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The small intestinal epithelium is regulated in response to various beneficial or harmful environmental information. Deoxynivalenol (DON), a mycotoxin widely distributed in cereal-based feeds, induces oxidative stress damage in the intestine due to the mitochondrial stress. As a functional nutrient, selenomethionine (Se-Met) is involved in synthesizing several antioxidant enzymes, yet whether it can replenish the intestinal epithelium upon DON exposure remains unknown. Therefore, the in vivo model C57BL/6 mice and the in vitro model MODE-K cells were treated with l-Se-Met and DON alone or in combination to confirm the status of intestinal stem cell (ISC)-driven epithelial regeneration. The results showed that 0.1 mg/kg body weight (BW) Se-Met reinstated the growth performance and integrity of jejunal structure and barrier function in DON-challenged mice. Moreover, Lgr5+ ISCs and PCNA+ mitotic cells in crypts were prominently increased by Se-Met in the presence of DON, concomitant with a significant increase in absorptive cells, goblet cells, and Paneth cells. Simultaneously, crypt-derived jejunal organoids from the Se-Met + DON group exhibited more significant growth advantages ex vivo. Furthermore, Se-Met-stimulated Keap1/Nrf2-dependent antioxidant system (T-AOC and GSH-Px) to inhibit the accumulation of ROS and MDA in the jejunum and serum. Moreover, Se-Met failed to rescue the DON-triggered impairment of cell antioxidant function after Nrf2 perturbation using its specific inhibitor ML385 in MODE-K cells. In conclusion, Se-Met protects ISC-driven intestinal epithelial integrity against DON-induced oxidative stress damage by modulating Keap1/Nrf2 signaling.

R-spondin 1 (RSPO1) is a ligand for the intestinal stem cell (ISC) marker Lgr5 in the crypt, which functions to amplify canonical Wnt signaling to stimulate the division of ISCs. Despite the crucial role of recombinant human RSPO1 (rhRSPO1) in homeostasis and regeneration, little is known about RSPO1 among different species. Here, we cloned the porcine RSPO1 (pRSPO1) gene and obtained rpRSPO1 protein through the expression system of the recombinant Escherichia coli Rosetta (DE3) chemical competent cells. Using the in vitro IPEC-J2 model that combines cell proliferation evaluation approaches, we identified the rpRSPO1 activity in stimulating jejunal epithelial cells. And upon deoxynivalenol challenge in mice, we found that rpRSPO1 ameliorated their growth retardation and jejunal epithelial integrity. Importantly, the ISCs in the jejunum had greater proliferation and differentiation potential that was accompanied by Wnt/β-catenin pathway activation after rpRSPO1 modulation. Subsequently, the jejunal organoids expanded from these ISCs ex vivo presented robust growth advantages. And the rpRSPO1 was able to guide Wnt/β-catenin activity to increase ISC activity. Our work systematically demonstrates that rpRSPO1 facilitates ISC expansion by potentiating Wnt/β-catenin signaling during homeostasis and responding to deoxynivalenol perturbations.

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