大脑小胶质细胞M1极化及外泌体释放在1-溴丙烷神经毒性中的作用

1-bromopropane (1-BP) is a common organic solvent, the occupational exposure to 1-BP is increasing rapidly. The poisoning cases and animal experiments indicated that 1-BP could induce neurotoxicity. However, the mechanism remains unclear. Our previous studies suggested that 1-BP induce microglia M1 polarization and neuronal loss. Further, the cytochrome P4502E1(CYP2E1) was significantly increased in the activated microglia of brains in 1-BP treated rats, and competitive inhibitor of CYP2E1-allyl sulfide protected against 1-BP-induced neurotoxicity. Thus, we hypothesized that the CYP2E1-dependent oxidation of 1-BP in brain might be an initially important event of the neurotoxicity of 1-BP. The present study will investigate the roles of CYP2E1 in microglia M1 polarization, neuronal MAPK apoptosis cascade, as well as the effects on the inflammatory components of exosome from 1-BP-trated rat’s brains. Following long-time exposure to low-dose 1-BP, we will observe the harmful effects and CYP2E1 activity in the rats’ brain. The neurotoxicity of active metabolites of 1-BP from the CYP2E1-dependent metabolism will be validated by using the primary neuron, astrocyte and microglia mixed cell culture system in vitro, and compared the protective effects by intervention on different target molecules. In addition, the propagation of 1-BP neurotoxicity by exosome secreted from activated microglia will be also studied using BV-2, which is mediated by P2X7 receptor. The present study would be helpful to find the therapeutic targets and potential preventive strategies for 1-BP poisoning, and provide the reliable data for the establishment of the protective measures for exposed population.

1-溴丙烷(1-BP)是常用有机溶剂，职业接触人群增长迅速。1-BP具有神经毒性，但毒作用机制仍不清楚。我们前期发现1-BP诱导大鼠大脑小胶质细胞M1极化、神经元丢失，活化的小胶质细胞中CYP2E1表达增强，CYP2E1干预剂对1-BP暴露动物有明显的保护作用。本研究以1-BP经脑内CYP2E1原位代谢活化为切入点，采用动物实验调控CYP2E1活性，观察不同活性下1-BP对大脑小胶质细胞M1极化、神经元MAPK凋亡通路、外泌体炎性成分的影响；研究1-BP暴露对大脑CYP2E1的诱导及神经毒性的影响。体外原代神经元-胶质细胞混合培养体系观察1-BP活性代谢产物的神经毒性，采用BV-2细胞研究P2X7受体介导活化小胶质细胞释放的外泌体对1-BP神经毒性的传播扩散作用；比较不同靶点干预对不良结局的逆转效果，以期发现治疗和预防1-BP中毒的有效干预靶点，为接触人群保护措施制定提供可靠数据。

1-溴丙烷（1-BP）是广泛应用的有机溶剂，中毒病例和实验室研究显示1-BP具有神经毒性。为探讨1-BP神经毒性确切的分子机制和有效防治靶点，借助该项目资助的研究观察到：①大鼠连续暴露800mg/kg.bw 1-BP 5d起可导致明显的认知功能损伤，伴随大脑小胶质细胞M1极化，氧化/氮化应激，NLRP3炎性小体增加，以上变化随暴露时间延长逐渐加重；NF-κB抑制剂PDTC能挽救1-BP引起的神经元炎性损伤及改善动物的认知功能。②大鼠连续暴露200mg/kg.bw～800mg/kg.bw的1-BP 6周，可发生后肢抓力降低为特征的周围神经损伤，病理形态学分析显示大脑皮层运动区小胶质细胞活化、神经元丢失，大脑NO水平升高、Trx1-ASK1-p38 MAPK凋亡信号传导通路激活，以上变化均呈现剂量效应关系；自由基清除剂依达拉奉在一定程度上逆转1-BP的周围神经毒性。③CYP2E1抑制剂烯丙基硫提前给予大鼠然后暴露800mg/kg.bw 1-BP连续9周，观察到抑制CYP2E1能够降低1-BP诱导动物后肢瘫痪，减轻大鼠大脑运动皮层小胶质细胞M1极化、脑内氧化/氮化应激，抑制Trx1-ASK1-p38 MAPK凋亡通路激活，逆转运动神经元凋亡。④GC-MS检测证实，大鼠暴露1-BP可升高大脑中1-BP氧化代谢活性产物1-溴-2-丙醇含量，抑制CYP2E1可明显降低大脑1-溴-2-丙醇水平。⑤体外培养体系中，1-BP、1-溴-2-丙醇均可导致BV-2小胶质细胞CYP2E1表达增加，M1/M2极化失衡；1-溴-2-丙醇激活BV2分泌外泌体中IL-1β表达明显升高；将1-溴-2-丙醇处理BV2小胶质细胞外泌体进行Dil荧光标记，加入原代大鼠神经元培养体系，观察到炎性外泌体极易被原代神经元吞噬，并呈现对神经元的损伤作用，表现为轴突缩短或消失、胞体分解。⑥高脂饲料诱导肥胖大鼠暴露100mg/kg.bw低剂量的1-BP连续10周，发现肥胖联合低剂量1-BP暴露导致大鼠脑内胰岛素信号通路障碍和炎性反应，P2X7表达增加，成熟IL-1β蛋白转化增加，伴随动物认知功能损伤障碍。⑦其它环境神经毒物如百草枯和代森锰诱导帕金森症状小鼠模型及微塑料神经损伤效应验证研究证实，小胶质细胞M1激活是介导神经毒物致脑内慢性炎症的早期事件，可作为干预人类慢性神经退行性疾病及环境神经毒物神经毒性的有效靶点。