ceRNA网络调控人工光环境下卵巢钟基因表达及中药干预的机制研究

Global light pollution is continuing to deteriorate. It disrupts the circadian rhythm of human life, resulting in dysfunction of the hypothalamic pituitary ovarian axis, female reproductive rhythm disorders, reducing fertility and affecting reproductive health of the offspring. Previous studies had shown that the estrous cycle of the female rats with light pollution was disturbed and the gene expression of miR-421-5P and clock in the ovary were abnormal. The sequential therapy of Chinese herbal medicine for nourishing yin and supplementing yang can reverse the above abnormality, improve the function of the ovary and reconstruct the rhythm of reproduction, but the mechanism of its action has not been elucidated. The competition of endogenous RNA (ceRNA) is a new mechanism of regulatory networks about ncRNA involved in the regulation of mRNA have received much concern in recent years. lncRNA as a ceRNA with the binding site of miRNA. lncRNA has combining pairs of complementary base of miRNA, thereby inhibits the silence effect of miRNA on the mRNA of the target, as to realize the regulation and control of gene expression. Based on our previous research, we put forward the hypothesis that this Chinese medicine could regulate the expression of the ovarian clock gene under light pollution in order to restore the reproductive rhythm of the ovary through the regulation of lncRNA-miR-421-5p-Clock ceRNA.

全球光污染正在持续恶化，它扰乱人类生命的昼夜节律，引起下丘脑-垂体-卵巢生殖轴功能异常，导致女性生殖节律紊乱，生育力降低，影响子代生殖健康。既往研究已证实，光污染雌性大鼠动情周期紊乱，卵巢miR-421-5P、钟基因表达异常。中药滋阴补阳方序贯治疗可逆转上述异常，改善卵巢功能，重建生殖节律，但其作用机制尚未能阐明。竞争性内源RNA（ceRNA）调控网络是近年倍受关注的ncRNA参与调控mRNA的新机制，lncRNA作为一种ceRNA，具有miRNA的结合位点，lncRNA通过与miRNA碱基互补配对的相结合，进而抑制miRNA对靶mRNA的沉默作用，实现对基因表达的调控。基于我们前期研究，提出中药滋阴补阳方序贯可能通过lncRNA-miR-421-5p-Clock ceRNA调控网络干预光污染下卵巢钟基因表达从而恢复卵巢生殖节律的设想，诠释中药调周的作用。

光污染严重扰乱人类生命的昼夜节律，诱导女性生殖节律紊乱、卵巢功能障碍，降低女性生育力，并影响子代生殖健康，但其作用机制和靶点尚未能阐明。本项目基于团队前期研究基础，提出光污染可能通过ceRNA调控网络干预卵巢生殖节律，影响卵巢功能，采用名医验方滋阴补阳方序贯可介导ceRNA网络恢复女性生殖节律，重建月经周期的科学假说。为阐明光污染扰乱女性生殖节律和卵巢功能的发病机制和天然中草药处方的研发提供新的思路和靶点。. 研究结果：①持续光照可诱导大鼠动情周期紊乱，体重下降，卵巢内有多个囊性扩张卵泡形成，颗粒细胞层数减少，排卵障碍，胚胎吸收率增加。滋阴补阳方序贯可以恢复大鼠动情周期、体质量、减少胚胎吸收率，恢复生殖周期节律紊乱；②持续光照导致大鼠性激素水平紊乱，FSH、LH、E2、AMH、INH-B水平显著降低。此外，持续光照扰乱了褪黑素分泌的昼低夜高节律。滋阴补阳方序贯干预后能显著增加FSH、LH、E2、AMH、INH-B水平，恢复褪黑素的节律分泌；③持续光照后大鼠卵巢lncRNA、circRNA、mRNA显著差异表达，导致大鼠日节律紊乱，LncRNA TCONS_00265853、circRNA_chr2表达显著下降；miR-3583-3p显著增加；钟基因Clock、Bmal1、Per1、Per2、RORA、NPAS2均呈现显著下降；凋亡因子Bax、Caspase3显著升高，Bcl-2表达下降；TUNEL显示细胞凋亡率显著增加。滋阴补阳方序贯可逆转上述因子的表达水平；④LncRNA TCONS_00265853可以竞争性结合miR-421-5p，circRNA_chr2竞争性结合miR-3583-3p，从而靶向调节钟基因Clock的表达，形成ceRNA网络参与调控生殖节律。滋阴补阳方序贯可通过lncRNA、circRNA_chr2参与的ceRNA调控网络调节卵巢功能。. 本课题研究结果证实ceRNA调控网络在光污染诱导的生殖节律紊乱中扮演重要角色，滋阴补阳方序贯可通过lncRNA、circRNA_chr2竞争性结合miR-421-5p、miR-4583-3p参与调控靶基因Clock的转录和表达，通过ceRNA网络调节性激素水平，恢复生殖节律。本项目为扩大名医验方的临床应用，挖掘中药恢复卵巢生殖节律的有效物质提供了科学依据。