Gao Ting, Zhong Jia-Wen, Qin Ling, Wang Xue-Yi, Li Xiao-Rong, Luo Yu-Xue
School of Traditional Chinese Medicine, Ningxia Medical University Yinchuan 750004, China Key Laboratory of Fertility Maintenance, Ningxia Medical University Yinchuan 750004, China Key Laboratory of the Ministry of Education for the Modernization of Ethnic Minority Medicine, Ningxia Medical University Yinchuan 750004, China.
Yinchuan Hospital of Traditional Chinese Medicine Yinchuan 750000, China.
Zhongguo Zhong Yao Za Zhi. 2023 Jun;48(12):3224-3234. doi: 10.19540/j.cnki.cjcmm.20230202.704.
This study aims to investigate the efficacy and possible mechanism of Liuwei Dihuang Pills in the treatment of diminished ovarian reserve(DOR) by using proteomic techniques. Firstly, cyclophosphamide(60 mg·kg(-1)) combined with busulfan(6 mg·kg(-1)) was injected intraperitoneally to establish the mouse model of DOR. After drug injection, the mice were continuously observed and the success of modeling was evaluated by the disturbance of the estrous cycle. After successful modeling, the mice were administrated with the suspension of Liuwei Dihuang Pills by gavage for 28 days. At the end of the gavage, four female mice were selected and caged together with males at a ratio of 2∶1 for the determination of the pregnancy rate. Blood and ovary samples were collected from the remaining mice on the next day after the end of gavage. Hematoxylin-eosin(HE) staining and transmission electron microscopy(TEM) were then employed to observe the morphological and ultrastructural changes in the ovaries. The serum levels of hormones and oxidation indicators were measured by enzyme-linked immunosorbent assay. Quantitative proteomics techniques were used to compare the ovarian protein expression before and after modeling and before and after the intervention with Liuwei Dihuang Pills. The results showed that Liuwei Dihuang Pills regulated the estrous cycle of DOR mice, elevated the serum levels of hormones and anti-oxidation indicators, promoted follicle development, protected the mitochondrial morphology of ovarian granulosa cells, and increased the litter size and survival of DOR mice. Furthermore, Liuwei Dihuang Pills negatively regulated the expression of 12 differentially expressed proteins associated with DOR, which were mainly involved in lipid catabolism, inflammatory response, immune regulation, and coenzyme biosynthesis. These differentially expressed proteins were significantly enriched in sphingolipid metabolism, arachidonic acid metabolism, ribosomes, ferroptosis, and cGMP-PKG signaling pathway. In summary, the occurrence of DOR and the treatment of DOR with Liuwei Dihuang Pills are associated with multiple biological pathways, mainly including oxidative stress response, inflammatory response, and immune regulation. "Mitochondria-oxidative stress-apoptosis" is the key to the treatment of DOR by Liuwei Dihuang Pills. YY1 and CYP4F3 may be the key upstream targets that trigger mitochondrial dysfunction and ROS accumulation, and the metabolism of arachidonic acid is the main signaling pathway of drug action.
本研究旨在运用蛋白质组学技术探讨六味地黄丸治疗卵巢储备功能下降（DOR）的疗效及可能机制。首先，腹腔注射环磷酰胺（60 mg·kg⁻¹）联合白消安（6 mg·kg⁻¹）建立DOR小鼠模型。注射药物后，持续观察小鼠并通过动情周期紊乱评估建模是否成功。建模成功后，给予小鼠灌胃六味地黄丸混悬液，持续28天。灌胃结束时，选取4只雌性小鼠，以2∶1的比例与雄性小鼠合笼，测定妊娠率。在灌胃结束后的次日，从其余小鼠采集血液和卵巢样本。随后采用苏木精-伊红（HE）染色和透射电子显微镜（TEM）观察卵巢的形态和超微结构变化。通过酶联免疫吸附测定法检测血清激素水平和氧化指标。运用定量蛋白质组学技术比较建模前后以及六味地黄丸干预前后卵巢蛋白质表达情况。结果显示，六味地黄丸可调节DOR小鼠的动情周期，提高血清激素水平和抗氧化指标，促进卵泡发育，保护卵巢颗粒细胞的线粒体形态，并增加DOR小鼠的产仔数和仔鼠存活率。此外，六味地黄丸对12种与DOR相关的差异表达蛋白的表达具有负调控作用，这些蛋白主要参与脂质分解代谢、炎症反应、免疫调节和辅酶生物合成。这些差异表达蛋白在鞘脂代谢、花生四烯酸代谢、核糖体、铁死亡和cGMP-PKG信号通路中显著富集。综上所述，DOR的发生及六味地黄丸对DOR的治疗与多种生物学途径相关，主要包括氧化应激反应、炎症反应和免疫调节。“线粒体-氧化应激-凋亡”是六味地黄丸治疗DOR的关键。YY1和CYP4F3可能是引发线粒体功能障碍和ROS积累的关键上游靶点，花生四烯酸代谢是药物作用的主要信号通路。
