Abstract Objective This study aimed to investigate the mechanisms through which Erzhi Tiangui Formula (EZTG) mitigates age-related ovarian aging in mice through the Nrf2/HO-1 antioxidant pathway. Methods A total of 100 C57BL/6J mice were divided into young control (YC, 6–8 weeks, n = 20) and aged groups (8–9 months, n = 80). Aged mice were randomized into four subgroups: control, low-(0.05 g/mL), medium- (0.2 g/mL), and high-dose (0.8 g/mL) EZTG groups ( n = 20/group). After 10 days of treatment, ovarian morphology and follicular counts were assessed. On day 11, the remaining mice received an intraperitoneal injection of human menopausal gonadotropin (hMG, 15 IU), followed by human chorionic gonadotropin (hCG, 12 IU) 48 h later. 14 h after hCG administration, the mice were euthanized under deep anesthesia via cervical dislocation, and granulosa cells were collected. The following parameters were evaluated: oxidative stress markers (ROS), Nrf2/HO-1 pathway components (Nrf2, HO-1, Keap1), apoptosis factors (Bax, Bcl-2, cleaved Caspase-3), antioxidant enzymes (SOD, CAT, GPX1), and mitochondrial function (mitochondrial membrane potential, MMP; ATP). In vitro, KGN human granulosa cells were subjected to H₂O₂-induced oxidative stress with or without EZTG-containing serum, and Nrf2 was knocked down by siRNA to validate pathway specificity. Results EZTG treatment significantly improved multiple parameters in a dose-dependent manner. Treated mice exhibited restored estrous cyclicity and enhanced follicular development with reduced atresia. At the cellular level, EZTG effectively decreased ROS accumulation while upregulating antioxidant enzymes, including SOD, CAT, and GPX1. Molecular analyses revealed activation of the Nrf2/HO-1 signaling pathway, as evidenced by increased Nrf2 and HO-1 expression alongside Keap1 downregulation. Additionally, EZTG exerted an anti-apoptotic effect, demonstrated by upregulated Bcl-2 expression and downregulated Bax and cleaved Caspase-3. Mitochondrial function assays further revealed that EZTG significantly improved MMP and ATP production in granulosa cells. Notably, the medium- and high-dose groups consistently outperformed the low-dose group across all measured outcomes, reinforcing a clear dose-response relationship. Conclusion EZTG attenuates ovarian aging by coordinated activation of the Nrf2/HO-1 pathway, enhancing antioxidant defenses, inhibiting apoptosis, and restoring mitochondrial function in a dose-dependent manner. These findings provide mechanistic insights into EZTG’s potential as a therapeutic intervention for age-related ovarian decline. Clinical trial number not applicable.
Xiufang et al. (Sat,) studied this question.