Mechanism Study of Cinnamomi Ramulus and Paris polyphylla Sm. Drug Pair in the Treatment of Adenomyosis by Network Pharmacology and Experimental Validation

Abstract

Objective: To explore the molecular mechanism of the Cinnamomi ramulus and Paris polyphylla Sm. (C-P) drug pair in the treatment of adenomyosis (AM) based on network pharmacology and animal experiments.

Methods: Via a network pharmacology strategy, a drug-component-target-disease network (D-C-T-D) and protein-protein interaction (PPI) network were constructed to explore the core components and key targets of C-P drug pair therapy for AM, and the core components and key targets were verified by molecular docking. Based on the results of network pharmacology, animal experiments were performed for further verification. The therapeutic effect of the C-P drug pair on uterine ectopic lesions was evaluated in a constructed AM rat model.

Results: A total of 30 components and 45 corresponding targets of C-P in the treatment of AM were obtained through network pharmacology. In the D-C-T-D network and PPI network, 5 core components and 10 key targets were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the PI3K signaling pathway was the most significantly enriched nontumor pathway. Molecular docking showed that most of the core components and key targets docked completely. Animal experiments showed that the C-P drug pair significantly ameliorated the pathological changes of endometriotic lesions in AM model rats and inhibited PI3K and Akt gene expression, and PI3K and Akt protein phosphorylation. In addition, treatment with the C-P drug pair promoted AM cell apoptosis; upregulated the protein expression of Bax, Caspase-3, and cleaved Caspase-9; and restrained Bcl-2 expression.

Conclusions: We propose that the pharmacological mechanism of the C-P drug pair in the treatment of AM is related to inhibition of the PI3K/Akt pathway and promotion of apoptosis in AM ectopic lesions.

Figure 1

Venn diagram and drug-active component-target-disease network. (a) The Venn diagram shows 45 overlapping targets between AM-related targets and active compound-related targets. (b) Drug-component-target-disease (D-C-T-D) network of C-P in the treatment of AM. The blue nodes represent drugs, the yellow nodes represent active components, the dark green nodes represent targets, and the red nodes represent disease.

Figure 2

PPI network and core targets. (a) PPI network of potential C-P therapeutic targets for AM. (b) 10 core targets in the PPI network.

Figure 3

Bubble charts. (a) Bubble chart for GO enrichment analysis. (b) Bubble chart for KEGG enrichment analysis. The closer the bubble color is to red, the smaller the P value is, and the bubble size reflects the number of enriched genes.

Figure 4

Results of molecular docking. (a) Heatmap of binding energy. Unit: kcal/mol. (b) Molecular docking of PIK3CA with 20-hydroxyecdysone. (c) Molecular docking of PIK3CA with diosgenin. (d) Molecular docking of PIK3CA with ecdysone. (e) Molecular docking of PIK3CA with pinnatasterone. (f) Molecular docking of PIK3CA with flavone. (g) Molecular docking of PTGS2 with diosgenin.

Figure 5

H&E staining and results of real-time PCR. (a) H&E staining. (b)The mRNA expression of PIK3CA and Akt1 (compared with control group, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. Compared with model group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001).

Figure 6

Results of western blotting assays. (a) The expression of p-PI3K and p-Akt. (b) The expression of Bax, Bcl-2, Caspase-9 and C-Caspase-9 (compared with control group, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. Compared with model group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001).

Figure 7

The results of immunohistochemistry. (a) Bax. (b) Bcl-2. (c) Caspase-3. (d) Caspase-9. (e) Quantitative analysis of immunohistochemistry (compared with control group, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. Compared with model group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001).