Fig. 7.

Blockade of KCa3.1 by its inhibitor TRAM-34 mitigates DSS-induced injury and inflammasome activation in the colon in mice. DSS was dissolved in drinking water and fed ad libitum. After 5 days of DSS treatment, the mice were fed DSS-free normal drinking water and observed for another 2 days. TRAM-34 (40 mg/kg) or vehicle (peanut oil) was administered twice daily by intraperitoneal injection. A, B TRAM-34 administration protected mice from DSS-induced weight loss (A) and the increased in disease activity index (DAI) (B). C, D TRAM-34 reversed colonic shortening. Representative photographs of colons from vehicle- or TRAM-34-treated mice with or without DSS treatment are shown (C), and the length of the colon in each group was compared (D). E, F TRAM-34 administration attenuated tissue injury and inflammatory cell infiltration in the DSS-exposed colon. Representative H&E staining of colon sections from vehicle- or TRAM-34-treated mice with or without DSS treatment is shown (E). Scale bars, 100 µm. The histology score (the sum of the inflammation and injury histology scores) of each colon section was quantified and compared among the groups (F). G, H TRAM-34 suppressed ASC speck formation in the colons of DSS-treated mice. Frozen colon sections were prepared and incubated with anti-ASC antibodies (red) and counterstained with Hoechst 33342 (blue). Representative images of colonic ASC immunofluorescence in each group are shown (G). Arrowheads indicate ASC specks. Scale bars, 50 µm. The number of ASC specks in colon sections was quantified (H). I, J TRAM-34 reduced the release of caspase-1p10 (Cas1p10), IL-1β and IL-18 from DSS-treated colons. IL-1β levels in the supernatants of colon organ cultures were measured by a CBA and normalized to the total protein concentration (pg/mg protein). The precipitated proteins from the supernatants of colon organ cultures were analyzed by Western blotting. The data are expressed as the mean ± SD. ns, not significant. *p < 0.05, **p < 0.01, ***p < 0.001