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

<b><i>Objective:</i></b> 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. <b><i>Methods:</i></b> 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. <b><i>Results:</i></b> 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. <b><i>Conclusion:</i></b> HLF manifest protective effects against PCOS progression in the animal model, which may hold great promise for future clinical applications.

<b><i>研究目的：</i></b> 本研究旨在评估山楂叶总黄酮（hawthorn leaf flavonoids, HLF）对多囊卵巢综合征（polycystic ovary syndrome, PCOS）大鼠模型的潜在防治效应，并阐明其潜在分子机制。<b><i>实验方法：</i></b> 连续21天皮下注射脱氢表雄酮（dehydroepiandrosterone, DHEA，60 mg/kg/天）构建PCOS大鼠模型。分别于造模同时或造模结束后，以200 mg/kg/天的剂量灌胃给予HLF。定期监测并记录大鼠体质量；称量卵巢重量，采用苏木精-伊红染色法进行组织学检查，在光学显微镜下计数卵泡囊肿数量；采用酶联免疫吸附试剂盒检测血清激素水平；通过公式HOMA-IR = 空腹胰岛素（µU/L）× 空腹血糖（mM）/22.5计算胰岛素抵抗（insulin resistance, IR）程度；采用阴道涂片法确定大鼠动情周期；通过实时聚合酶链式反应检测肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）与白细胞介素-6（interleukin-6, IL-6）的相对表达量；采用商品化试剂盒检测超氧化物歧化酶（superoxide dismutase, SOD）活性与丙二醛（malondialdehyde, MDA）含量。<b><i>实验结果：</i></b> 脱氢表雄酮诱导造模可显著升高大鼠体质量、卵巢重量、卵泡囊肿数量、血清激素水平、胰岛素抵抗指数、炎症因子水平及氧化应激指标水平，同时破坏大鼠动情周期。灌胃给予HLF可显著缓解上述各类并发症，且本大鼠模型中未观察到HLF存在明显毒性。<b><i>研究结论：</i></b> HLF对该动物模型中的PCOS进展具有保护作用，有望为未来临床应用提供良好前景。