Daphnetin attenuates intestinal inflammation, oxidative stress, and apoptosis in ulcerative colitis via inhibiting REG3A‐dependent JAK2/STAT3 signaling pathway

Daphnetin is a natural coumarin compound with anti-inflammatory, anti-oxidant, and anti-apoptotic effects, which has been previously demonstrated to ameliorate DSS-induced ulcerative colitis (UC). However, the molecular mechanism involved in the daphnetin-mediated pathological process of UC remains unclarified. The current study used DSS-induced mice and LPS-challenged Caco-2 cells as UC models. Bodyweight, disease activity index (DAI) score, and colon length were used to evaluate the severity of colitis. The histological changes in colon tissues were observed using H&E and PAS staining. Protein levels were detected by western blot. The malondialdehyde (MDA) and superoxide dismutase (SOD) activities were used to assess oxidative stress. Inflammatory responses were evaluated by detecting the levels of inflammatory cytokines (IFN-r, IL-1β, IL-6, and TNF-α) using flow cytometry. CCK-8 and TUNEL assay were employed to determine cell growth and cell death, respectively. The results showed that daphnetin could ameliorate the severity of colitis and attenuate the damage to intestinal structure in DSS-induced mice. Compared with the DSS group, the expression of ZO-1, occludin, and anti-apoptotic protein (BCL-2) was increased while the level of pro-apoptotic proteins (Bax and cleaved caspase 3) was decreased in DSS + daphnetin group. The activity of MDA and SOD, as well as the levels of inflammatory cytokines were substantially suppressed by daphnetin. In consistency, in vitro assays indicated that daphnetin protected Caco-2 cells from LPS-stimulated viability impairment, apoptosis, oxidative stress, and inflammation. Furthermore, daphnetin suppressed the activity of JAK2/STAT signaling in LPS-induced Caco-2 cells in a REG3A-dependent manner. REG3A overexpression abated the ameliorative effects of daphnetin while JAK2/STAT signaling inhibition functioned synergically with daphnetin in LPS-stimulated Caco-2 cells. Collectively, this study deepened the understanding of the therapeutic effects of daphnetin on UC and uncovered for the first time that daphnetin functioned through REG3A-activated JAK2/STAT3 signaling in UC, which may provide novel insights for the treatment of UC.