Epicatechin alleviates ulcerative colitis by restoring intestinal mucosa via regulating the NCR⁺ILC3 cell counts

Promoting damaged intestinal mucosal repair and restoring epithelial barrier function are critical strategies for treating ulcerative colitis (UC). Interleukin-22 (IL-22), a key cytokine for intestinal epithelial homeostasis, is mainly secreted by group 3 innate lymphoid cells (ILC3s). ILC3s exhibit substantial plasticity: NCR⁻ILC3 preferentially secrete IL-17A, whereas NCR⁺ILC3 primarily produce IL-22; under inflammatory conditions, NCR⁺ILC3 and ILC1 can undergo reciprocal conversion. Hypoxia-inducible factor-1α (HIF-1α) signaling is involved in regulating ILC3 subset balance and influences intestinal immune homeostasis. Epicatechin, a common dietary flavonoid, possesses antioxidant and anti-inflammatory activities; however, whether it modulates ILC3 biological functions through the HIF-1α pathway remains unknown.

To investigate whether epicatechin ameliorates UC through HIF-1α-mediated restoration of NCR⁺ILC3 cells and to identify its direct molecular target.

Primary lymphocytes isolated from mesenteric lymph nodes of C57BL/6 mice and the ILC3-derived MNK3 cell line were used to validate the effect of HIF-1α modulation on NCR⁺ILC3 numbers. An acute UC model was induced by 3 % DSS in C57BL/6 mice. Mice were treated with epicatechin (37.5, 75, or 150 mg/kg/d) or mesalazine (500 mg/kg/d) by gavage, or with roxadustat (25.0 mg/kg) by intraperitoneal injection. Intestinal epithelial barrier function was assessed by Western blot, immunofluorescence, and histopathology. Flow cytometry was used to detect changes in HIF-1α levels and NCR⁺ILC3 abundance. The direct target of epicatechin was identified by integrating DARTS, CETSA, and mass spectrometry, and the mechanism was validated by shRNA-mediated knockdown combined with co-immunoprecipitation.

HIF-1α levels correlated positively with NCR⁺ILC3 numbers. In DSS-induced acute colitis, epicatechin dose-dependently alleviated body weight loss, colon shortening, and systemic inflammation; improved histopathological injury; restored tight junction protein expression (Occludin, ZO-1) and mucin secretion; and reduced intestinal permeability. Epicatechin restored NCR⁺ILC3 numbers in the colonic lamina propria and mesenteric lymph nodes while concomitantly restoring HIF-1α levels. In vitro, epicatechin dose-dependently upregulated the mRNA expression of Il22, Rorc, HIF-1A, and Ncr1, as well as NICD and HIF-1α protein levels in MNK3 cells, whereas total NLE1 protein remained unaffected. Mechanistically, epicatechin bound NLE1 (binding energy -8.5 kcal/mol), enhanced the interaction between NLE1 and NICD, and simultaneously disrupted the binding of NICD to FBXW7, the substrate recognition subunit of the E3 ubiquitin ligase complex, thereby reducing NICD ubiquitination and stabilizing HIF-1α signaling. NLE1 knockdown completely abolished epicatechin-induced activation of the NICD/HIF-1α pathway, IL-22 secretion, and target gene upregulation, confirming NLE1 as its critical molecular target.

Epicatechin alleviates DSS-induced colitis by directly targeting NLE1, enhancing NLE1-NICD binding and reducing NICD-FBXW7 interaction to stabilize NICD protein, thereby activating the NLE1/NICD/HIF-1α signaling axis and restoring NCR⁺ILC3 numbers and IL-22 secretion. These findings establish a molecular link between dietary flavonoids and ILC3-mediated immunity, positioning epicatechin as a phytochemical lead for UC mucosal repair.