Supplementary Material for: Effect of Hawthorn Leaf Flavonoids in Dehydroepiandrosterone-Induced Polycystic Ovary Syndrome in Rats

Objective: To evaluate the potential beneficial effects of hawthorn leaf flavonoids (HLF) against polycystic ovary syndrome (PCOS) in a rat model of disease and elucidate the underlying molecular mechanism. Methods: The PCOS model was established by subcutaneous injection of dehydroepiandrosterone (DHEA, 60 mg/kg/day) for 21 consecutive days. HLF (200 mg/kg/day) were orally administered simultaneously or after the injection. The body weight was regularly monitored and recorded. The ovaries were weighed and histologically examined via hematoxylin and eosin staining. The number of follicular cysts was counted under a light microscope. The serous hormones were measured using enzyme-linked immunosorbent assay kits. Insulin resistance (IR) was calculated as HOMA-IR = fasting insulin (µU/L) × fasting glucose (mM)/22.5. The estrous cycle was determined by vaginal smear. The relative expression of tumor necrosis factor-α and interleukin-6 was measured by real-time polymerase chain reaction. The superoxide dismutase activity and malondialdehyde content was determined using commercially available kits. Results: DHEA induced a significant increase of body weight, ovary weight, number of follicular cysts, serous hormones, IR, inflammatory cytokines, and oxidative stress, and it also impaired the estrous cycle. Oral administration of HLF greatly alleviated these complications. Little toxicity of HLF was observed in our rat model. Conclusion: HLF manifest protective effects against PCOS progression in the animal model, which may hold great promise for future clinical applications