Fig 1.
Chemical structure of nornidulin.
Fig 2.
Modulation of cAMP-mediated Cl− efflux and cell viability by nornudulin in T84 cells.
(A) Demonstrates nornidulin’s impact on cAMP-mediated Cl- efflux stimulated by 20 μM forskolin (FSK). Various concentrations of nornidulin were applied to the apical solutions (n = 5). The inset graph shows nornidulin’s effect on basal current at different concentrations (n = 5). (B) Cell viability after 24-hour exposure to varying concentrations of nornidulin in apical solutions (n = 5). A 10% DMSO solution serves as the positive control. Results are presented as percentage of cell viability relative to the control ± standard deviation (S.D.). ****, p<0.0001 compared to control, while "ns" denotes non-significant differences from the control.
Fig 3.
The polarity of inhibition by nornidulin on cAMP-induced Cl− secretion in T84 cells.
(A) Shows a representative short-circuit current (Isc) trace. In this experiment, nornidulin was applied at various concentrations to both the basolateral and apical sides of the cell layer after stimulation with FSK (n = 5). (B) Illustrates the dose-dependent response of nornidulin on CFTR-mediated apical chloride current (ICl-) in T84 cells. Before activating CFTR with FSK, the basolateral membrane was first made permeable by using amphotericin B (250 μg/mL for 30 minutes). Nornidulin was then added to the apical side at different concentrations. The graph shows a representative trace of the apical chloride current (ICl-) (n = 5). (C) Western blot analysis of CFTR protein expression in T84 cells after 24-h exposure to nornidulin (5 μM) (n = 8). ns, non-significant compared with control.
Fig 4.
Mechanisms of CFTR inhibition by nornidulin in T84 cells.
(A) A representative trace of apical chloride current (ICl-) is shown. CFTR was first stimulated with 20 μM genistein, followed by the addition of nornidulin at various concentrations to the apical side (n = 5). (B–E) Examine how upstream regulatory proteins affect nornidulin’s inhibition of CFTR-mediated apical chloride current (ICl-). T84 cells were pre-treated with specific activators or inhibitors: CPT-cAMP (100 μM) for PKA, IBMX (100 μM) for PDE, NaF plus Na3VO4 (1 mM) for PP, and Compound C (50 μM) for AMPK. After pre-treatment, dose-inhibition studies with nornidulin were performed, with each experiment repeated five times (n = 5). (F) Compiles dose-response curves from all the dose-inhibition studies. The data are fitted to Hill’s equation and presented as means of percentage of agonist-stimulated apical chloride current (ICl-), with standard error of the mean (S.E.M.) indicated (n = 5).
Fig 5.
The effect of nornidulin on intracellular cAMP level.
The experiment involved pretreating T84 cells for one hour with different conditions: vehicle (control), nornidulin, N9 (5 μM), nornidulin (5 μM) with FSK (5 μM), or FSK (5 μM). Following treatment, intracellular cAMP levels were determined using a cAMP assay kit. The results are expressed as cAMP concentrations with standard deviation (S.D.) (n = 5–6). ns, not significantly different from the control group. **, p <0.01 compared to the indicated group. ****, p <0.0001 compared to the indicated group.
Fig 6.
The effect of nornidulin on FSK-induced fluid secretion in human colonoid models.
Effect of nornidulin on fluid secretion in human colonoids after 60 min of FSK treatment. (A) Bright-field images of colonoids after FSK (5 μM) with or without treatment with nornidulin (10 μM) at indicated time points (scale bar: 50 μm). (B) Swelling of colonoids was evaluated by relative area increased from 0 min to 60 min. (C) The area under the curve (AUC) of each condition is calculated from (B.) Each experiment was repeated independently (n = 6). **, p <0.01 versus FSK alone.
Fig 7.
The effect of nornidulin on cholera toxin-induced fluid secretion in human colonoid models.
Effect of nornidulin on fluid secretion in human colonoids after 240 min of cholera toxin treatment. (A) Bright-field images of colonoids after cholera toxin (2 μg/mL) with or without treatment with nornidulin (40 μM) at indicated time points (scale bar: 50 μm). (B) Swelling of colonoids was evaluated by relative area increased from 0 min to 240 min. (C) The area under the curve (AUC) of each condition is calculated from (B.) Each experiment was repeated independently (n = 3).***, p <0.001 compared to the control group. *, p <0.05 compared to cholera toxin-treated group.