DNMT3a-Mediated Enterocyte Barrier Dysfunction Contributes to Ulcerative Colitis via Facilitating the Interaction of Enterocytes and B Cells

Figure 3

DNMT3a regulates intestinal epithelial barrier function and differentiation of B cells. The primary IECs were isolated from intestinal epithelial tissues of mice. B cells were isolated from the spleens of wide-type mice. Then, the si-DNMT3a or si-NC was transfected into IECs and then treated with LPS (1 μg/mL) for 48 h. Cells were divided into 4 groups: IECs, IECs+LPS, IECs-si-NC+LPS, and IEC-si-DNMT3a+LPS. (a) DNMT3a mRNA level and protein level in each group of IECs were measured. (b) After transfection of si-DNMT3a or si-NC for 24 h, IECs were cocultured with B cells in a cocultural system, and LPS (1 μg/mL) was added into IECs and cultured for 48 h. Four groups were assigned as follows: IECs+B, IECs+B+LPS, IEC-si-NC+B+LPS, and IEC-si-DNMT3a+B+LPS. TEER of IECs in each group was detected. (c) and (d) ZO-1 and occludin protein levels were measured by western blot and immunofluorescence staining. (e) The CD1d+ B cells and CD138+ B cells were detected by flow cytometry. (f) IL-10, TGF-β, IL-6, and TNF-α levels in cell supernatants were examined. vs. IECs or IECs+B group; ^##, ^### vs. IEC-si-NC+LPS or IEC-si-NC+B+LPS group; .

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