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. 2025 Dec;63(1):201-217.
doi: 10.1080/13880209.2025.2485905. Epub 2025 Apr 12.

Integrated pharmacoanalysis, bioinformatics analysis, and experimental validation to identify the ingredients and mechanisms of Xiao-Luo-Wan in uterine fibroids treatment

Yonghui Yu  1 , Haojun Zhang  2 , Fang Yang  1 , Hong Liu  1
Affiliations

Integrated pharmacoanalysis, bioinformatics analysis, and experimental validation to identify the ingredients and mechanisms of Xiao-Luo-Wan in uterine fibroids treatment

Yonghui Yu et al. Pharm Biol. 2025 Dec.

Abstract

Context: Xiao-Luo-Wan (XLW), a classical prescription in traditional Chinese medicine, has therapeutic effects on uterine fibroids (UFs). Herein, its anti-UF effects were examined using a systematic pharmacological method.

Objective: To explore the active ingredients of XLW via mass spectrometry and its potential effects on UFs by network pharmacology, molecular docking, and experimental validation.

Materials and methods: A mass spectrometer was used to scrutinize the composition of the XLW drug-containing serum. The critical targets and potential mechanisms of XLW against UFs were predicted by network pharmacology and molecular docking. Next, human uterine leiomyoma cells (UMCs) were treated with 20%, 30%, or 40% XLW serum for 24 h, 48 h or 72 h. Cell viability was analyzed via a CCK-8 assay, and cell apoptosis and the cell cycle were examined via flow cytometry. The predicted targets were further identified by RT-PCR and western blotting.

Results: There were 16 chemical components identified in XLW drug-containing serum, with 53 target genes predicated in the treatment of UFs. The molecular binding of core targets, including TRIM9, NF-κB and p38MAPK, was relatively stable to components, especially buergerinin B, cedrol and ent-15B-16-epoxy- kauan-17-ol. The in vitro experiments revealed that the IC50 of XLW in UMCs was 63.21%, and the anti-UF effects of XLW may be closely associated with targets that inhibit cell proliferation and promote cell apoptosis by regulating TRIM9, NF-κB and p38MAPK expression.

Discussion and conclusions: The integration of mass spectrometry, network pharmacology, molecular docking and biological experiments revealed the key constituents of XLW and its pharmacological mechanism in UFs, which may help in the discovery of therapeutic agents for treating UFs.

Keywords: Xiao-Luo-wan (XLW); human uterine leiomyoma cells; mass spectrometry; molecular docking; network pharmacology; uterine fibroids.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Chemical components of the XLW decoction and XLW drug-containing serum. (A) Total ion chromatograph (TIC) of XLW in positive ion mode. (B) TIC image of XLW in negative ion mode. (C) Total ion flow diagram of the 18 F-FDG serum sample after electrospray ionization.
Figure 2.
Figure 2.
(A) Edwards’ Venn diagrams and (B, C) multivariate Venn diagram of the different components in the XLW decoction and XLW drug-containing serum. Herb-X (Xuanshen), Herb-S (Shengmuli) and Herb-Z (Zhebeimu) represent identified components of the XLW decoction in both positive and negative ion modes. Melt-X (Xuanshen), Melt-S (Shengmuli) and Melt-Z (Zhebeimu) represent identified components in the total ion model of XLW drug-containing serum. (D) Network relationships of XLW components in the decoction and drug-containing serum samples. The blue diamonds represent the components in the XLW decoction. The violet circles represent the 16 identified components in both the XLW decoction and the XLW drug-containing serum.
Figure 3.
Figure 3.
Chemical structures of the 16 identified chemical components in both the XLW decoction and the XLW drug-containing serum. (The chemical structures were purchased from PubChem at https://pubchem.ncbi.nlm.nih.gov/)
Figure 4.
Figure 4.
Network pharmacology analysis of XLW in the treatment of UFs. (A) Flower plot showing the number of potential targets for each detected active ingredient in XLW drug-containing serum. The petals show the number of target genes associated with each chemical ingredient in the XLW drug-containing serum. (B) The number of targets of XLW drug-treated serum in UFs is shown in a Venn diagram. The lilac circles represent targets of the XLW drug-containing serum in Figure 5(A) after eliminating duplicates, and the blue circles represent UF targets found in the DigSee, DrugBank and OMIM databases. (C) PPI network of the targets of XLW in treating UFs. The combined cores increase from white to dark blue in nodes and yellow to purple in edges, and larger nodes suggest more combined cores.
Figure 5.
Figure 5.
GO enrichment and KEGG pathway analyses of therapeutic targets of XLW in the treatment of UFs. (A) Color gradient bar chart of the results of the GO enrichment analysis. The P value of the enrichment analysis increased from blue to red, and the number of genes enriched in the GO term increased from short to long. Positive regulation of the MAPK Cascade is marked in the red box. (B) Bar graph of KEGG pathway annotations. The number of genes enriched in the KEGG pathway increased from short to long. (C) Circle diagram of the GO enrichment analysis results. From outside to inside, the first ring indicates the top 10 GO terms, the second ring indicates the total number of genes enriched in the specified GO terms, the third ring indicates the number of enriched genes in XLW drug-containing serum, the fourth ring indicates the rich factor (the number in the third ring/the number in the second ring) for each GO term, and the taller bar indicates a greater rich factor. (D) Sankey and dot plots of the results of the KEGG pathway analysis. The left Sankey diagram shows the genes contained in each pathway. The size of the bubble represents the number of genes, and the P value increases from red to green in the right bubble diagram. (E) Network visualization of 53 putative genes associated with GO terms and KEGG pathways. The red hexagons represent the genes. The purple, blue and green V-shaped labels on the left represent GO terms in the biological process (BP), cellular component (CC), and molecular function (MF) categories. The orange V-shaped label on the right represents the KEGG pathway.
Figure 6.
Figure 6.
The binding activities of 16 chemical components with each candidate protein target are shown in (A) a violin plot and (B) a matrix heatmap.
Figure 7.
Figure 7.
Molecular docking models of XLW components that bind to potential targets in 3-dimensional stereoimages. Molecular docking pattern diagrams of (A) TRIM9 and buergerinin B, (B) TRIM9 and cedrol, (C) TRIM9 and ent-15B-16-epoxy-kauan-17-ol, (D) NF-κB p65 and buergerinin B, (E) NF-κB p65 and cedrol, (F) NF-κB p65 and ent-15B-16-epoxy-kauan-17-ol, (G) p38MAPK and buergerinin B, (H) p38MAPK and cedrol, and (I) p38MAPK and ent-15B-epoxy-kauan-17-ol.
Figure 8.
Figure 8.
Effect of XLW drug-containing serum on UMC proliferation. (A) The viability of UMCs treated with different concentrations of XLW drug-containing serum for 24 h was examined via CCK-8 assays. (B) IC50 values of XLW drug-containing serum in UMCs were determined via a nonlinear regression analysis. (C) The effects of XLW drug-containing serum on the proliferation of UMCs at 24 h, 48 h and 72 h were examined via CCK-8 assays. *p < 0.05 compared with the control. RU-486: mifepristone; XLW-L: 20% XLW drug-containing serum; XLW-M: 30% XLW drug-containing serum; XLW-H: 40% XLW drug-containing serum.
Figure 9.
Figure 9.
Effects of XLW decoction-containing serum on UMC apoptosis and cell cycle progression. (A) XLW drug-containing serum affects the apoptosis of UMCs, as examined by annexin V-FITC/PI staining. (B) Effect of XLW drug-containing serum treatment on the percentage of UMCs in G0/G1, S and G2/M phases. *p < 0.05 compared with the control. #p < 0.05 compared with RU-486. RU-486: mifepristone; XLW-L: 20% XLW drug-containing serum; XLW-M: 30% XLW drug-containing serum; XLW-H: 40% XLW drug-containing serum.
Figure 10.
Figure 10.
Effects of XLW decoction-containing serum on the expression of mRNAs and proteins related to proliferation and apoptosis progression in UMCs. *p < 0.05 compared with the control. #p < 0.05 compared with RU-486. RU-486: mifepristone; XLW-L: 20% XLW drug-containing serum; XLW-M: 30% XLW drug-containing serum; XLW-H: 40% XLW drug-containing serum.
Figure 11.
Figure 11.
Effects of XLW decoction-treated serum on the (A) mRNA and (B) protein expression of TRIM9, NF-κB and p-p38MAPK in UMCs. *p < 0.05 compared with the control. #p < 0.05 compared with RU-486. RU-486: mifepristone; XLW-L: 20% XLW drug-containing serum; XLW-M: 30% XLW drug-containing serum; XLW-H: 40% XLW drug-containing serum.
Figure 12.
Figure 12.
The mechanistic diagram shows the active ingredients of XLW and their potential effects on UFs identified via a systematic pharmacological method.
See this image and copyright information in PMC

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