基于Trem2介导的小胶质细胞对α-突触核蛋白的清除作用探讨补肾益智方治疗帕金森病的机制研究

The abnormal aggregation and deposition of α-synuclein (α-syn) is the main cause of the progressive dopaminergic neurons degeneration of Parkinson's disease (PD). Enhancing the phagocytosis of α-syn by microglia is one of the main ways to eliminate the α-syn deposition in the brain. However, it is a big problem to avoid the massive phagocytosis of α-syn-induced neuroinflammation by microglia in the current treatment of PD. Recent studies have shown that activating of myeloid cell-expressing receptor 2 (Trem2) can not only enhance the ability of microglia to clear of abnormally aggregated proteins in the brain, but also can regulate microglia from pro-inflammatory M1 type to anti-inflammatory M2 type. Therefore, activation of Trem2 may be an effective strategy to promote microglia clearance of α-syn and inhibition of neuroinflammation. The pathogenesis of PD in Chinese medicine is Kidney and medulla empty and blood stasis. Previous studies have found that Bushen Yizhi Formula (BSYZ), has the effect of supplementing kidney and promoting blood circulation and removing blood stasis, can attenuates microglia activation and the dopaminergic neurons loss in PD animal model, in addition, it can increase the expression of Trem2 protein in BV2 microglia. In this study, we aim to explore the mechanism of BSYZ in the treatment of PD both in vivo and in vitro, it will provide the evidence of Chinese medicine, which have effect on enhancement of kidney, on treatment of neurodegenerative diseases.

α-突触核蛋白（α-syn）的异常聚集和沉积是帕金森病（PD）多巴胺能神经元退行性变的主要原因。增加小胶质细胞对α-syn的吞噬是清除脑内α-Syn的主要途径之一，然而，如何避免小胶质细胞因大量吞噬α-syn造成的多巴胺能神经元炎性损伤，是目前PD治疗的一个难点。激活髓样细胞表达的触发受体2（Trem2）可以增加小胶质细胞清除脑内异常聚集的蛋白，又能调节小胶质细胞由促炎的M1型向抗炎的M2型转化，可能是清除PD脑内α-syn同时抑制神经炎症发生的有效策略。PD中医学病机为肾虚髓空，瘀血阻络，前期研究发现具有补肾活血益智功效的补肾益智方（BSYZ）能够减轻PD模型动物黑质小胶质细胞过度激活，缓解多巴胺能神经元丢失，并且能够增加BV2小胶质细胞Trem2蛋白表达。本课题拟在前期研究基础上，通过体内外实验，探讨BSYZ治疗PD的机制，为补肾中药复方治疗神经退行疾病提供依据。

背景：增加小胶质细胞对α-突触核蛋白（α-syn）的清除同时避免小胶质细胞因大量吞噬α-syn造成的多巴胺能（DA）神经元炎性损伤是目前PD治疗的难点。中药复方补肾益智方（BSYZ）具有广泛的神经保护作用和抗炎活性，本研究在验证Trem2对小胶质细胞清除α-syn的作用的基础上，探讨了BSYZ对α-syn诱导的DA神经元损伤和α-syn沉积的作用，以期为PD的治疗提供新的靶点，为PD的药物开发提供证据支持。. 方法：用Trem2过表达慢病毒载体和Trem2siRNA评价Trem2对小胶质细胞极化的影响，采用RT-qPCR、免疫荧光法检测M1、M2小胶质细胞标记物和Trem2mRNA水平。用AAV注射Trem2 shRNA建立trem2-/-模型。采用旷场、爬杆和转棒试验评价小鼠的运动功能。采用免疫荧光染色法观察DA神经元存活、α-syn沉积和小胶质细胞极化情况。采用westren blotting、RT-qPCR和免疫荧光法检测Trem2蛋白和mRNA水平。. 结果：（1）Trem2可诱导小胶质细胞由M1型向M2型转换。Trem2缺失会导致小胶质细胞向M1型活化，激活Trem2可以诱导小胶质细胞向M2型活化，α-syn诱导刺激会导致小胶质细胞向M1型转化，补肾益智方能够抑制α-syn诱导的BV2小胶质细胞M1型炎症因子分泌，增加M2型抗炎因子表达。.（2）shTrem2会引起A53T小鼠中脑DA神经元丢失和α-syn的沉积。BSYZ能够增加A53T小鼠脑内小胶质细胞Trem2表达，抑制A53T小鼠脑内小胶质细胞过度激活，减少M1型小胶质细胞炎症因子分泌，增加M2型小胶质细胞抗炎因子的表达，并能起到缓解A53T小鼠的运动功能障碍，减轻A53T小鼠中脑黑质致密部DA神经元损伤和α-syn在脑内的沉积的作用。. 结论及意义：（1）激活Trem2可以抑制α-syn诱导的小胶质细胞由促炎M1型向抑制炎症的M2型转化，抑制Trem2会加重PD模型小鼠DA神经元损伤和α-syn沉积，靶向Trem2可能是PD治疗的新靶点，可进一步研究。（2）BSYZ可以激活小胶质细胞Trem2,缓解α-syn诱导的DA神经元损伤和神经炎症，减少PD病理产物α-syn在脑内的沉积，可进一步开发。