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. 2022 Nov 18:13:1038656.
doi: 10.3389/fphar.2022.1038656. eCollection 2022.

Combined metabolomics and network pharmacology to elucidate the mechanisms of Dracorhodin Perchlorate in treating diabetic foot ulcer rats

Pin Deng  1   2 , Huan Liang  3 , Shulong Wang  1   2 , Ruinan Hao  4   5 , Jinglu Han  1   2 , Xiaojie Sun  1   2 , Xuyue Pan  2 , Dongxiao Li  1   2 , Yinwen Wu  1 , Zhichao Huang  1   2 , Jiajia Xue  4   5 , Zhaojun Chen  2
Affiliations

Combined metabolomics and network pharmacology to elucidate the mechanisms of Dracorhodin Perchlorate in treating diabetic foot ulcer rats

Pin Deng et al. Front Pharmacol. .

Abstract

Background: Diabetic foot ulcer (DFU) is a severe chronic complication of diabetes, that can result in disability or death. Dracorhodin Perchlorate (DP) is effective for treating DFU, but the potential mechanisms need to be investigated. We aimed to explore the mechanisms underlying the acceleration of wound healing in DFU by the topical application of DP through the combination of metabolomics and network pharmacology. Methods: A DFU rat model was established, and the rate of ulcer wound healing was assessed. Different metabolites were found in the skin tissues of each group, and MetaboAnalyst was performed to analyse metabolic pathways. The candidate targets of DP in the treatment of DFU were screened using network pharmacology. Cytoscape was applied to construct an integrated network of metabolomics and network pharmacology. Moreover, the obtained hub targets were validated using molecular docking. After the topical application of DP, blood glucose, the rate of wound healing and pro-inflammatory cytokine levels were assessed. Results: The levels of IL-1, hs-CRP and TNF-α of the Adm group were significantly downregulated. A total of 114 metabolites were identified. These could be important to the therapeutic effects of DP in the treatment of DFU. Based on the network pharmacology, seven hub genes were found, which were partially consistent with the metabolomics results. We focused on four hub targets by further integrated analysis, namely, PAH, GSTM1, DHFR and CAT, and the crucial metabolites and pathways. Molecular docking results demonstrated that DP was well combined with the hub targets. Conclusion: Our research based on metabolomics and network pharmacology demonstrated that DP improves wound healing in DFU through multiple targets and pathways, and it can potentially be used for DFU treatment.

Keywords: Dracorhodin Perchlorate; diabetic foot ulcer; mechanisms; metabolomics; network pharmacology.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The flowchart of the integrated methods. The mechanisms of DP in treating DFU was explored with metabolomics of skin tissues (Part 1). Network pharmacology was performed to extract hub gene (Part 2). Through Part 1 and 2, important targets and metabolites were identified and connected. Molecular docking was conducted to further verify these core targets (Part 3).
FIGURE 2
FIGURE 2
Effect of DP with different concentration on fibroblast viability in different days. *p<0.05, **p<0.01 and ***p<0.001.
FIGURE 3
FIGURE 3
DP improves the wounds of DFU rats. (A) The time diagram of the experiment. (B) Representative figures of different time points of healing area of DFU rats. (C) Wound closure (%) of different groups at days 0, 3, 7, 14, 21 and 28. *p < 0.05 represent significance. (D) Images present the levels of FBG on 0th, 3th, 7th, 10th, 14th, 21st and 28th day.
FIGURE 4
FIGURE 4
H&E staining, Masson staining and cytokine assay of different groups of wound tissues. (A–B) Representative images of wound sections with H&E staining on day 28 (scale bar = 5 mm, scale bar = 500 μm). Red arrow indicates regenerating epidermis. (C) Thickness of epidermis (*p < 0.05, **p < 0.01 and ***p < 0.001). (D–E) Representative images of wound sections with Masson’s trichrome staining on day 28 (Masson’s trichrome: scale bar = 5 mm, scale bar = 1,000 μm). The blue colored ones are collagen fibres and collagen bundles, and the red ones are muscle fibres. (F) Fraction of collagen volume in wound tissue (*p < 0.05, **p < 0.01 and ***p < 0.001). (G) The level of TNF-α of in each group. (H) The level of IL-1 of in each group. (I) The level of hs-CRP of in each group.
FIGURE 5
FIGURE 5
Score chart of PCA, PLS-DA model and PLS-DA model response permutation testing (RPT). (A) Score chart of PCA analysis model pos and neg (B). Score chart of PLS-DA analysis model pos and neg. (C) PLS-DA model validation diagram.
FIGURE 6
FIGURE 6
The differential metabolites in DFU rats treated with DP. (A) Venn diagrams of the common metabolites related to DFU and DP treatment. (B) The heat map of potential metabolites.
FIGURE 7
FIGURE 7
The metabolic pathways enriched by significant metabolite. (A) Metabolic pathway of DP in the treatment of DFU. (B) Metabolic pathway disorder caused by DFU.
FIGURE 8
FIGURE 8
Correlation matrix of interaction in 37 differential metabolites. Coefficients of negative correlation (blue) and positive correlation (red) were plotted. The closer that the correlation coefficient was to 1, the redder the colour was, and the greater the positive correlation was. The closer that the correlation coefficient was to -1, the bluer the colour and the greater the negative correlation were. (A) The correlation heatmap of Adm and Mod groups. (B) The correlation heatmap of Mod and Con groups.
FIGURE 9
FIGURE 9
The enrichment analysis of GO and KEGG pathway of DP for the treatment of DFU. Enriched GO terms are for (A) BP analysis (B) CC analysis (C) MF analysis; and (D) KEGG pathway analysis.
FIGURE 10
FIGURE 10
The compound-reaction-enzyme-gene networks of the crucial targets and metabolites. The red hexagons represent the active compounds. The grey diamonds represent the reaction. The green round rectangle represent enzyme. The light purple circles present general genes, and the blue circles represent key genes. (A) Leukotriene metabolism. (B) Pyrimidine metabolism. (C) Amino sugars metabolism. (D) Tyrosine metabolism. (E) Vitamin B9 (folate) metabolism. (F) Histidine metabolism. (G) Urea cycle and metabolism of arginine, proline, glutamate and asparagine.
FIGURE 11
FIGURE 11
Molecular docking. Note. Binding mode of proteins and ligands. (A) Binding mode of GSTM1 with DP. (B) Binding mode of TYMS with DP. (C) Binding mode of PAH with DP. (D) Binding mode of DDC with DP. (E) Binding mode of DHFR with DP. (F) Binding mode of CAT with DP. (G) Binding mode of PGM1 with DP.
FIGURE 12
FIGURE 12
The mechanism diagram of DP in treating DFU rats.
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