基于中医“嫉妒不孕”理论研究心理应激所致非可控性炎症对卵泡SIRT1介导的“自噬-凋亡稳态”的影响

In today's society, prolonged exposures to stressful situations of women have tremendous impacts on their reproductive health. Theory of Jealousy Infertility in TCM introduced that stagnation of liver-qi will result in infertility, but the specific mechanism is not yet clear. Based on this, the research hypothesizes that long-term psychological stress can lead to nonresolving inflammation on female body, inflammatory factors downregulate SIRT1, and the autophagy- apoptosis homeostasis are damaged in the process of follicular growth, leading to apoptosis pathway activated, which induce granule cells and oocyte apoptosis; While Kaiyu Zhongyu Decoction can inhibits inflammation through the regulation of SIRT1-mediated autophagy- apoptosis homeostasis, reduce the apoptosis of germ cells, and treat infertility. We confirm the hypotheses by testing protein and gene expression levels of cfDNA in follicular fluid, autophagic Atg12-Atg5 compound , LC3, Beclin1, SIRT1, AMPK, FoxO, Caspase-3, Bcl-2, Bax and other factor in ovarys, and apoptosis conditions of granule cells and oocytes in psychological stress murine model, three-dimensional constructs system of preantral follicles, and SIRT1 gene silencing murine model. We aim to supply new ideas for the treatment of infertility in the clinic, and provide scientific data support for TCM theory of emotional diseases.

当今社会女性长期处于高压力、高应激状态中，严重影响其生殖健康。中医学“嫉妒不孕”理论提出肝郁可导致不孕症，但具体机制尚未明确。基于此，本研究提出“长期心理应激可导致女性机体处于非可控性炎症状态，炎症因子下调SIRT1，破坏卵泡发育过程中“自噬-凋亡稳态”，导致细胞凋亡通路激活，诱导颗粒细胞及卵母细胞凋亡；而开郁种玉汤可通过调控SIRT1介导的“自噬-凋亡稳态”抑制炎症反应，减少生殖细胞凋亡，改善生殖结局”的假说。以心理应激小鼠模型、腔前卵泡三维培养体系、SIRT1基因沉默小鼠模型为研究对象，检测卵泡液中cfDNA、卵巢中自噬Atg12-Atg5复合物、LC3、Beclin1、SIRT1、AMPK、FoxO、Caspase-3、Bcl-2、Bax等因子蛋白及基因表达水平，颗粒细胞、卵母细胞的凋亡情况来验证该假说。为临床不孕症的治疗提供新思路，为中医“情志致病”理论提供科学数据支持。

当今社会女性长期处于高压力、高应激状态中，严重影响其生殖健康。中医学“嫉妒不孕”理论提出肝郁可导致不孕症，但具体机制尚未明确。基于此，本研究提出“长期心理应激可导致女性机体处于非可控性炎症状态，炎症因子下调SIRT1，破坏卵泡发育过程中“自噬-凋亡稳态”，导致细胞凋亡通路激活，诱导颗粒细胞及卵母细胞凋亡；而开郁种玉汤可通过调控SIRT1介导的“自噬-凋亡稳态”抑制炎症反应，减少生殖细胞凋亡，改善生殖结局”的假说。.本课题通过心理应激小鼠模型，研究发现心理应激导致小鼠血清IL-1β、IL-6、IL-8、TNF-α等炎症因子水平显著增加，抑制生殖激素FSH、LH、E2、AMH的产生，导致小鼠卵泡发育障碍，卵巢中Beclin1、LC3、Atg12 等自噬蛋白表达降低，凋亡蛋白Bax、Caspase3表达增加，抗凋亡蛋白 Bcl-2表达降低， SIRT1和FOXO表达显著下调。通过腔前卵泡三维培养体系以及SIRT1激活剂和抑制剂，研究发现，应激抑制卵泡生长，发育成熟以及排卵，并导致颗粒细胞凋亡蛋白表达增加，下调自噬蛋白表达，抑制SIRT1、AMPK、FOXO的表达。SIRT1激活剂可促进“自噬-凋亡稳态”恢复，从而改善应激导致的卵泡生长受限，促进卵泡发育成熟。通过SIRT1基因沉默小鼠模型，给予开郁种玉汤干预，研究证实开郁种玉汤通过上调SIRT1通路，改善心理应激介导的炎症，恢复卵泡发育过程中“自噬-凋亡稳态”，发挥治疗“嫉妒不孕”的作用。