Fig. 6

TRPV4 regulates cell death in enteric glial cells (EGCs) induced by C. difficile toxins. A) Inhibition of TRPV4 by the antagonist RN-1734 significantly reduced cell death in EGCs exposed to TcdA and TcdB over an 18-hour incubation period. Apoptosis was assessed using the Glo-annexin V real-time assay. Data are presented as mean±SEM (n = 5) of the relative luminescence unit (RLUs), proportional to the amount of phosphatidylserine-annexin V binding. Statistical significance was determined using ANOVA and Tukey tests. ***p < 0.0001. B) Inhibition of TRPV4 by the antagonist RN-1734 significantly reduced the cleaved caspase-3 protein expression in EGCs exposed to TcdA and TcdB over an 18-hour incubation period. Western Blotting detected protein expression in EGCs. Data are presented as mean ± SEM (n = 5) of cleaved Caspase-3 protein expression relative to β-actin. Statistical significance was determined using one-way ANOVA and Tukey tests. ***p < 0.0001. C) Inhibition of TRPV4 by the antagonist RN-1734 significantly upregulates bcl-2 gene expression in EGCs exposed to TcdA and TcdB over an 18-hour incubation period compared to the control group. Gene expression of bcl-2 in EGCs was assessed by qPCR. Data are presented as mean±SEM of relative bcl-2 gene expression. Statistical significance was determined using one-way ANOVA and Tukey tests. **p < 0.001 and ***p < 0.0001. D) The inhibition of TRPV4 by the antagonist RN-1734 significantly reduced EGC death following simultaneous exposure to TcdA and TcdB over an 18-hour incubation period. Apoptosis was assessed using the Glo-Annexin V Real-Time Assay, with data presented as mean±SEM (n = 3–5) of relative luminescence units (RLUs), proportional to phosphatidylserine-annexin V binding. Statistical significance was determined using ANOVA followed by Tukey’s post hoc test. EGCs following 18 h of incubation with either TcdA (50ng/mL), TcdB (1ng/mL), only EGCs with only DMEM (control group), RN -1734 alone (100µM), RN-1734 plus TcdA or RN-1734 plus TcdB. The entire blotting membranes are available in the supplementary material (Figure S4–S8)