Huyang Yangkun Formula Enhances Ovarian Function and Delays Reproductive Aging by Influencing Hypothalamic GnRH/LH Pulse Release Through GAT-1/GABA/GABAAR2

Yang Li,1 Yin Peng,1 Guangning Nie,1 Fangping Cheng,1 Yuling Zhou,1 Jian Liu,1 Hongyan Yang1,2 1Department of Gynaecology, The Second Clinical Medical College, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510120, People’s Republic of China; 2Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, Guangzhou, Guangdong, 510120, People’s Republic of ChinaCorrespondence: Hongyan Yang; Jian Liu, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, No. 111 Dade Road, Yuexiu District, Guangzhou, 510120, People’s Republic of China, Tel +86-20-81887233, Email [email protected]; [email protected]: Huyang Yangkun Formula (HYF), based on Yin and Yang principles, is effective in treating Premature Ovarian Insufficiency (POI) and enhancing reproduction, but its mechanism needs to be clarified.Aim: This study investigates HYF’s impact on enhancing ovarian follicles development via GABA/GnRH neuron regulation.Materials and Methods: The chemical components of water extract of HYF were analyzed by combining the ultra-high performance liquid chromatography coupled with Q-Exactive mass spectrum analysis (UHPLC-QE-MS). Female SD rats received HYF for 90 days to assess changes in the ovarian function. In addition, female SD rats underwent ovariectomy to evaluated the impact of HYF or GABA treatments on LH release induced by E2 to address age-related neuroendocrine damage. Reproductive neuroendocrine markers were analyzed through immunohistochemistry and immunofluorescence.Results: HYF treatment improved the disrupted estrous cycle in aged female rats, breaking the continuous keratinized epithelial cell state and alternating between estrous periods. Aging reduces ovarian volume and follicle numbers, but after 3 months of HYF treatment, ovarian follicle development increased, and mature follicle and luteum numbers changed significantly. HYF improved sex hormone levels, advancing and enhancing the LH peak in older rats, thus boosting total 24-hour LH release. GABA injection into the lateral ventricle increased GnRH release in aging rats, highlighting GABA’s excitatory role. HYF also increased GnRH secretion by reducing GAT-1, leading to more GABA release, which activates GABAAR2 receptors in GnRH neurons, enhancing their function in the hypothalamic arcuate nucleus of elderly rats.Conclusion: HYF shows promise as a treatment for improving ovarian and reproductive neuroendocrine function in early reproductive aging, providing insights into its effects on hypothalamic GnRH/LH pulse release via the GAT-1/GABA/GABAAR2/GnRH pathway.Keywords: traditional Chinese medicine, Huyang yangkun formula, delays reproductive aging, GnRH/LH pulse, GABA neurons