双酚A对雄激素调节神经行为和突触可塑性的影响及其机制研究

The effects of bisphenol A (BPA), an environmental endocrine disruptor, on the brain have attracted a growing attention. The previous researches have found that BPA sex-specifically affects the brain and behaviors of animal, and the male brain is more sensitive to BPA than that of females. Whereas earlier studies focused on the estrogenic properties of BPA. Recent studies revealed that BPA antagonizes androgen receptor (AR)-mediated transcriptional activities. Because androgens are just as critical in the cognitive functions and synaptogenesis in males as estrogens are in females, the potential exists that BPA also interferes with the physiology and morphology of the adult male brain. Thus, using castrated male rats, the following experiments were performed to investigate whether BPA interfered in androgen on the regulation of behaviours, including memory, anxiety, and depression, and synaptic plascity in hippocampus of adult male rats, brain areas with crucial influence on cognitive functions. The effects of BPA and androgen on structure modification of synapses will be examined using electron microscope. The protein expression and phosphorylation of synaptic proteins, NMDA and AMPA receptors, and signaling moleculars will be analyzed by Western bloting. Time-lapse imaging of living neurons and quantification will be used to examine the influence of BPA and androgen on growth of dendritic spin in the cultured hippocampus neurons expressing enhanced GFP. And changes of synaptogenesis were examed by double fluorescent labeling the cultured hippocampus neurons. In addition, the effect of BPA and androgen on efficiency of synaptic transmission will be measured by electrophysiological analyses of LTP of hippocampal slice. Furthermore, the signaling pathways through which BPA interferes in androgen on the regulation of behaviors and synaptic plasticity will be investigated using estrogen and androgen receptor antagonists, impermeable membrane BSA-BPA, inhibitors of MAPK/ERK, MAPK/p38, and CaMKII,respectively. The results of the present study will provide a scientific basis for prevention and control of adverse effects of BPA on brain.

环境内分泌干扰物双酚A对脑的影响引起广泛关注。双酚A影响脑和行为存在性别差异，雄性脑对双酚A更敏感。雄激素对雄性脑的调节与雌激素对雌性脑一样非常重要。但目前有关双酚A脑作用机制的研究主要聚焦在其雌激素活性上。本课题拟在前期研究基础上，以去势雄鼠模型研究双酚A对雄激素调节认知和情绪行为的影响；通过①检测去势雄鼠海马突触蛋白和突触形态可塑性，②用感染GFP病毒的海马神经元活细胞成像动态分析神经元树突生长，③双荧光标记分析海马神经元突触形成，④电生理技术检测海马脑片LTP，多层面分析无芳香化作用的5α-双氢睾酮对海马突触可塑性的影响以及双酚A的抗雄激素作用；最后，结合雌、雄激素受体拮抗剂、不透膜的BSA-双酚A、MAPK/ERK、MAPK/p38和CaMKII抑制剂，以及信号分子磷酸化检测等分析双酚A影响雄激素调节突触可塑性的信号通路，阐明双酚A影响雄性脑的分子机制，为双酚A毒性防治提供科学依据

环境内分泌干扰物双酚A(bisphenol A，BPA)的神经毒性已引起人们极大关注，鉴于BPA的弱雌激素活性(约为雌二醇的1/100-1/1000)，以往的研究主要聚焦在其拟雌激素和抗雌激素作用。但前期研究发现，雄性个体的许多神经行为对BPA 更敏感，因而推测BPA可能会干扰雄激素对脑的调节作用。.本课题参考人和野生动物日常暴露水平，设计了0.04、0.4、4 mg/kg/day 3个BPA暴露剂量。首先建立10周龄睾丸摘除的去势(GDX)雄鼠模型，通过对假手术(Sham)、GDX、去势后丙酸睾酮补充(GDX+TP)的雄鼠BPA暴露8周，发现0.4-4 mg/kg/day的BPA降低脑和血清睾酮水平，下调海马和杏仁核雄激素受体(AR)表达，抑制睾酮对去势雄鼠焦虑抑郁情绪和空间记忆损伤的改善，同时伴随海马突触界面结构的负性改变，突触蛋白、NMDA受体亚基下调，海马和杏仁核GABA(A)α受体水平和ERK1/2磷酸化水平下调。.应用离体培养的海马神经细胞和脑片进一步研究发现，10-100 nM的BPA增加树突和突触密度、高频刺激诱导的长时程增强(LTP)，该作用主要经雌激素受体(ERs)而不是AR介导的ERKs和p38信号通路进行；10-100 nM的BPA还可通过雌激素相关γ受体(EERγ)经ERKs和p38通路促进NMDA受体介导的Ca2+内流，这可能是低剂量BPA促进突触可塑性的一个重要机制。但在雌激素或雄激素环境中，10-100 nM的BPA却可拮抗17β-雌二醇(17β-E2)或二氢睾酮(DHT)对Ca2+内流、突触结构和活性的促进作用。.综上分析，本研究结果首次表明，环境暴露水平(nM级)的BPA可因环境条件而表现出不同的性激素干扰作用。在无性激素环境中，主要激活EERγ以及ERs而表现出拟雌激素作用,促进海马神经元谷氨酸诱导的Ca2+内流，促进突触结构和功能可塑性；而在雌激素或雄激素环境下，BPA表现出抗雌激素或抗雄激素活性，拮抗17β-E2或DHT对突触结构和功能可塑性的调节作用。这使长期BPA暴露的人或野生动物因雌激素或雄激素对神经元突触可塑性调节作用的扰乱而表现出脑功能损伤和神经行为改变。高剂量BPA(1-10 μM)抑制海马神经元NMDA受体介导的Ca2+内流、降低树突和突触密度以及LTP诱导，但由于环境中的BPA水平远低于这个剂量范围，对非职业