TMEM166介导的颈动脉狭窄在缺血性脑中风中的作用及机制研究

Stroke and its sequelae continue to be a leading cause of disability and mortality worldwide. Despite interventional treatments that significantly decrease the general incidence of stroke, a majority of patients suffer from serious ischemia/reperfusion injury in the process. Our previous study showing TMEM166’s involvement in brain damage following ischemia/reperfusion in rats suggests TMEM166 to be a potential novel therapeutic in human stroke. In this proposal, we will explore the role of TMEM166 after balloon injury with TMEM166 knock-out mice and focus on the possible mechanisms regulated by TMEM166. .ApoE-ε4 and wnt/β-catenin signaling pathway is related with wall thickness of carotid arteries. In our preliminary tests, ApoE-ε4-TMEM166 compound experienced a significance increase and wnt/β-catenin signaling pathway was activated in patients with carotid artery stenosis. In the mouse carotid artery stenosis model, similar results were obtained. Besides, ApoE-ε4 expression and wnt/β-catenin signaling pathway were both inhibited, and the restenosis was significantly improved in the TMEM166 knock-out mice, and when ApoE-ε4shRNA or wnt specific inhibitor IWR-1-endo was administrated. However, wnt/β-catenin signaling pathway was activated and carotid artery stenosis rate increased after Lv-TMEM166 treatment. Thus, we hypothesized that TMEM166 regulates carotid artery stenosis and leads to ischemic stroke through combination with ApoE-ε4 and the subsequent activation of wnt/β-catenin signaling pathway.

脑中风是导致全球病人致死和致残的重要原因。我们研究发现TMEM166与脑缺血损伤后的神经损伤密切相关。本研究在TMEM166基因敲除鼠颈动脉狭窄模型的基础上观察其在该过程中的作用并重点探讨其机制。.ApoE-ε4和wnt/β-catenin通路与内膜厚度显著相关，我们的预实验证实颈动脉狭窄患者TMEM166-ApoE-ε4复合物明显增加，wnt/β-catenin通路激活。在小鼠颈动脉狭窄模型中也有类似发现，且上述指标在TMEM166基因敲除鼠，或在给予ApoE-ε4shRNA或wnt特异性抑制剂IWR-1-endo后则明显被抑制，同时管腔狭窄改善；而给予Lv-TMEM166后，wnt/β-catenin通路被激活，管腔狭窄显著。因此我们提出科学假说：TMEM166与ApoE-ε4结合后通过激活并放大wnt/β-catenin信号通路，介导管腔狭窄，最终导致缺血性脑中风发生。

脑中风是导致全球病人致死和致残的重要原因，约25%~50%的缺血性脑卒中是由颈动脉狭窄(CAS)引起。颈动脉内膜剥脱(CEA)是目前临床上治疗CAS的有效手段之一，但最近的研究发现，CEA是CAS患者围术期卒中的重要因素。CEA后中风是一种不常见但危险的并发症。尽管在过去的几年中，我们对这一疾病过程的了解有所增加，但其机制仍有待明确。我们研究发现TMEM166与脑缺血损伤后的神经损伤密切相关。TMEM166是目前通过高通量功能筛选出的一个明显下调内参活性同时诱导细胞死亡的跨膜蛋白。研究发现，过表达TMEM166可以抑制HeLa细胞的克隆形成能力，最终引起HeLa细胞的自噬性死亡。本课题组分别收集我院择期行颈动脉内膜剥脱术后无明显神经系统并发症患者（对照组）的剥脱处内膜、外周血及术后出现围术期脑卒中患者（脑卒中组）的外周血；并构建TMEM166基因敲除鼠，建立小鼠颈动脉狭窄后大脑中动脉闭塞(MCAO)动物模型。结果发现术后并发脑卒中患者的TMEM166、IL-6和CRP含量明显高于对照组；与脑卒中后幸存者相比，死亡患者的这些指标明显更高。与临床研究结果类似，CAS小鼠中上述蛋白表达水平略有升高，MCAO后显著升高，敲除TMEM166后则明显降低。此外，转染Ad5-TMEM166可通过诱导MCAO后小鼠脑小胶质细胞自噬激活和IL-6等炎症因子释放，加重缺血性脑损伤。敲除TMEM166可通过哺乳动物雷帕霉素靶点(mTOR)通路减轻炎症和抑制小胶质细胞过度自噬来改善脑损伤。总之，这些发现表明TMEM166可能是CEA后中风的生物标志物，并在这一过程中发挥关键作用。