Thymosin β4 released by mast cells under stress conditions impairs intestinal epithelial barrier via IL22RA1/JAK1/STAT3 signaling in irritable bowel syndrome

Figure 9 Thymosin β4 regulates the Janus kinase 1/signal transducer and activation of transcription 3 pathways in epithelial cells. Caco2 cells were treated with thymosin β4 (Tβ4). A: Heatmap of differentially expressed mRNAs; B: Volcano plot of RNA-seq results (n = 3 biological replicates/group; differential expression defined as |log₂ (fold change)| > 1 and FDR < 0.05); C: Top 20 enriched pathways by KEGG analysis; D: GSEA analysis of Janus kinase 1 (JAK1)/signal transducer and activation of transcription 3 (STAT3) pathway enrichment in Tβ4-positive vs -negative Caco2 cell; E: Representative western blot images and biochemical quantification of p-JAK1, JAK1, p-STAT3, STAT3. Bar, SD, ^cP < 0.05 vs Control; F: Immunofluorescence staining of p-JAK1 in Caco2 cells. Scale bar, 100 μm; G: Activated mast cells [HMC-1 and HMC-1^shTβ4 treated with corticotropin-releasing hormone (CRH)] stimulated with SHR0302 and co-cultured with Caco2 cells, and representative western blot images and biochemical quantification of p-JAK1, JAK1, p-STAT3, STAT3, zonulin 1, myosin light chain kinase and GAPDH. Bar, SD, ^dP < 0.05 vs “HMC-1 with CRH”, ^eP < 0.05 vs “HMC-1^shTβ4 with CRH”. Tβ4: Thymosin β4; JAK1: Janus kinase 1; STAT3: Signal transducer and activation of transcription 3; MC: Mast cell; ZO-1: Zonulin 1; MLCK: Myosin light chain kinase; CRH: Corticotropin-releasing hormone.