Heat-stable enterotoxin inhibits intestinal stem cell expansion to disrupt the intestinal integrity by downregulating the Wnt/ß-catenin pathway.

Enterotoxigenic Escherichia coli causes severe infectious diarrhea with high morbidity and mortality in newborn and weanling pigs mainly through the production of heat-stable enterotoxins (STs). However, the precise regulatory mechanisms involved in ST-induced intestinal epithelium injury remain unclear. Consequently, we conducted the experiments in vivo (mice), ex vivo (mouse and porcine enteroids), and in vitro (MODE-K and IPEC-J2 cells) to explore the effect of STp (one type of STa) on the integrity of the intestinal epithelium. The results showed that acute STp exposure led to small intestinal edema, disrupted intestinal integrity, induced crypt cell expansion into spheroids, and downregulated Wnt/ß-catenin activity in the mice. Following a similar trend, the enteroid-budding efficiency and the expression of Active ß-catenin, ß-catenin, Lgr5, PCNA, and KRT20 were significantly decreased after STp treatment, as determined ex vivo. In addition, STp inhibited cell proliferation, induced cell apoptosis, destroyed cell barriers, and reduced Wnt/ß-catenin activity by downregulating its membrane receptor Frizzled7 (FZD7). In contrast, Wnt/ß-catenin reactivation protected the IPEC-J2 cells from STp-induced injury. Taking these findings together, we conclude that STp inhibits intestinal stem cell expansion to disrupt the integrity of the intestinal mucosa through the downregulation of the Wnt/ß-catenin signaling pathway.

Wnt/ß-catenin-mediated heat exposure inhibits intestinal epithelial cell proliferation and stem cell expansion through endoplasmic reticulum stress.

Heat stress induced by continuous high ambient temperatures or strenuous exercise in humans and animals leads to intestinal epithelial damage through the induction of intracellular stress response. However, the precise mechanisms involved in the regulation of intestinal epithelial cell injury, especially intestinal stem cells (ISCs), remain unclear. Thereby, in vitro a confluent monolayer of IPEC-J2 cells was exposed to the high temperatures (39, 40, and 41°C), the IPEC-J2 cell proliferation, apoptosis, differentiation, and barrier were determined, as well as the expression of GRP78, which is a marker protein of endoplasmic reticulum stress (ERS). The Wnt/ß-catenin pathway-mediated regenerative response was validated using R-spondin 1 (Rspo1). And ex-vivo, three-dimensional cultured enteroids were developed from piglet jejunal crypt and employed to assess the ISC activity under heat exposure. The results showed that exposure to 41°C for 72 hr, rather than 39°C and 40°C, decreased IPEC-J2 cell viability, inhibited cell proliferation and differentiation, induced ERS and cell apoptosis, damaged barrier function and restricted the Wnt/ß-catenin pathway. Nevertheless, Wnt/ß-catenin reactivation via Rspo1 protects the intestinal epithelium from heat exposure-induced injury. Furthermore, exposure to 41°C for 24 hr reduced ISC activity, stimulated crypt-cell apoptosis, upregulated the expression of GRP78 and caspase-3, and downregulated the expression of ß-catenin, Lgr5, Bmi1, Ki67, KRT20, ZO-1, occludin, and claudin-1. Taken together, we conclude that heat exposure induces ERS and downregulates the Wnt/ß-catenin signaling pathway to disrupt epithelial integrity by inhibiting the intestinal epithelial cell proliferation and stem cell expansion.

mTORC1 signaling activation increases intestinal stem cell activity and promotes epithelial cell proliferation.

The crypt-villus axis of the intestine undergoes a continuous renewal process that is driven by intestinal stem cells (ISCs). However, the homeostasis is disturbed under constant exposure to high ambient temperatures, and the precise mechanism is unclear. We found that both EdU+ and Ki67+ cell ratios were significantly reduced after exposure to 41°C, as well as the protein synthesis rate of IPEC-J2 cells, and the expression of ubiquitin and heat shock protein 60, 70, and 90 were significantly increased. Additionally, heat exposure decreased enteroid expansion and budding efficiency, as well as induced apoptosis after 48 hr; however, no significant difference was observed in the apoptosis ratio after 24 hr. In the process of heat exposure, the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway was significantly inhibited in both IPEC-J2 cells and enteroids. Correspondingly, treatment of IPEC-J2 and enteroids with the mTORC1 agonist MHY1485 at 41°C significantly attenuated the inhibition of proliferation and protein synthesis, increased the ISC activity, and promoted expansion and budding of enteroid. In summary, we conclude that the mTORC1 signaling pathway regulates intestinal epithelial cell and stem cell activity during heat exposure-induced injury.

Zinc L-Aspartate enhances intestinal stem cell activity to protect the integrity of the intestinal mucosa against deoxynivalenol through activation of the Wnt/ß-catenin signaling pathway.

The micronutrient, zinc, plays a vital role in modulating cellular signaling recognition and enhancing intestinal barrier function. However, the precise mechanisms underlying the zinc regulation of intestinal stem cell (ISC) renewal and regeneration ability, which drive intestinal epithelial turnover to maintain the intestinal barrier, under physiological and pathological conditions are unknown. In this study, we used in vivo mouse plus ex vivo enteroid model to investigate thoroughly the protection efficacy of zinc L-aspartate (Zn-Asp) on intestinal mucosal integrity exposed to deoxynivalenol (DON). The results showed that 10 rather than 20 mg/kg body weight (BW) Zn-Asp (calculation in zinc) significantly increased the jejunum mass and ameliorated mucosa injury caused by 2 mg/kg BW DON treatment, including improvement of the intestinal morphology and barrier, as well as enteroid-forming and -budding efficiency, which was expanded from crypt cells isolated from jejunum of mice in each group. The repair process stimulated by Zn-Asp was also accompanied by increased fluorescence signal intensity of KRT20 and Villin; increased numbers of MUC2+, CAG+, LYZ+, BrdU+ and Ki67+ cells in mouse jejunum; and protein expression of Ki67 and PCNA in the jejunum, crypt and enteroid. Simultaneously, Zn-Asp increased ISC activity to promote intestinal epithelial renewal even under physiological conditions. These results were further verified in ex vivo enteroid culture experiments, which were treated with 100 µmol/L Zn-Asp (calculation in zinc) and 100 ng/mL DON for 72 h. Furthermore, we demonstrated that Zn-Asp improved intestinal integrity or accelerated wound healing along with Wnt/ß-catenin signaling upregulation or reactivation. Our findings indicate Zn-Asp, especially Zn, enhances ISC activity to maintain the intestinal integrity by activating the Wnt/ß-catenin signaling, which sheds some light upon effective preventive strategies for intestinal injury induced by mycotoxin based on ISCs with exogenous zinc preparations in the proper drugs, health foods or qualified feed.