四物汤介导Nrf2/p62/PINK1通路调控线粒体功能改善卵巢功能减退的作用机制研究

Diminished ovarian reserve(DOR) is associated with oxidative damage. Mitochondria are the main source of free radicals and reactive oxygen species in cells. Autophagy can eliminate the waste generated by cellular metabolism and facilitate cell survival. Mitochondria autophagy is a biological process that by self-degrading of mitochondria so as to keep the steady state of cells against external stimulate. PINK1/ Parkin pathway is the main pathway of mitochondrial autophagy, p62 protein is one of the molecular markers of autophagy, Nrf2 can induce the expression of p62 at the transcriptional level, So it is a completely new approach to study the mechanism of anti DOR drugs from the pathway of Nrf2/p62/PINK1.Siwutang is a classic prescription. It has sufficient theoretical and clinical basis for the prevention and treatment of DOR. It can significantly improve the symptoms of ovarian dysfunction and avoid the side effects caused by estrogen replacement therapy. Previous studies have shown that Siwutang can regulate the expression of Nrf2 protein and has obvious anti-oxidation effects in vitro and in vivo, but the regulation mechanism of Nrf2/p62/PINK1 signaling pathway remains to be further studied. This project used D-galactose and cyclophosphamide to establish an DOR model to study whether Siwutang affects mitochondrial function through Nrf2/p62/PINK1 signaling pathway to counteract DOR caused by oxidative stress, so as to provide new ideas for the research of anti-ovarian aging drugs and provide a reference for the modern interpretation of the efficacy mechanism of the classic prescription- Siwu Decoction.

卵巢功能减退(DOR)与氧化损伤相关，线粒体是细胞中自由基和活性氧的主要来源，自噬可以清除细胞代谢中产生的废物而利于其生存，线粒体自噬是其应对外界刺激自我降解维持细胞稳态的生物过程，PINK1/Parkin通路是线粒体自噬的主要途径，p62蛋白是自噬的分子标志物之一，Nrf2可诱导p62的表达，从Nrf2/p62/PINK1通路入手研究抗DOR药物的作用机制是一个全新的思路方法。四物汤在DOR的防治上具有充分的理论和临床依据，能显著改善DOR症状，且避免激素替代疗法带来的副作用。前期研究显示四物汤能调控Nrf2蛋白的表达，具有明显的体内外抗氧化作用，但对Nrf2/p62/PINK1信号通路的调控机制还未知。本项目研究四物汤是否通过Nrf2/p62/PINK1信号通路影响线粒体功能以对抗氧化应激带来的卵巢功能衰退，为抗DOR药物的研究提供新思路，为经典名方四物汤功效机制的现代阐释提供参考。

氧化应激导致的线粒体功能异常是卵巢衰老过程中主要的影响因素，Nrf2不仅自身具有调控内源性抗氧化因子的转录而发挥抗氧化的作用，还能通过诱导p62的表达参与线粒体自噬而维持线粒体质量的稳态，发挥细胞保护作用，但是在过度刺激或慢性激活之下，Nrf2可能将其保护细胞的作用转变为对细胞的毒性作用，最后会导致退行性病变和组织的过早衰老甚至癌症的发生。四物汤在卵巢功能减退的治疗中具有深刻的传统理论和临床实践依据，但具体的作用机制尚不清楚。自噬是细胞降解细胞器和大分子等物质的主要手段，对于细胞的存活和凋亡具有双重调控作用，适度的自噬水平具有促进细胞存活的作用，过度的自噬诱导则能促进细胞的凋亡。PINK1/parkin通路是调控线粒体自噬最主要的通路，本项目基于Nrf2/p62/PINK1通路，推测四物汤可能通过介导Nrf2/p62/PINK1通路改善线粒体功能，促进卵巢发育而发挥改善卵巢功能减退的作用。研究结果表明：1）四物汤可提高卵巢功能减退模型大鼠卵巢组织及体外培养的卵巢颗粒细胞中雌激素的水平，降低促卵泡素和黄体生成素水平，促进卵巢颗粒细胞的发育；2）四物汤具有明显的体内外抗氧化作用，明显提高SOD和GSH-Px水平，降低MDA水平；3）四物汤可通过增强Nrf2，p62的表达，降低PINK1的表达抑制卵巢颗粒细胞的过度自噬，调节线粒体自噬平衡，控制线粒体质量，进而抵抗氧化应激对卵巢颗粒细胞及卵母细胞的损伤，改善卵巢衰老状态，验证了本项目所提出的科学假说，为四物汤改善卵巢功能减退作用机制的阐明提供了科学依据。