Hebei Key Laboratory of Integrative Medicine on Liver-Kidney Patterns, Institute of Integrative Medicine, College of Integrative Medicine, Hebei University of Chinese Medicine, Shijiazhuang, 050200, China.
Department of Biochemistry and Molecular Biology, College of Basic Medicine, Key Laboratory of Medical Biotechnology of Hebei Province, Key Laboratory of Neural and Vascular Biology of Ministry of Education, Hebei Medical University, Shijiazhuang, 050017, China.
J Ethnopharmacol. 2023 Oct 5;314:116608. doi: 10.1016/j.jep.2023.116608. Epub 2023 May 5.
Zigui-Yichong-Fang (ZGYCF) is a traditional Chinese medicine prescription for the treatment of infertility and premature ovarian insufficiency (POI). It is clinically used to regulate hormone levels, improve ovarian reserve and increase pregnancy rate. However, the exact mechanism of action is not yet clear.
This study aimed to explore the potential impact and mechanism of ZGYCF on POI, and provide a scientific basis for its clinical application.
UHPLC‒MS/MS was used to identify the main compounds of ZGYCF. Female 8-week-old C57BL/6N mice were randomized into four group containing the vehicle control (Veh) group, the cyclophosphamide (CTX) model group, the low-dose ZGYCF (CTX-ZG-L) group and the high-dose ZGYCF (CTX-ZG-H) group. A mouse POI model was induced with a single intraperitoneal injection of CTX, and the therapeutic effects of different doses of ZGYCF on POI were evaluated according to the ovarian weight coefficient, serum AMH, serum E, ovarian histomorphology and follicle counts. After the dose screening experiment, the CTX-ZG-L group was renamed the CTX-ZG group and subjected to follow-up experiments. RNA-seq was used to explore the mechanism of POI and the therapeutic mechanism of ZGYCF on POI in Veh group, CTX group and CTX-ZG group. The mechanism of action of ZGYCF on POI were determined by measuring serum hormone level, histomorphology, follicle counts, protein expression and acetylation modification in groups of Veh, CTX, CTX-ZG and CTX-ZG-Nam (SIRT1 inhibitor).
A total of 37 compounds in ZGYCF were identified. ZGYCF attenuated the morphological changes in ovarian tissue in POI model mice, increased serum AMH and E levels, reduced the damage to primordial follicles and other follicles at all stages, and protected ovarian reserve. RNA-seq results suggested that the genes expression of the PI3K signaling and apoptosis signaling pathways was increased in POI mice, while ZGYCF upregulated SIRT1 gene and the expression of estradiol, apoptosis inhibition and other signaling pathway genes. Immunohistochemical staining, TUNEL staining, Western blot analysis and immunoprecipitation results showed that in CTX group, SIRT1 expression and Foxo3a nuclei localization were decreased, while Ac-Foxo3a, p-AKT, p-Foxo3a and apoptotic markers were upregulated. After administration of ZGYCF, these conditions were reversed, however, after treatment with the SIRT1 inhibitor, the results were opposite to those of ZGYCF.
Acetylated Foxo3a plays an important role in the occurrence of POI. ZGYCF improves the ovarian reserve of CTX-induced POI mice by activating SIRT1-mediated deacetylation of Foxo3a, and played a role in the treatment of POI. SIRT1 may be a novel target for ZGYCF to ameliorate POI.
自拟益智充风方（ZGYCF）是一种治疗不孕症和卵巢早衰（POI）的中药方剂。临床上用于调节激素水平、改善卵巢储备功能并提高妊娠率。然而，其确切的作用机制尚不清楚。
本研究旨在探讨 ZGYCF 对 POI 的潜在影响及其作用机制，为其临床应用提供科学依据。
采用 UHPLC-MS/MS 鉴定 ZGYCF 的主要成分。将 8 周龄雌性 C57BL/6N 小鼠随机分为 4 组： vehicle 对照组（Veh）、环磷酰胺（CTX）模型组、低剂量 ZGYCF（CTX-ZG-L）组和高剂量 ZGYCF（CTX-ZG-H）组。通过单次腹腔注射 CTX 诱导小鼠 POI 模型，根据卵巢重量系数、血清 AMH、血清 E、卵巢组织形态和卵泡计数评估不同剂量 ZGYCF 对 POI 的治疗效果。在剂量筛选实验后，将 CTX-ZG-L 组更名为 CTX-ZG 组并进行后续实验。采用 RNA-seq 技术探讨 POI 的发生机制以及 ZGYCF 对 POI 的治疗机制。通过测量各组血清激素水平、组织形态学、卵泡计数、蛋白表达和乙酰化修饰，来确定 ZGYCF 对 POI 的作用机制。
ZGYCF 中鉴定出 37 种化合物。ZGYCF 可减轻 POI 模型小鼠卵巢组织的形态学变化，提高血清 AMH 和 E 水平，减少各期原始卵泡和其他卵泡的损伤，保护卵巢储备功能。RNA-seq 结果表明，PI3K 信号通路和细胞凋亡信号通路的基因表达在 POI 小鼠中增加，而 ZGYCF 上调了 SIRT1 基因和雌二醇、细胞凋亡抑制等信号通路基因的表达。免疫组织化学染色、TUNEL 染色、Western blot 分析和免疫沉淀结果显示，CTX 组 SIRT1 表达和 Foxo3a 核定位减少，而 Ac-Foxo3a、p-AKT、p-Foxo3a 和凋亡标志物上调。给予 ZGYCF 后，这些情况得到逆转，然而，在用 SIRT1 抑制剂治疗后，结果与 ZGYCF 相反。
乙酰化 Foxo3a 在 POI 的发生中起重要作用。ZGYCF 通过激活 SIRT1 介导的 Foxo3a 去乙酰化，改善 CTX 诱导的 POI 小鼠的卵巢储备功能，发挥 POI 治疗作用。SIRT1 可能是 ZGYCF 改善 POI 的一个新靶点。
