[Therapeutic mechanism of Cynanchum wilfordii for ulcerative colitis: an analysis using UPLC-QE-MS, network pharmacology and metabolomics]

Abstract

Objective: To explore the targets and pathways of Cynanchum wilfordii for treatment of ulcerative colitis (UC).

Methods: UPLC-QE-MS was used to identify the components of Cynanchum wilfordii ethanol extract, and their targets were screened using public databases for construction of the core protein-protein interaction (PPI) network and GO and KEGG enrichment analyses. Forty male C57 mice were randomized into normal control group, model group, mesalazine group and Cynanchum wilfordii group (n=10), and in the latter 3 groups, mouse UC models were established by treatment with 2.5% DSS and the latter 2 groups drug interventions by gavage. The therapeutic effect was evaluated by recording body weight changes and DAI score. Pathological changes of the colon tissue were observed with HE and AB-PAS staining, and JAK2 and STAT3 protein expressions were detected with Western blotting. The metabolites and metabolic pathways were identified by metabonomics analysis.

Results: We identified 240 chemical components in Cynanchum wilfordii alcoholic extracts, including 19 steroids. A total of 177 Cynanchum wilfordii targets, 5406 UC genes, and 117 intersection genes were obtained. JAK2 and STAT3 were the core targets and significantly enriched in lipid and atherosclerosis pathways. Cynanchum wilfordii treatment significantly increased the body weight and decreased DAI score of UC mice (P < 0.05), alleviated intestinal pathologies, and decreased JAK2 and STAT3 protein expressions in the colon tissues. Most of the 83 intersecting differential metabolites between the control, model and Cynanchum wilfordii groups were identified as glycerophospholipids, arachidonic acid, and amino acids involving glycerophospholipid metabolism and other pathways. Correlation analysis suggested that the core targets of Cynanchum wilfordii for UC participated in regulation of the metabolites.

Conclusion: Cynanchum wilfordii alleviates lipid and amino acid metabolism disorders to lessen UC in mice by regulating the core targets including JAK2 and STAT3 and the levels of endogenous metabolites.

Keywords: Cynanchum wilfordii (Maxim.) Hook. F; UPLC-QE-MS; metabolomics; steroids; ulcerative colitis.

图1

Venn图、网络调控图及核心PPI Fig.1 Venn diagrams, network regulation charts and core PPI. A: Venn diagram. B: Network regulation charts. C: PPI network. D: Core PPI. CW: Cynanchum wilfordii.

图2

GO富集分析 Fig.2 GO enrichment analysis. A: Biological process. B: Cellular components. C: Molecular function.

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KEGG富集分析 Fig.3 KEGG enrichment analysis.

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体质量变化率及DAI评分 Fig.4 Weight change rate and DAI score of the mouse models of UC. A: Weight change rate. B: DAI score. N: Normal group. M: Model group. Y: Mesalazine group. G: Cynanchum wilfordii Group. ^# P<0.05 vs normal group; *P<0.05 vs model group.

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HE染色及AB-PAS染色 Fig.5 HE staining and AB-PAS staining of the colon tissue of the mice. A: Normal group. B: Model group. C: mesalazine group. D: Cynanchum wilfordii Group.

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各组小鼠结肠组织中JAK2和STAT3蛋白表达 Fig.6 Expression of JAK2 and STAT3 proteins in the colon tissue of the mice detected by Western blotting. CW: Cynanchum wilfordii Group. ^# P<0.05 vs normal group; *P<0.05 vs model group;^△ P<0.05 vs mesalazine group.

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基峰图 Fig.7 Base peak chromatogram. A: Normal group. B: Model group. C: mesalazine group. D: Cynanchum wilfordii Group.

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代谢组学多元统计分析 Fig.8 Metabolomics multivariate statistical analysis. A: PCA. B, C: OPLS-DA.

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差异代谢物火山图 Fig.9 Volcano map based on different metabolites. A:M vs N; B: M vs G.

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差异代谢物热图 Fig.10 Heatmaps based on different metabolites.

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差异代谢物富集代谢通路 Fig.11 Differential metabolite-enriched metabolic pathways.

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代谢物-靶点基因网络 Fig.12 Metabolite-target gene network. Squares represent differential genes, circles represent the core target. The darker the color, the stronger the correlation.