Exploration of the Danggui Buxue Decoction Mechanism Regulating the Balance of ESR and AR in the TP53-AKT Signaling Pathway in the Prevention and Treatment of POF

Abstract

Objective: The purpose of this study was to explore the molecular mechanism of Danggui Buxue Decoction (DBD) intervening premature ovarian failure (POF).

Methods: 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.

Results: 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 E₂ and AMH, and protein expression levels of AKT1, ESR1, AR, and BCL2.

Conclusions: DBD and its active components could treat POF by regulating the balance of ESR and AR in TP53-AKT signaling pathway.

Figure 1

Active compounds-targets network diagram. Light blue represents targets, pink represents active compounds of Radix Astragali, and orange represents active compounds of Radix Angelicae sinensis.

Figure 2

Venn diagram of DBD and the intersection genes in POF. DBD: Danggui Buxue Decoction; POF, premature ovarian failure.

Figure 3

Active compounds-POF-targets network diagram. The light green circles represent targets, green V shapes represent Radix Astragali, orange V shapes represent Radix Angelicae sinensis, and the blue hexagon represents POF.

Figure 4

The PPI network of DBD-intermediating POF targets. (a) The PPI network of 36 targets of DBD intervening POF targets. The size and depth of the node color represent the scale of the degree value. (b) The PPI network of five core targets.

Figure 5

The histogram of the GO function of the POF targets mediated by DBD. The red indicates biological processes (BP), green indicates cell compositions (CC), and blue indicates molecular functions (MF).

Figure 6

KEGG pathway bubble chart of DBD intervening POF targets.

Figure 7

Molecular docking diagram of the core active compounds and the core targets.

Figure 8

(a) Standard quercetin chromatogram. (b) Quercetin chromatogram in DBD. (c) Standard kaempferol chromatogram. (d) Kaempferol chromatogram in DBD.

Figure 9

Effect of DBD on the estrous cycle in the rat POF model (n = 10). ^∗P < 0.05, compared with model group.

Figure 10

Effect of DBD on organ index in rat POF model. (a) Ovary index (n = 10). (b) Uterine index (n = 10). ^∗P < 0.05, compared to the model group.

Figure 11

Effect of DBD on serum levels of E₂, FSH, AMH, and LH in POF model rats. (a) Serum estradiol (E₂) (n = 10). (b) Serum antimullerian hormone (AMH) (n = 10). (c) Serum follicular stimulating hormone (FSH) (n = 10). (d) Serum luteinizing hormone (LH) (n = 10). ^∗P < 0.05, compared to model group.

Figure 12

Effects of DBD on the expression of AKT1, p53, ESR1, IL6, AR, BCL2, BAX, and Cytochrome C proteins in ovary tissues. (a) Western blot analysis of the proteins AKT1, p53, ESR1, and IL6 proteins in the ovaries tissue. (b) Western blot analysis of the proteins AR, BCL2, BAX, and Cytochrome C in the ovary tissue; (c-j): Protein expressions of AKT1, p53, ESR1, IL6, AR, BCL2, BAX, and Cytochrome C (n = 3); ^∗P < 0.05, compared to model group.