Tian Li ¹ Yao Wei ² Beibie Jiao ² Rui Hao ² Beibei Zhou ² Xinlan Bian ¹ Peijuan Wang ² Yahong Zhou ³ Xia Sun ¹ Jian Zhang ^2,4*

• ¹ Nanjing Lishui District Hospital of Traditional Chinese Medicine, Nanjing, Liaoning Province, China
• ² Affliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Liaoning Province, China
• ³ Wuxi Hospital Affiliated to Nanjing University of Chinese Medicine, Wuxi, China
• ⁴ Jiangsu Provincial Hospital of Traditional Chinese Medicine, Nanjing, China

Premature ovarian insufficiency (POI) has affected about 3.7% of women of reproductive age and is a major factor contributing to infertility. Bushen Huoxue formula (BHF), a traditional Chinese medicine prescription, is clinically used to treat POI in China. This study aims to investigate the potential mechanisms of BHF in combating POI using corticosterone-induced rats and palmitic acid (PA)-challenged human ovarian granulosa cells (GCs). Initially, ultra performance liquid chromatography tandem mass spectrometry was employed to analyze the components of BHF. The effects and mechanisms of BHF were then assessed in rats and GCs. Our results shown that BHF effectively normalized serum hormone levels, including follicle-stimulating hormone (FSH), anti-Müllerian hormone (AMH), estradiol (E2), and luteinizing hormone (LH). Concurrently, BHF also significantly reduced follicular atresia and promoted cell proliferation while inhibiting apoptosis in POI rats.Furthermore, BHF mitigated ovarian lipid accumulation by modulating lipid metabolism, which included reducing lipid synthesis (expression of peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α), increasing lipid catabolism (expression of adipose triglyceride lipase), and enhancing lipid oxidation (expression of carnitine palmitoyl transferase 1A). Mechanistically, the therapeutic effects of BHF on POI were linked with alleviation of lipid depositioninduced reactive oxygen species (ROS) accumulation and excessive autophagy, corroborating the results in PA-challenged GCs. After treatment with elesclomol (a ROS inducer) and rapamycin (an autophagy inducer) in GCs, the effects of BHF were almost counteracted under model conditions. These findings suggest that BHF alleviates the symptoms of POI by altering lipid metabolism and reducing lipid accumulation-induced ROS and autophagy, offering evidence for BHF's efficacy in treating POI clinically.