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

Figure 3 Thymosin β4 is sufficient to drive intestinal permeability and dysbiosis in vitro. A: The epithelial barrier function of Caco2 was measured after treatment with thymosin β4 (Tβ4) (100 nmol/L, 200 nmol/L, 400 nmol/L). Each line represents the mean of 3 wells per condition. The cell index serves as a real-time value reflecting changes in barrier properties; B and C: Caco2 cells were treated with Tβ4 (25 nmol/L, 50 nmol/L, 100 nmol/L, 200 nmol/L, 400 nmol/L) for 24 hours, and the protein levels of (B) zonulin 1 (ZO-1), Occludin, (C) interleukin 22 receptor A1 (IL22RA1), IL-10Rβ, Reg3γ, and myosin light chain kinase (MLCK) were detected by western blot assay. Bar, SD, ^aP < 0.001, ^bP < 0.01, ^cP < 0.05 vs the Control group; D: Representative immunofluorescence images of MLCK (green) and DAPI (blue). Scale bar, 100 μm. Bar, SD, ^cP < 0.05 vs the Control group; E: After treatment with Tβ4 (100 nmol/L, 200 nmol/L, 400 nmol/L) for 24 hours, the mRNA levels of ZO-1, IL22RA1, MLCK, and Reg3γ were evaluated by qPCR. Bar, SD, ^aP < 0.001, ^cP < 0.05 vs the Control group (0 nmol/L); F: Cells were treated with Tβ4 (200 nmol/L) for 24 h in MLCK inhibitor (ML-7)-induced Caco2 (prepared 30 minutes in advance), and then the protein levels of MLCK and ZO-1 were evaluated by western blotting; G and H: After treatment with ML-7 in Tβ4-treated Caco2, the mRNA level of (G) ZO-1 and (H) MLCK were evaluated by qPCR. Bar, SD, ^cP < 0.05 vs the Control group, ^dP < 0.001 vs the Tβ4 group, ^eP < 0.05 vs the Tβ4 group. MLCK: Myosin light chain kinase; ZO-1: Zonulin 1; Tβ4: Thymosin β4; IL: Interleukin; IL-10R: Interleukin-10 receptor.