Active compounds in FC
A total of 20 FC active compounds were obtained from TCMSP database (Table S1). Although do not meet the criterion of ADME-related properties (oral bioavailability ≥ 30% and drug-likeness ≥ 0.18), several compounds indcluding ursolic acid, fraxetin and esculetin were included in candidate components due to their poly-pharmacology features in FC. These 20 active compounds have 115 potential targets after remove the duplicates (Table S2). Compound-target network showed that ursolic acid (F14) possessed the most target genes (49 targets), followed by beta-sitosterol (F8) with 37 targets, caffeic acid (F3) with 21 targets and fraxetin (F12) with 19 target genes (Fig. 2A).
Retrieval Of Uc Genes And Screening Of Fc-uc Targets
The UC-related genes were retrieved from the GeneCards database and DisGeNet database. Then, the FC potential targets were matched with these UC-related genes via the Venny software, and a total of 43 FC-UC common targets were acquired (Fig. 2B).
Construction Of Ppi Network
A network with 43 nodes (FC-UC common genes) and 459 edges was constructed basing on the results of the protein-protein interactions (PPI) analyzed by STRING database. According to topological analysis and the criteria of 2-fold median of DC, CC and BC, a core network with 28 nodes and 318 edges was generated (Fig. 2C). TP53, CASP3, IL6, PTGS2, TNF, IL1B, STAT3, MMP9 and NFKBIA have higher degree scores, which indicated that they might be the core targets of FC in UC treatment. Besides, these 28 key targets regulated by 16 compounds of FC (Fig. 2D). Among them, ursolic acid had the most targets, followed by fraxetin, beta-sitosterol, and esculetin, suggesting their potentially important roles in anti-UC function (Fig. 2E).
Go And Kegg Enrichment Analysis
GO enrichment analysis of 28 hub genes in the core PPI network totally got 269 GO items, including 214 items related to biological processes (BP), 15 items to cellular components (CC), and 40 items to molecular function (MF) (Table S3-S5). In the BP category, the genes were mainly enriched in cytokine-mediated signaling pathway, positive regulation of apoptotic process, and inflammatory response. In the CC, these genes were primarily enriched in cytosol and nucleus. In the MF, they enriched in enzyme binding and identical protein binding (Fig. 3A). KEGG pathway enrichment analysis showed that these hub genes highly participated in IL-17 signaling pathway, TNF signaling pathway and Pathways in cancer, indicating the multi-targets and multi-channels regulation of FC in UC treatment (Fig. 3B and Table S6).
Compound-target-pathway Network Construction
A compound-target-pathway network was built by matching the nine key inflammatory and immune response-related pathways with the hub genes and FC components. This network consists of 35 nodes (4 compounds, 22 targets, 9 pathways) and 117 edges (Fig. 4A). Specifically, ten major targets of FC against UC were identified with a higher degree ≥ 5, i.e., MAPK8, IL6, RELA, TNF, IL1B, NFKBIA, CASP3, FOS, PTGS2 and TGFB1(Fig. 4B), and IL-17 signaling pathway was discerned with the highest degree (degree = 14) (Fig. 4C). The results provided a potential direction for molecular docking and future experimental validation.
Molecular Docking Analysis
The molecular docking analyses were conducted between the four main compounds and the 22 target proteins. A binding energy less than − 5.0 kcal/mol indicates strong binding activity [28]. In present docking study, 35% (31/88) targets had a binding energy less than − 5.0 kcal/mol (Fig. 5). Ursolic acid binds to PTGS2 with a binding energy of -7.98 kcal/mol, binds to IL1B at -7.86 kcal/mol, and FOS at -7.73 kcal/mol. Fraxetin binds to PTGS2 with a binding energy of -5.31 kcal/mol. Beta-sitosterol binds to PTGS2 at -5.45 kcal/mol, and CASP3 at -6.39 kcal/mol (Table S7). The docking views showed that hydrophobic and hydrogen bond interactions are the primary modes of binding between compounds and proteins (Fig. 6A-D). Thus, we proposed that targeted regulation of these genes may be the mechanism of action of FC for UC treatment.
The Expression Patterns Of Fc Target Genes In Uc Colonic Tissues
The mRNA expression data from two independent datasets (GSE22619 and GSE37283) were analyzed after normalization and batch removal (Fig. S1). A total of 164 differentially expressed genes (DEGs) were identified in UC colonic samples compared to the normal mucosal tissues, with 114 up-regulated and 50 down-regulated genes (Fig. 7A-B). Among of these DEGs, IL1B, PTGS2, MAPK8, MMP1, MMP3, and MMP9 were highly expressed in the colonic tissues of UC patients compared to that in the heathy controls (Fig. 7D-I). Notably, these six genes are the FC-UC common genes and enriched in IL-17 signaling pathway (Fig. 7C), which implied that these genes and IL-17 signaling pathway may be the targets of FC for UC treatment (Fig. 8, Table S8).
Fc Attenuated Dss-induced Colitis
To explore the therapeutic effect of FC on UC, a mouse colitis model was established and treated with FC. In comparison to the control group, mice induced with DSS had significant shortened colon, increased DAI and colon index, and higher histological score (Fig. 9A-D). The colon tissues had obvious edema and epithelial destruction in the model group compared with that in the control group (Fig. 9E). However, these colitis symptoms and pathological changes were mitigated after FC treatment. The DAI, histological score, and colon length shortening were lower in FC-M and FC-H groups than that in the model group (Fig. 9A-D). All of these data suggested that FC ameliorated DSS-induce colitis.
Fc Alleviated Dss-induced Inflammation
The serum contents of TNF-α, IL-1β and IL-6 were higher in the model group than that in the control group (Fig. 10A-C). While, the level of IL-10 was lower in the model group compared with that in the control group (Fig. 10D). After FC administration, the serum levels of TNF-α and IL-6 were decreased in FC-H group, and IL-1β was downregulated in FC-M and FC-H groups. Besides, IL-10 was upregulated in FC-H group compared with that in the model group (Fig. 10A-D). These results suggested that FC alleviated DSS-induced systemic inflammatory response in mice.
Fc Modulated The Expression Of Fc-uc Common Genes In Colitis Mice
Several key targets including IL1β, COX2, IL-17, RORγt, MMP1 and MMP3 expression levels were validated by western blot. The protein levels of IL1β, PTGS2, IL-17 and RORγt were obvious decreased in FC-M and FC-H groups (Fig. 11A-D), and MMP3 was reduced in FC-H group (Fig. 11E-F), compared to that in the model group.