脑水肿、血脑屏障(BBB)损伤等EBI，并分别伴随着自噬水平和氧化应激水平的降低和升高。最后，我们利用缺失E3泛素连接酶活性的TRAF6突变体GFP-TRAF6-C70A探讨了TRAF6在SAH中的作用机制，结果显示，TRAF6-C70A过表达有效改善EBI和氧化应激，同时自噬水平升高。综上表明：TRAF6通过抑制自噬和促进氧化应激参与SAH后EBI；适度调节TRAF6的蛋白水平可能有利于缓解SAH后脑损伤。