Journal: World Journal of Gastroenterology

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

doi: 10.3748/wjg.v31.i42.111706

Figure Lengend Snippet: 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, a P < 0.001, b P < 0.01, c P < 0.05 vs the Control group; D: Representative immunofluorescence images of MLCK (green) and DAPI (blue). Scale bar, 100 μm. Bar, SD, c P < 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, a P < 0.001, c P < 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, c P < 0.05 vs the Control group, d P < 0.001 vs the Tβ4 group, e P < 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.

Article Snippet: The membranes were incubated with primary antibodies after being blocked with 5% skimmed milk, including ZO-1 (Proteintech, Cat#21773-1-AP), Occludin (Abcam, Cat# Ab216327 ), IL22RA1 (Proteintech, Cat#13462-1-AP), Reg3γ (Abcam, Cat# Ab198216 ), β-actin (Proteintech, Cat#60008-1-Ig) and GAPDH (Proteintech, Cat#60004-1-Ig).

Techniques: Permeability, In Vitro, Western Blot, Control, Immunofluorescence

Journal: World Journal of Gastroenterology

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

doi: 10.3748/wjg.v31.i42.111706

Figure Lengend Snippet: Thymosin β4 drives intestinal permeability increase in vivo . Thymosin β4 (Tβ4) (3 mg/kg) was intraperitoneally injected into C57BL/6 mice every day for a week. A-C: Representative intestinal immunofluorescence images of (A) zonulin 1 (ZO-1) (Scale bar, 50 μm), (B) myosin light chain kinase (MLCK) and (C) IL22RA1. Scale bar, 100 μm; D: Representative FISH staining for bacterial infiltration using EUB338 probe (red) and DAPI (blue). Scale bar, 100 μm. Tβ4 (3 mg/kg) was intraperitoneally injected into Kit W-sh/W-sh mice every day for a week; E and F: Representative intestinal immunofluorescence images of (E) ZO-1, Scale bar, 100 μm, and (F) MLCK, Scale bar, 100 μm; G: Representative FISH staining for bacterial infiltration using EUB338 probe (red) and DAPI (blue). Scale bar, 100 μm; H-J: The level of (H) diamine oxidase, (I) D-lactate and (J) bacterial endotoxin in serum. Bar, SD, b P < 0.01 vs the Control group. MLCK: Myosin light chain kinase; ZO-1: Zonulin 1; Tβ4: Thymosin β4.

Article Snippet: The membranes were incubated with primary antibodies after being blocked with 5% skimmed milk, including ZO-1 (Proteintech, Cat#21773-1-AP), Occludin (Abcam, Cat# Ab216327 ), IL22RA1 (Proteintech, Cat#13462-1-AP), Reg3γ (Abcam, Cat# Ab198216 ), β-actin (Proteintech, Cat#60008-1-Ig) and GAPDH (Proteintech, Cat#60004-1-Ig).

Techniques: Permeability, In Vivo, Injection, Immunofluorescence, Staining, Control

Journal: World Journal of Gastroenterology

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

doi: 10.3748/wjg.v31.i42.111706

Figure Lengend Snippet: Thymosin β4 deficiency protects rats from stress-induced injury in the intestine. Thymosin β4 (Tβ4) (3 mg/kg) was injected into stress-induced Tβ4 deficiency ( Tβ4 −/− ) rats. A: Representative intestinal immunofluorescence images of zonulin 1. Scale bar, 50 μm; B: Representative TEM images of the colon. Magnification: 8000 ×, Scale bar, 2 μm. Magnification: 25000 ×, Scale bar, 500 nm; C: Immunohistochemistry staining of myosin light chain kinase. Scale bar, 100 μm; D: FITC-dextran 4000 (FD4, 4 kDa, 5 mg/kg) was orally administered, and the flux of FITC-dextran in rat serum was observed 3 hours later. a P < 0.05 vs the Tβ4 −/− group, b P < 0.001 vs the WT group; E and F: The colonic tissue mRNA levels of (E) IL22RA1 and (F) Reg3γ. Bar, SD, c P < 0.05 vs the Tβ4 −/− under stress group; G: Representative FISH staining for bacterial infiltration using EUB338 probe (red) and DAPI (blue). Scale bar, 50 μm. Bar, SD, c P < 0.05 vs the Tβ4 −/− under stress group; H: Representative intestinal immunofluorescence images of mast cell (Tryptase, red) and DAPI (blue), Scale bar, 50 μm. Bar, SD, c P < 0.05 vs the Tβ4 −/− under stress group. MLCK: Myosin light chain kinase; Tβ4: Thymosin β4; IL22RA1: Interleukin 22 receptor A1; WT: Wild-type.

Article Snippet: The membranes were incubated with primary antibodies after being blocked with 5% skimmed milk, including ZO-1 (Proteintech, Cat#21773-1-AP), Occludin (Abcam, Cat# Ab216327 ), IL22RA1 (Proteintech, Cat#13462-1-AP), Reg3γ (Abcam, Cat# Ab198216 ), β-actin (Proteintech, Cat#60008-1-Ig) and GAPDH (Proteintech, Cat#60004-1-Ig).

Techniques: Injection, Immunofluorescence, Immunohistochemistry, Staining

Journal: World Journal of Gastroenterology

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

doi: 10.3748/wjg.v31.i42.111706

Figure Lengend Snippet: Graphical abstract. Mast cell-released thymosin β4 drives intestinal barrier dysfunction in stress-induced irritable bowel syndrome. Tβ4: Thymosin β4; CRH: Corticotropin-releasing hormone; CRHR1: Corticotropin-releasing hormone receptor 1; JAK1: Janus kinase 1; STAT3: Signal transducer and activation of transcription 3; MC: Mast cell; MLCK: Myosin light chain kinase; IL22RA1: Interleukin 22 receptor A1; IBS: Irritable bowel syndrome.

Article Snippet: The membranes were incubated with primary antibodies after being blocked with 5% skimmed milk, including ZO-1 (Proteintech, Cat#21773-1-AP), Occludin (Abcam, Cat# Ab216327 ), IL22RA1 (Proteintech, Cat#13462-1-AP), Reg3γ (Abcam, Cat# Ab198216 ), β-actin (Proteintech, Cat#60008-1-Ig) and GAPDH (Proteintech, Cat#60004-1-Ig).

Techniques: Activation Assay