Fig. 4

Blocking TRPV4 with RN-1734 partially prevents the activation of NFκB induced by C. difficile toxins A (TcdA) and B (TcdB) in enteric glial cells (EGCs). A) The data represent a quantitative analysis of the percentage of EGCs showing positive nuclear immunostaining for NFκBp65 after 18 h of incubation with either TcdA (50ng/mL), TcdB (1ng/mL), EGCs with only DMEM (control group), RN -1734 alone (100µM), RN-1734 plus TcdA or RN-1734 plus TcdB. TcdA and TcdB activate TRPV4, leading to increased translocation of NFκBp65 to the nuclei of EGCs. Conversely, blocking the receptor with RN-1734 decreases this nuclear translocation compared to the toxin groups. Data are presented as mean ± SEM of the percentage of nuclei with positive immunostaining for NFκBp65. Statistical significance was determined using the one-way ANOVA and Tukey tests. **p < 0.001 and ***p < 0.0001. B) Photomicrographs illustrate the nuclear immunostaining of NFκBp65 (green), the nuclear staining with DAPI (blue), and the merged images (MERGED). Red arrows indicate the nuclear translocation of NFkB. The percentage of cells positive for NFkBp65 was evaluated in every 100 cells counted (n = 5), specifically targeting the hot areas of each group (areas showing intense staining). Scale bars = 50 μm