TNFAIP1在增塑剂DEHP诱发学习记忆障碍中的作用与机制研究

Exposure to di-(2-ethylhexyl) phthalate (DEHP) plasticizer is closely related to learning and memory impairment. Our previous studies found that oxidative stress-induced upregulation of tumor necrosis factor α-induced protein 1 (TNFAIP1) mediated DEHP-induced neurotoxicity; DEHP downregulated estrogen receptor β (ERβ), and inhibited protein kinase CK2β and Akt/CREB pathway by upregulating TNFAIP1; TNFAIP1 interacted with CK2β, and TNFAIP1 promoted ubiquitin degradation of its interacting proteins. It is reported that antagonizing ERβ promoted expression of TNFAIP1; CK2 activated Akt. Based on these points, we proposed the four-stage mechanism hypothesis that DEHP firstly induced oxidative stress and inhibited ERβ, thereby upregulated TNFAIP1 expression and promoted its binding protein CK2 ubiquitin degradation. Then Akt/CREB pathway was inhibited, which resulted in synaptic plasticity reduction and neuronal apoptosis, ultimately induced learning and memory impairment. We will use molecular event blocking technology to explore the validity of the four-stage mechanism in vivo based on C57BL/6 mice and in vitro based on three-dimensional culture model of hippocampal slice. Our study will be helpful to further clarify the molecular mechanisms of environmental endocrine disruptors-induced learning and memory impairment and related neurological disorders. It will also provide a reference for developing new therapeutic targets of these diseases.

DEHP暴露与学习记忆障碍密切相关。我们前期发现氧化应激使TNFAIP1上调介导了DEHP诱发的神经毒性；DEHP下调ERβ，通过上调TNFAIP1抑制CK2β和Akt/CREB通路；TNFAIP1与CK2β存在相互作用，TNFAIP1促进其相互作用蛋白泛素化降解。同行发现拮抗ERβ促进TNFAIP1表达；CK2激活Akt。因此我们提出DEHP诱发学习记忆障碍的“四阶段机制”假说：DEHP诱导氧化应激和抑制ERβ，从而上调TNFAIP1表达，促进与其结合的CK2泛素化降解，进而抑制Akt/CREB通路，造成突触可塑性降低和神经元凋亡，最终诱发学习记忆障碍。本项目将在体内整体水平（C57BL/6小鼠）和体外器官水平（海马脑片三维培养模型）利用分子事件阻断技术探讨“四阶段机制”的正确性。这将有利于阐明环境内分泌干扰物诱发学习记忆障碍和相关神经疾病的分子机制，也为开发新的治疗靶点提供借鉴和参考。

邻苯二甲酸二（2-乙基己）酯（DEHP）是被广泛使用的增塑剂之一。作为一种环境内分泌干扰物，DEHP具有神经毒性。暴露于DEHP导致神经发育障碍、神经行为和认知受损，但其具体的分子机制尚不是很清楚。本项目首先完善了DEHP对青少年期C57BL/6小鼠学习记忆影响的研究，从行为学、组织病理和细胞分子水平评价了DEHP对C57BL/6小鼠的学习记忆障碍程度。Morris水迷宫实验结果发现暴露于0.5 mg/kg/day DEHP四周即能损伤小鼠的学习记忆能力，造成海马组织突触相关蛋白synapsin1、PSD95和BDNF表达下调。5 mg/kg/day DEHP可造成神经元数目减少、诱发神经细胞凋亡。接下来在小鼠体内整体水平、体外细胞水平和人3D类脑器官水平分析了肿瘤坏死因子α诱导蛋白1（TNFAIP1）在DEHP诱发神经毒性中的作用，结果发现一定暴露浓度的DEHP均能显著上调TNFAIP1的表达。在细胞水平深入研究了TNFAIP1在DEHP诱发神经毒性中的具体分子机制：DEHP通过下调miR-93的表达水平促进TNFAIP1的表达，进而通过泛素-蛋白酶体途径降解其相互作用蛋白CK2β和CAMKK1从而抑制Akt/CREB和CAMK4/CREB通路，造成神经细胞凋亡和突触可塑性的破坏，最终诱发神经毒性，引起学习记忆障碍。最后我们以TNFAIP1为治疗靶点寻找到潜在药物重楼皂苷II。动物体内实验表明重楼皂苷II可上调miR-93并抑制TNFAIP1的表达，降低血清中的炎症因子水平，在DEHP诱发的学习记忆障碍中有着很好的治疗作用。本项目的研究结果有助于阐明环境内分泌干扰物诱发学习记忆障碍和相关神经疾病的分子机制，并为开发相关神经疾病的新治疗靶点提供借鉴和参考。