基于雌雄激素受体信号通路研究双酚A致生精障碍与肾虚不育的相关性

The spermatogenic impairment caused by classic environmental estrogen (EEs) : bisphenol A (BPA) , has become a research highlight both at home and abroad. Owing to many similarity ways were found between BPA-induced spermatogenic impairment and kidney deficiency infertility. Meanwhile, the specific protein of androgen and estrogen receptor signaling pathway have an important regulating role in spermatogenesis. What’s more, our study on the testicular of Kidney-yang deficiency infertility mice, differential proteins were found in estrogen and androgen receptor signaling pathways, and had some specific function. Accordingly, we propose the hypothesis that BPA-induced spermatogenic impairment is belong to the category of kidney deficiency infertility, and that the protein expression abnormality and function disorder in estrogen and androgen receptors signaling pathway is one of the important pathological mechanism of kidney deficiency infertility. The project is planned on the basis of our previous study, by using iTRAQ labeling, LC-ESI-MS/MS analysis and gene silencing in vivo and vitro experiments. Firstly, differential proteins would be identified in androgen and estrogen signaling pathway between the two model mice testes. Secondly, the appropriate database would be established, and marker proteins would be found. Thirdly, after Jingui Shenqi pill and Liuwei Dihuang pill intervention, the effects of marker protein in spermatogenesis would be observed in model mice. Hence， the relationship between BPA-induced spermatogenic impairment and kidney deficiency infertility would be clarified. In this way, not only the new connotation of Kidney controlling reproduction will be illustrated, but also new evidence will be provided for Kidney-tonifying Herbs prevention and treatment EEs-induced spermatogenesis impairment.

经典环境雌激素（EEs）双酚A（BPA）引起的生精障碍，已成为国内外研究热点。由于BPA致生精障碍与肾虚不育在诸多方面相似，且雌、雄激素及受体信号通路中特异蛋白在精子生成中具有重要调控作用，及我们在肾阳虚不育小鼠睾丸的雌、雄激素受体信号通路中发现有差异蛋白表达，并具有特定功能。故我们提出“BPA诱导的生精障碍属于肾虚不育范畴，雌、雄激素受体信号通路中标志蛋白表达异常及功能紊乱是肾虚不育的重要病理机制之一”的假说。本项目拟在前期研究基础上，通过体内外实验，采用iTRAQ标记、质谱分析及基因沉默技术，筛选BPA致生精障碍和肾虚不育模型小鼠睾丸中雌、雄激素信号通路的差异蛋白，建立相应数据库，寻找标志蛋白；观察金匮肾气丸和六味地黄丸对标志蛋白表达和功能的的影响，以期明确BPA致生精障碍与肾虚不育的关系，不仅为诠释“肾主生殖”的新内涵，而且为寻找补肾中药防治EEs引起生精障碍的新靶标，提供实验依据。

随着工业化进程的加剧，大量环境雌激素( environmental estrogens，EEs ) 释放造成的雄性生精功能障碍，因尚无行之有效的防治方法，已引起国内外生态学家及毒理学家的极大关注。由于典型 EEs--双酚A（Bisphenol -A, BPA）导致的生精障碍与肾虚不育不仅在性腺轴形态和功能的异常，尤其在行为学改变和补肾中药疗效等方面存在相似点，因此，研究 BPA 引起生精障碍与肾虚不育的关系，对探索补肾中药的防治作用及机制具有十分重要的意义。.本项目进行了3大部分研究：⑴ BPA染毒有与肾虚对雄性小鼠生殖功能影响的相关性研究，初步证实，100mg/kg BPA导致的生精障碍属于肾虚不育的范畴，且与肾阳虚更为相关。⑵ BPA 染毒有与肾虚对雄性小鼠生殖功能障碍的机制研究的相关性研究：进一步证实，① BPA染毒和肾虚均可通过引起睾丸Orm1蛋白表达上调及Apoa1、B2m、Svs5、Svs2、Svs4、Svs3a、Svs1、Svs6等蛋白在睾丸表达的下调，导致的小鼠生殖障碍；其中，Apoa1蛋白可能是其中最为重要的蛋白之一。② G-蛋白偶联雌激素受体（ G protein-coupled estrogen receptor , GPER）和雄激素受体（Androgen recepto , AR）在BPA和肾阳虚组小鼠睾丸的表达在均阳性表达上调，而肾阴虚组与BPA组相比明显减弱，肾阴虚小鼠睾丸丸间质细胞AR明显高于BPA组。③ 100mg/kg BPA可通过AR和AR磷酸化及其激酶Akt的磷酸化以显著诱导睾丸组织的生精细胞凋亡，与肾阳虚模型相关。进一步证实，100mg/kg BPA导致的生精障碍属于肾虚不育的范畴，且与肾阳虚更为相关。且睾丸GPER和AR及AR磷酸化表达改变是重要的病理机制之一。⑶ 补肾药物对BPA 染毒小鼠生殖功能的保护及机制研究：进一步反证，100mg/kg BPA导致的生精障碍属于肾虚不育的范畴，且与肾阳虚更为相关； 睾丸GPER和AR及AR磷酸化表达改变是重要的病理机制之一。.总之，本研究不仅为诠释“肾主生殖”的新内涵，而且为寻找补肾中药治疗 EEs 引起生精障碍的新靶标及其机制提供可靠的实验依据。