Background

Ulcerative colitis (UC) is a chronic and recurrent bowel disease, seriously impairs the quality of life, and aggravates the economic burden on patients [1]. In the last two decades, the incidence and prevalence of UC have continued to increase globally [2, 3]. The causes of UC still remain unclear, but the occurrence and progression of UC involves interactions among genetic, microbial, auto immune and environmental factors, making it challenging to develop effective drugs [3, 4]. Although several different types of drugs, such as 5-aminosalicylic acid, TNF-α blockers, and glucocorticoids, have been commonly used to treat UC [5, 6]. But these drugs are usually associated with high recurrence rates and adverse effects. Therefore, develo** novel and safe strategies for UC treatment is urgently needed.

Fraxini Cortex (FC) is the dry barks of Fraxinus rhynchophylla Hance, Fraxinus chinensis Roxb, Fraxinus szaboana Lingelsh, or Fraxinus stylosa Lingelsh [7, 8]. It has been used as Chinese herbal medicine (THM) for various medical disorders due to its anti-inflammation [9, 10], anti-apoptosis [11] and antifibrotic effects [12]. Accumulated evidences have highlighted the beneficial roles or its ingredients of FC on prevention and treatment of UC [13]. In a previous study, the ethanol extract of FC was shown to exhibit anti-diarrheal function by affecting the transport of chloride ions in the rat intestinal epithelia [14]. Fraxinellone, a natural compound isolated from FC, was demonstrated to reduce weight loss and diarrhea in DSS-induced colitis mice, suppress the activities of myeloperoxidase and alkaline phosphatase, and increase the levels of glutathione in colitis tissues [15]. Besides, this compound also decreased the colonic levels of IL-1β, IL-6, IL-18 and TNF-α, and inhibited CD11b (+) macrophage infiltration [15]. Moreover, aesculin and aesculetin, another two natural compounds of FC, were proved to relieve the symptoms of DSS-induced colitis, restrain the secretion of TNF-α, IL-1β through inhibiting the activation of NF-κB and MAPKs pathway in colonic tissues and macrophages [16, 17]. However, the mechanisms of action of FC on UC treatment still remain elusive.

This study aimed to uncover the pharmacological mechanism of FC for UC treatment through a systems approach. Firstly, the active ingredients of FC were screened out based on the existing databases and pharmacokinetic characteristics. Then, the targets of the compounds, and the compound-target interactions were identified using comprehensive methods. The hub targets of FC against UC were obtained through the PPI network analysis. GO and KEGG pathway enrichment analyses were performed to predict the potential function of hub genes. Molecular docking and GEO microarray dataset were further used to identify the core targets, and in vivo studies were performed to assess the therapeutic effect of FC on DSS-induced colitis and validate the results of network pharmacology. The schematic overview of the process is summarized in Fig. 1.

Fig. 1
figure 1

The framework of this study for exploring the pharmacological mechanisms of FC against UC

Full size image

Methods

FC active ingredients collection and screening of FC-UC targets

A Traditional Chinese Medicine Systems Pharmacology Database (TCMSP, version: 2.3) was used to collect the compounds contained in FC. The name and molecular structure of the compounds were verified by PubChem database. Predicted targets of all compounds were collected from Swiss Target Prediction and TCMSP databases. The official target names were standardized by the UniProt database (release 2022_02), and duplicates were excluded to obtain potential targets of FC against UC.

UC-related genes were collected from DisGeNET database (version 7.0) and GeneCards database (Version 5.8) with the keyword “ulcerative colitis, UC”, as our previous studies [18, 19]. The therapeutic targets of FC for UC were obtained by an intersection between FC potential targets and UC-related genes, and a Venn diagram was achieved by Venny 2.1.0 software.

Protein-protein interaction (PPI) network construction

PPI network of the FC-UC targets was built via STRING database (version 11.5), and visualized by Cytoscape software (version 3.8.0). The topology analyses of the networks were conducted using CytoNCA plugin. The hub targets were obtained based on three topological parameters, including degree centrality (DC), betweenness centrality (BC) and closeness centrality (CC), according to network pharmacology evaluation method guidance [20].

Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment and analysis

GO function and KEGG pathway enrichment analyses of hub genes were conducted on the online platform of DAVID 2021(https://david.ncifcrf.gov/home.jsp) to uncover the detailed events of these genes involved in UC pathogenesis. The inclusion criteria was set as p-value < 0.05.

Molecular docking

Four key pharmacodynamic molecules including ursolic acid, fraxetin, beta-sitosterol, and esculetin were selected to dock with 22 targets (degree score ≥ 5). The SDF format of compounds were obtained from the PubChem database (2021 update), and the target molecular structures of the proteins were derived from the RCSB protein database. The docking study was employed with a semi-flexible docking system, and conducted via AutoDock v4.2.6. PyMOL (version 2.5.4) was used to generate docking conformation, and analyze the lowest binding energy.

Gene expression omnibus (GEO) database analysis

To study the expression levels of FC targeted UC genes, the expression profiling data from GSE22619 [21] and GSE37283 [22] were downloaded from the GEO database. The LIMMA package in the R software was used to filter the differentially expressed genes (DEGs) with the criteria of |Log2FC| > 1 and the p-value< 0.05. The expression distributions of DEGs in different tissue were showed as box plots. Statistical difference of two groups was analyzed by Wilcox test, and a p-value< 0.05 was considered statistically significant.

Fraxini cortex extract preparation

Fraxini cortex extract was provided by ** of systematic reviews and meta-analysis on traditional Chinese medicine for ulcerative colitis. BMC Complement Med Ther. 2021;21(1):228." href="/article/10.1186/s12906-023-03983-0#ref-CR31" id="ref-link-section-d84801428e1878">31]. Recently, it has drawn more attention to develop potential new UC therapeutics, attributing to its efficacy and low side effects [32, 33]. As one of the commonly used TCMs, FC is usually applied for treating diarrhea, bacillary dysentery, arthritis and cancer [34]. Identification of the pharmacological mechanisms of FC against UC will facilitate the development of novel therapies in UC treatment.

In this study, we used systems pharmacology to uncover the active compounds and potential targets of FC, and establish compound-target and target-disease relationships. Then, an in vivo animal experiment was used to validate the reliability of findings in the network pharmacology and molecular docking [35]. As the results achieved here, 16 active compounds and 28 hub target genes were identified, suggesting that FC exerted its anti-UC effects through multi-compounds and multi-targets. Ursolic acid, beta-sitosterol, fraxetin and esculetin were identified as the pivotal components with the highest degrees. In previous studies, ursolic acid has been proved to relive DSS-induced colitis in mice by reducing the content of malondialdehyde, IL-1β and TNF-α and increasing superoxide dismutase activity in mice colon tissues [36]. It also could attenuate experimental colitis by restoring intestinal flora homeostasis and regulating fatty acid metabolism [37]. In a Drosophila ulcerative colitis model, ursolic acid is demonstrated to restore the proliferation and differentiation of intestine stem cells and prevent intestine injury via inhibiting the JNK/JAK/STAT signaling pathway [38]. Beta-sitosterol is reported to alleviate microscopic appearances of DSS-induced colitis and increase the expression of antimicrobial peptides [39], and it could promote tissue repair by enhancing antioxidant defenses [40]. Fraxetin has been confirmed that it exhibits its intestinal anti-inflammatory activity through antioxidant property [41]. Esculetin has been demonstrated to ameliorate TNBS-induced rat colitis, and attenuate the expression of pro-inflammatory mediators TNF-α and IL-1β [42]. Overall, FC has a variety of active substances, and its anti-UC effect may be achieved by a variety of active ingredients acting on multiple targets, which is worthy of further study.

The PPI network analysis revealed that TP53, CASP3, IL6, PTGS2, TNF, IL1B, STAT3, MMP9 and NFKBIA were the core targets of FC. Ursolic acid was proved to exhibit anti-inflammation activities by acting on CASP3, ERK1 and JNK2 targets, inhibiting activation of inflammation-associated downstream factors ERK1, NF-κB and STAT3. It also downregulated the activities of IL-1β, IL-6, and TNF-α as well as expression of caspase-3 and caspase-9 [43]. Beta-sitosterol was demonstrated to reduce the expression levels of PTGS2 and NF-κB, and downregulate TNF-α, IL-1β and IL-6 [44]. Fraxetin was reported to mitigate the levels of IL-1β, IL-6, TNF-α and prostaglandin E2, improve the content of superoxide dismutase (SOD) and IL-10 in rats with enteritis [45]. Esculetin could attenuate iNOS and COX2 protein expression by inhibiting NF-κB pathway, and reduce LPS-induced elevated levels of IL-6, IL-1β and TNF-α in mice [46].

GO analysis revealed that FC was primarily associated with the biological processes of cytokine-mediated signaling pathway, positive regulation of apoptotic process, and cellular response to lipopolysaccharide. KEGG pathway enrichment analysis showed that FC exerted its therapeutic effects on UC by regulating IL-17 signaling pathway, TNF signaling pathway and Pathways in cancer. IL-17 is a proinflammatory factor in intestinal inflammation and is closely related to the pathogenesis of UC [47, 48]. IL-17A is the most important factor in the IL-17 family, which were highly expressed in biopsies of UC [49, 50]. IL-17A acts on immune and non-immune cells [51], actives NF-κB, MAPKs and C/EBP cascades [52,53,54], which induces the production of chemokines, inflammatory cytokines and acute phase reactive proteins, thus promotes the occurrence of inflammation. Inhibition of IL-17 signaling pathway by multi-targets and multi-channel inhibitors is an important approach for the treatment of UC [55].

In present study, IL-17 pathway was identified as the crucial pathway of FC in the UC treatment with the highest degree score via the compound-target-pathway network analysis. Molecular docking speculated that FC main components had high affinity with IL-17 signaling pathway related genes, and six of them (IL1B, MAPK8, PTGS2, MMP1, MMP3 and MMP9) were highly expressed in UC clinical samples validated by analysis of GEO expression datasets. Previous studies also demonstrated that THMs conquer UC via interfering IL-17 signaling pathway. For example, Qing Chang Suppository Powder improved DSS-induced colitis, and modulated the expression of mediators in IL-17 signaling pathway [56]. As the main components of FC, ursolic acid was previously reported to ameliorate the symptoms of autoimmune myasthenia gravis via inhibiting IL-17 and shifting Th17 to Th2 cytokines [57]. Ursolic acid also could alleviate autoimmune arthritis, decrease the levels of inflammatory cytokines, and reduce the number of Th17 cells [58]. Moreover, esculetin relived the lipopolysaccharide-induced acute lung injury, and inhibited the activation and/or expression of IL-17, AKT, ERK and RORγt [59]. In line with the results of network pharmacology approach, in vivo studies confirmed that FC alleviated DSS-induced colitis, reduced systemic inflammatory response, and diminished the expression of IL-17 signaling pathway mediators, IL-17A, RORγt, and tissue remodeling factors MMP1, MMP3 and MMP9.

Recently, network pharmacology has been widely applied to explore the action mechanism of THMs on various disease. This approach can be used to screen active compounds, describe the interaction between compounds and targets, and predict the molecular mechanism of THMs [29]. In addition to network pharmacology approach, we also used molecular docking and experimental studies to validate the predictive mechanism of FC for UC. However, there are some limitations in this research. For example, the predictive research relies on the various databases which maybe result in missing of important compounds and targets. The FC active compounds were retrieved from databases which are probably inconsistent with the ingredients in blood of UC patients. In addition, there are multiple targets and pathways were predicted in this study, but only IL-17 signaling pathway and related targets were validated in vivo studies. Therefore, the experimental studies for verification of the predicted molecular mechanisms of FC against UC are needed in future researches.

Conclusions

In this research, ursolic acid, fraxetin, beta-sitosterol, and esculetin were identified as the main compounds, and MAPK8, IL6, RELA, TNF, IL1B, FOS, PTGS2, and MMP3 were considered as major targets of FC in UC treatment. Molecular docking verified that these compounds showed good binding interaction with these target proteins. FC exerted its therapeutic effect on UC primarily through IL-17 signaling pathway, TNF signaling pathway and Pathways in cancer. In vivo studies demonstrated that FC exerted its therapeutic effect on UC by inhibiting inflammatory cytokines release, and modulated IL-17 signaling pathway. Future studies are needed to determine the detailed molecular mechanism in mammals and human cell lines.