Erzhi Tiangui formula ameliorates ovarian aging by enhancing antioxidant defense via Nrf2/HO-1 signaling.

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.

Key Findings

• Erzhi Tiangui Formula (EZTG) treatment dose-dependently improved ovarian morphology and follicular development in aged mice.
• EZTG reduced reactive oxygen species (ROS) accumulation and upregulated antioxidant enzymes (SOD, CAT, GPX1).
• EZTG activated the Nrf2/HO-1 signaling pathway, increasing Nrf2 and HO-1 expression while decreasing Keap1 levels.
• EZTG exerted anti-apoptotic effects by increasing Bcl-2 and decreasing Bax and cleaved Caspase-3 expression.
• Mitochondrial function was improved with EZTG treatment, as indicated by enhanced mitochondrial membrane potential and ATP levels.

Clinical Significance

Citation

Li Xiufang, Wang Zhongqing, Zhang Xiuqinget al.. Erzhi Tiangui formula ameliorates ovarian aging by enhancing antioxidant defense via Nrf2/HO-1 signaling. Journal of ovarian research. 2026-Jul-18.