Electroacupuncture ameliorates visceral hypersensitivity and negative emotions by regulating paraventricular hypothalamic nucleus and colonic corticotropin-releasing factor signaling

Figure 4 The peripheral corticotropin-releasing factor-colonic mast cell/transient potential receptor vanilloid type 1 pathway is involved in the therapeutic effects of electroacupuncture. A: Statistical graph of the serum corticotropin-releasing factor (CRF) levels detected by enzyme-linked immunosorbent assay in each group of mice; B: Representative plots of CRF in the colon of each group of mice determined by an immunofluorescence assay. Scale bar, 124.5 μm; C: Statistical graph of the average fluorescence intensity of CRF expression levels in the colons of the mice in each group using immunofluorescence detection; D: Representative image of the Western blot of corticotropin-releasing factor-receptor 1 (CRF-R1); E: Statistical graph of the relative expression level of colonic CRF-R1 detected by Western blot in each group of mice; F: Immunofluorescence plots in the colons of each group of mice. (CRF-R1 is red, CD117 is green, and 4,6-diamidino-2-phenylindole is blue). Scale bar, 124.5 μm; G: Statistical plot of the CRF-R1 and CD117 co-labelling rates in the colons of the mice in each group; H: Mast cell (MC) degranulation rate of the colon in each group of mice detected by toluidine blue staining. Black arrows represent steady state MCs, orange arrows represent degranulated MCs. Scale bar, 50 μm; I: Statistical graph of the MC degranulation rate in each group of mice; J: Representative image of the Western blot of MC tryptase (MCT), protease-activated receptor 2 (PAR2) and transient potential receptor vanilloid type 1 (TRPV1) in mice colons were detected by Western blot; K: Statistical graph of the relative expression levels of MCT; L: Statistical graph of the relative expression levels of PAR2; M: Statistical graph of the relative expression levels of TRPV1; N: Statistical graph of the average fluorescence intensity of TRPV1 expression levels in the colons of the mice in each group; O: Representative plots of TRPV1 in the colon of each group of mice following an immunofluorescence assay. Scale bar, 124.5 μm; Each group n = 6, model group vs control group, model + EA group vs model group; ^aP < 0.05, ^bP < 0.01; CRF: Corticotropin-releasing factor; MC: Mast cell; TRPV1: Transient potential receptor vanilloid type 1; CRF-R1: Corticotropin-releasing factor-receptor 1; MCT: Mast cell tryptase; PAR2: Protease-activated receptor 2; EA: Electroacupuncture; DAPI: 4,6-diamidino-2-phenylindole.