Fig. 5. Oral administration of CT induces enhancement of intestinal cell death, cytoplasmic HMGB1 expression in IECs, and increased HMGB1 release.

B6 mice were orally administered 10 μg of CTA, CTB, or intact CT. a Representative photographs of the intestine 16 h after oral administration. b IECs were isolated from the mice 16 h after oral administration and viable cells were counted using Trypan blue and microscopy. c IECs were stained with APC-labeled anti-EpCAM Ab and FITC-labeled anti-CD45 Ab. d, e IECs were stained with PE-labeled anti-EpCAM Ab and the APC Annexin V/7-AAD staining kit. d Annexin V⁺ 7-AAD⁺ cells were identified as dying cells and EpCAM expression was analyzed. e Data (%) for Annexin V⁺ 7-AAD⁺ EpCAM⁺ cells as described in (e) are shown as the mean + SE in three mice. The asterisk (*) indicates a statistically significant difference (P < 0.05) between the mice group treated with PBS ( × ), CTA (●), or CTB (▲); Welch’s t-test. f Feces and blood were collected 0, 6, 16, 24, and 48 h after the oral administration, and HMGB1 levels in fecal extracts and plasma were measured by ELISA assay. The results are shown as the mean + SE in four to seven mice per group. The asterisk (*) indicates statistically significant differences (P < 0.05) between the mouse groups treated with PBS ( × ) at 6 and 16 h after oral administration; Welch’s t-test. g–j IECs were isolated from mice 16 h after oral administration, stained with APC-labeled anti-EpCAM Ab and FITC-labeled anti-CD45 Ab, and then subjected to cytoplasmic staining with PE-labeled anti-HMGB1 Ab. g Cytoplasmic HMGB1⁺ cells were gated and their scatter properties (FSC/SSC) were analyzed. h Data (%) for cytoplasmic HMGB1⁺ cells among the total cells and HMGB1⁺ cells gated with the HMGB1⁺ cell-rich scatter gate as described in (h) are shown as the mean + SE in four to five mice. The single asterisk (*) and double asterisk (**) indicate statistically significant differences (P < 0.05 and P < 0.005, respectively) between the mouse groups treated with PBS ( × ), CTA (●), or CTB (▲); Welch’s t-test. i The HMGB1⁺ cell-rich population as described in (h) was gated and the expression of EpCAM and CD45 was analyzed. j Data (%) for the total cells gated with the HMGB1⁺ cell-rich scatter gate and cytoplasmic HMGB1⁺ EpCAM⁺ CD45⁻ cells as described in (j) are shown as the mean + SE in four to five mice. The asterisk (*) indicates a statistically significant difference (P < 0.05) between the mouse groups treated with PBS ( × ), CTA (●), or CTB (▲); Welch’s t-test