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

Figure 8 Thymosin β4 release from mast cells is driven by corticotropin-releasing hormone/corticotropin-releasing hormone receptor 1 signaling, bypassing degranulation. A: HMC-1 cells or Thymosin β4 knock down HMC-1 cells (shTβ4) were co-cultured with Caco2 cells monolayers. The immunofluorescence staining of zonulin 1 (ZO-1) was evaluated. Scale bar, 50 μm; B and C: After treatment of HMC-1 cells with corticotropin-releasing hormone (CRH) (500 nmol/L) for 15 minutes, (B) the level of degranulation and (C) the secretion of Tβ4 were determined; D and E: The cells were treated with DSCG before CRH, and (D) the level of degranulation and (E) the secretion of Tβ4 were determined; F: The concentration of Tβ4 in HMC-1 after pre-administration with CRH receptor 1 antagonist (R121919) followed by CRH treatment; G: Stressed rats were injected with R121919 (10 mg/kg), and the intestinal barrier permeability was assessed by FD4 assay; H and I: Representative immunofluorescence of the intestine for (H) Tβ4 (Scale bar, 100 μm) and (I) ZO-1, Scale bar, 50 μm; J: Representative FISH staining for bacterial infiltration using EUB338 probe (red) and DAPI (blue). Scale bar, 100 μm. Bar, SD, ^aP < 0.001 vs Control, ^bP < 0.01 vs Control, ^cP < 0.05 vs Control, ^dP < 0.001 vs CRH group, ^eP < 0.05 vs CRH group, ^fP < 0.05 vs stress group. MC: Mast cell; ZO-1: Zonulin 1; CRH: Corticotropin-releasing hormone; Tβ4: Thymosin β4.