Research and experimental verification of the molecular mechanism of berberine in improving premature ovarian failure based on network pharmacology

Based on the research methods of network pharmacology, this study analyzed the improvement effect of berberine (BBR) on premature ovarian failure (POF) and its molecular mechanism. Carry out GO and KEGG enrichment analysis by R language to obtain the potential targets and pathways of BBR in the improvement of POF. Use SD rats and ovarian granulosa cells (GCs) for experimental verification. ELISA was used to measure the content of related hormones in the serum, CCK-8 was used to measure cell viability, western blot was used to measure the content of the target protein in the ovaries and GCs, and q-RT-PCR was used to detect the expression of the target genes in the ovaries and GCs. Predicted by network pharmacology: PTEN, AKT1, FoxO1, FasL, and Bim are the targets with the highest relative correlation between BBR and POF. The results of experiments show that the treatment of low and medium doses of BBR can increase the ovarian index of rats; BBR can increase the levels of Estradiol (E₂) and Anti-Mullerian hormone (AMH) in the serum of rats and reduce the levels of Follicle stimulating hormone (FSH) and Luteinizing hormone (LH). BBR can increase the cell viability of GCs; BBR can inhibit the PTEN/AKT1/FoxO1 signaling pathway and its phosphorylation level and reduce the expression of Fas/FasL and Bim mRNA. Overall, BBR can promote the ovarian to maintain normal hormone levels, protect GCs, and enhance the function of POF.

本研究基于网络药理学研究方法，分析小檗碱（berberine, BBR）对早发性卵巢功能不全（premature ovarian failure, POF）的改善作用及其分子机制。通过R语言开展GO与KEGG富集分析，以获取BBR改善POF的潜在靶点与通路。采用SD大鼠及卵巢颗粒细胞（ovarian granulosa cells, GCs）进行实验验证：运用酶联免疫吸附实验（enzyme-linked immunosorbent assay, ELISA）检测血清相关激素含量，CCK-8法检测细胞活力，蛋白质印迹法（western blot）检测卵巢与GCs中的靶蛋白水平，实时荧光定量聚合酶链式反应（quantitative real-time polymerase chain reaction, q-RT-PCR）检测卵巢与GCs中的靶基因表达量。经网络药理学预测，PTEN、AKT1、FoxO1、FasL及Bim为BBR与POF关联度最高的靶点。实验结果表明：低、中剂量BBR干预可提升大鼠卵巢指数；BBR可升高大鼠血清雌二醇（Estradiol, E₂）与抗缪勒管激素（Anti-Mullerian hormone, AMH）水平，降低卵泡刺激素（Follicle stimulating hormone, FSH）与黄体生成素（Luteinizing hormone, LH）水平；BBR可提升GCs的细胞活力；BBR可抑制PTEN/AKT1/FoxO1信号通路及其磷酸化水平，并下调Fas/FasL与Bim的mRNA表达。综上，BBR可促进卵巢维持正常激素水平、保护GCs，进而改善POF的相关功能。