基于LHR-cAMP信号的LPS间接作用抑制鹅等级卵泡P4合成的机制研究

We observed that P4 synthesis in goose hierarchiacal follicular granulose cell was indirectly inhibited at 6 h after lippolysaccharide (LPS) treatment during the same reproductive period. However, the underlying mechanisms are unknown. LHR-cAMP signaling pathway is essential for avian follicular granulose cell P4 synthesis. It is proposed that LPS inhibits expression of key enzymes associated with P4 synthesis through LH receptor signaling due to changed circulating LH and proinflammatory cytokines levels, which results in decreased P4 synthesis. In view of the above, this project planes to further investigate the plasma concentrations of LH and proinflammatory cytokines, expression level of LHβ in pituitary, and expression levels of LHR, proinflammatory cytokine receptors, and key enzymes associated with cAMP signaling pathway and P4 synthesis in hierarchical follicular granulosa layer. Furthermore, granulosa cells from goose hierarchiacal folicles treated by LPS for 0 and 6 h would be cultured with and without the addition of proinflammatory cytokine, respectively. The capacity of P4 synthesis in granulosa cells would be determined after gradually enhancing the LHR-cAMP signaling. These works will elucidate the LHR-cAMP signaling pathway in which LPS inhibits goose hierarchiacal follicular P4 synthesis. Finally, the molecular mechanisms of indirect inhibitory effect of LPS on goose hierarchiacal follicular P4 synthesis would be discovered according to the results of epigenetic modification of promoter region of key enzymes associated with P4 synthesis. This project will provide theoretical foundations for preventing ovarian dysfunction induced by gram-negative bacterium.

申请人发现在鹅同一生殖生理期处理6h，存在LPS间接作用抑制等级卵泡颗粒层P4合成的现象，但其作用机制不清楚。禽类卵泡颗粒细胞LHR-cAMP信号通路对P4合成至关重要。LPS可能影响了LH和炎性因子生成，间接通过颗粒细胞LHR-cAMP信号抑制P4合成限速酶表达，最终抑制P4合成。本项目拟进一步检测血液LH和炎性因子水平、垂体LHβ表达以及不同等级卵泡颗粒层LHR、炎性因子受体、cAMP信号和P4合成关键酶表达；再培养体内LPS处理的颗粒细胞和体外炎性因子处理的颗粒细胞，并梯度增强LHR-cAMP信号，检测P4合成能力，证明LHR-cAMP信号介导LPS间接作用抑制颗粒细胞P4合成；最后结合P4合成限速酶基因启动子区表观遗传修饰水平，揭示LPS间接作用抑制鹅等级卵泡颗粒细胞P4合成的分子机制。预期结果为预防LPS诱发的鹅卵巢功能紊乱提供理论依据。

脂多糖（LPS）是革兰氏阴性菌细胞壁的主要成分，细菌和LPS感染已被证明是高密度养殖和夏季种鹅产蛋性能下降的重要原因。类固醇激素P4和E2参与调控种鹅的产蛋性能，前期研究表明LPS处理显著抑制血液P4和E2水平，但是其中的分子机制不明。因此，本项目拟从LPS处理后HPG轴基因表达和促性腺激素分泌、卵泡颗粒细胞全转录组（lncRNA、miRNA和mRNA）以及LPS-miRNA轴调控颗粒细胞功能等三方面开展工作，以期揭示LPS抑制鹅卵泡类固醇激素合成和种鹅产蛋性能的分子机制。首先，HPG轴基因表达和血液内分泌激素的研究表明，LPS处理提高种鹅血液的FSH和LH浓度，与之对应的是垂体FSHβ和LHβ基因表达显著上升；LPS处理显著提高GnIH和GnIHR的表达，抑制GnRH和GnRHR的表达；在卵巢方面，LPS处理显著抑制卵泡FSHR、LHR和类固醇合成酶的表达。其次，LPS处理后0、6 、12、24、36 h等5个时间点的鹅卵泡全转录组分析表明，差异表达的lncRNA和miRNA的靶基因显著富集于细胞凋亡、胆固醇合成、免疫、生殖过程和生殖等过程。再次，生物信息学分析结果表明miR-10a是LPS处理后靶向类固醇合成酶和类固醇转录因子的差异miRNA，在鹅颗粒细胞过表达miR-10a可以抑制P4和E2合成限速酶和相关转录因子，干扰miR-10a则可缓解LPS抑制的P4和E2合成。以上研究从不同角度初步阐明了LPS影响鹅类固醇激素合成的细胞和分子机制。