Liu Huaiquan, Yang Hong, Qin Zhong, Chen Yunzhi, Yu Haiyang, Li Wen, Zhu Xing, Cai Jingwen, Chen Jing, Zhang Mengzhi
Shandong Xiandai University, Jinan, Shandong 250104, China.
Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou 550025, China.
Evid Based Complement Alternat Med. 2021 Dec 30;2021:4862164. doi: 10.1155/2021/4862164. eCollection 2021.
The purpose of this study was to explore the molecular mechanism of Danggui Buxue Decoction (DBD) intervening premature ovarian failure (POF).
The active compounds-targets network, active compounds-POF-targets network, and protein-protein interaction (PPI) network were constructed by a network pharmacology approach: Gene Ontology (GO) function and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway analysis by DAVID 6.8 database. The molecular docking method was used to verify the interaction between core components of DBD and targets. Then, High-Performance Liquid Chromatography (HPLC) analysis was used to determine whether the DBD contained two key components including quercetin and kaempferol. Finally, the estrous cycle, organ index, ELISA, and western blot were used to verify that mechanism of DBD improved POF induced by cyclophosphamide (CTX) in rats.
Based on the network database including TCMSP, Swiss Target Prediction, DisGeNET, DrugBank, OMIM, and Malacard, we built the active compounds-targets network and active compounds-POF-targets network. We found that 2 core compounds (quercetin and kaempferol) and 5 critical targets (TP53, IL6, ESR1, AKT1, and AR) play an important role in the treatment of POF with DBD. The GO and KEGG enrichment analysis showed that the common targets involved a variety of signaling pathways, including the reactive oxygen species metabolic process, release of Cytochrome C from mitochondria and apoptotic signaling pathway, p53 signaling pathway, the PI3K-Akt signaling pathway, and the estrogen signaling pathway. The molecular docking showed that quercetin, kaempferol, and 5 critical targets had good results regarding the binding energy. Chromatography showed that DBD contained quercetin and kaempferol compounds, which was consistent with the database prediction results. Based on the above results, we found that the process of DBD interfering POF is closely related to the balance of ESR and AR in TP53-AKT signaling pathway and verified animal experiments. In animal experiments, we have shown that DBD and its active compounds can effectively improve estrus cycle of POF rats, inhibit serum levels of FSH and LH, protein expression levels of Cytochrome C, BAX, p53, and IL6, and promote ovary index, uterine index, serum levels of E and AMH, and protein expression levels of AKT1, ESR1, AR, and BCL2.
DBD and its active components could treat POF by regulating the balance of ESR and AR in TP53-AKT signaling pathway.
本研究旨在探讨当归补血汤（DBD）干预卵巢早衰（POF）的分子机制。
采用网络药理学方法构建活性成分-靶点网络、活性成分-POF靶点网络及蛋白质-蛋白质相互作用（PPI）网络：通过DAVID 6.8数据库进行基因本体（GO）功能和京都基因与基因组百科全书（KEGG）通路分析。运用分子对接方法验证DBD核心成分与靶点之间的相互作用。然后，采用高效液相色谱（HPLC）分析确定DBD是否含有槲皮素和山柰酚这两种关键成分。最后，通过动情周期、脏器指数、酶联免疫吸附测定（ELISA）及蛋白质免疫印迹法验证DBD改善环磷酰胺（CTX）诱导的大鼠POF的机制。
基于包括中药系统药理学数据库与分析平台（TCMSP）、瑞士靶点预测、疾病基因数据库（DisGeNET）、药物银行（DrugBank）、在线孟德尔遗传性人类疾病数据库（OMIM）和人类疾病数据库（Malacard）在内的网络数据库，构建了活性成分-靶点网络和活性成分-POF靶点网络。我们发现2种核心化合物（槲皮素和山柰酚）及5个关键靶点（TP53、白细胞介素6（IL6）、雌激素受体1（ESR1）、蛋白激酶B1（AKT1）和雄激素受体（AR））在DBD治疗POF中发挥重要作用。GO和KEGG富集分析表明，共同靶点涉及多种信号通路，包括活性氧代谢过程、细胞色素C从线粒体的释放及凋亡信号通路、p53信号通路、磷脂酰肌醇-3激酶-蛋白激酶B（PI3K-Akt）信号通路和雌激素信号通路。分子对接显示槲皮素、山柰酚与5个关键靶点在结合能方面具有良好结果。色谱分析表明DBD含有槲皮素和山柰酚化合物，这与数据库预测结果一致。基于上述结果，我们发现DBD干预POF的过程与TP53-AKT信号通路中ESR和AR的平衡密切相关，并通过动物实验进行了验证。在动物实验中，我们已表明DBD及其活性成分可有效改善POF大鼠的动情周期，抑制血清卵泡刺激素（FSH）和黄体生成素（LH）水平、细胞色素C、Bax、p53和IL6的蛋白表达水平，并提高卵巢指数、子宫指数、血清雌二醇（E）和抗苗勒管激素（AMH）水平以及AKT1、ESR1、AR和B细胞淋巴瘤/白血病-2（BCL2）的蛋白表达水平。
DBD及其活性成分可通过调节TP53-AKT信号通路中ESR和AR的平衡来治疗POF。
