Isoorientin attenuates Crohn’s disease-associated intestinal fibrosis by inhibiting the differentiation of inflammatory fibroblasts

BACKGROUNDCrohn's disease (CD)-associated intestinal fibrosis is the most common complication of CD.Although isoorientin (ISO), a main flavonoid in bamboo leaves, has been validated to have significant anti-inflammatory efficacy, the effect on CD-associated intestinal fibrosis and the associated molecular alterations remain unclear. AIMTo explore the potential anti-fibrotic effects of ISO on inflammatory fibroblasts (iFib) and associated hub genes. METHODSPotential targets for CD, fibrosis and ISO were retrieved from public databases.Hub genes associated with the ameliorative effects of ISO on CD-associated intestinal fibrosis (ISO-CDIF-HGs) were identified by three machine learning algorithms, receiver operating characteristic curves and artificial neural network models.The relationships between ISO-CDIF-HGs and effector cells were determined using pseudotime analysis at single-cell resolution.The in vivo therapeutic efficacy of ISO and the expression levels of ISO-CDIF-HGs were verified by quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry in a 2,4,6-trinitrobenzene sulfonic acidinduced murine model (n = 6 per group).In vitro, an iFib model was established using M1-polarized macrophage-conditioned medium (M1-CM) to evaluate the anti-fibrotic effects of ISO. RESULTSA total of 14 overlapping genes related to ISO, CD, and fibrosis were screened, which were mainly enriched in the interleukin-17 and relaxin signaling pathways as shown by Kyoto Encyclopedia Genes and Genomes pathway analysis.Furthermore, the ISO-CDIF-HGs (MMP3, MMP7 and STAT1) were screened and evaluated via machine learning algorithms and artificial neural network models.At single-cell resolution, it was observed that ISO-CDIF-HGs were also heterogeneously expressed in the constructed differentiation trajectories of fibroblast subtypes, which may influence the differentiation of fibroblast subtypes and then lead to their dysregulation.In the in vivo experiments, Masson's trichrome staining, quantitative real-time polymerase chain reaction and western blotting assays further verified that ISO treatment significantly reduced intestinal collagen deposition in the TNBS-induced intestinal fibrosis mouse model, accompanied by reduced expression of ISO-CDIF-HGs.In vitro, ISO significantly inhibited the upregulation of iFib characteristic markers (CXCL2, CXCL3 and interleukin-6) accompanied by decreased expression of ISO-CDIF-HGs (P < 0.05). CONCLUSIONISO attenuated CD-associated intestinal fibrosis, with reduced expression of MMP3, MMP7 and STAT1 and decreased pro-inflammatory fibroblasts-related features, highlighting its promise as a candidate anti-fibrotic agent.