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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

Intestinal stem cells (ISCs) decode and coordinate various types of nutritional information from the diet to support the crypt-villus axis architecture, but how specific dietary molecules affect intestinal epithelial homeostasis remains unclear. In the current study, L-glutamate (Glu) supplementation in either a nitrogen-free diet (NFD) or a corn-soybean meal diet (CSMD) stimulated gut growth and ISC expansion in weaned piglets. Quantitative proteomics screening identified the canonical Wnt signalling pathway as a central regulator of intestinal epithelial development and ISC activity in vivo. Importantly, the Wnt transmembrane receptor Frizzled7 (FZD7) was upregulated in response to dietary Glu patterns, and its perturbations in intestinal organoids (IOs) treated with a specific inhibitor and in FZD7-KO IPEC-J2 cells disrupted the link between Glu inputs and β-catenin signalling and a subsequent reduction in cell viability. Furthermore, co-localization, coimmunoprecipitation (Co-IP), isothermal titration calorimetry (ITC), and microscale thermophoresis (MST) revealed that Glu served as a signalling molecule directly bound to FZD7. We propose that FZD7-mediated integration of the extracellular Glu signal controls ISC proliferation and differentiation, which provides new insights into the crosstalk of nutrients and ISCs.

Herbivore-associated molecular patterns (HAMPs) enable plants to recognize herbivores and may help plants adjust their defense responses. Here, we report on herbivore-induced changes in a protein disulfide isomerase (PDI) widely distributed across arthropods. PDI from the spider mite Tetranychus evansi (TePDI), a mesophyll-feeding agricultural pest worldwide, triggered immunity in multiple Solanaceae plants. TePDI-mediated cell death in Nicotiana benthamiana required the plant signaling proteins SGT1 (suppressor of the G2 allele of skp1) and HSP90 (heat shock protein 90), but was suppressed by spider mite effectors Te28 and Te84. Moreover, PDIs from phylogenetically distinct herbivorous and nonherbivorous arthropods triggered plant immunity. Finally, although PDI-induced plant defenses impaired the performance of spider mites on plants, RNAi experiments revealed that PDI genes are essential for the survival of mites and whiteflies. Our findings indicate that plants recognize evolutionarily conserved HAMPs to activate plant defense and resist pest damage, pointing to opportunities for broad-spectrum pest management.

Enterotoxigenic Escherichia coli (ETEC) in humans and animals colonizes the intestine and thereafter secrets heat-stable enterotoxin (ST) with or without heat-labile enterotoxin (LT), which triggers massive fluid and electrolyte secretion into the gut lumen. The crosstalk between the cyclic nucleotide-dependent protein kinase/cystic fibrosis transmembrane conductance regulator (cAMP or cGMP/CFTR) pathway involved in ETEC-induced diarrhea channels, and the canonical Wnt/β-catenin signaling pathway leads to changes in intestinal stem cell (ISC) fates, which are strongly associated with developmental disorders caused by diarrhea. We review how alterations in enterotoxin-activated ion channel pathways and the canonical Wnt/β-catenin signaling pathway can explain inhibited intestinal epithelial activity, characterize alterations in the crosstalk of cyclic nucleotides, and predict harmful effects on ISCs in targeted therapy. Besides, we discuss current deficits in the understanding of enterotoxin-intestinal epithelial cell activity relationships that should be considered when interpreting sequelae of diarrhea.

Vertebrate embryogenesis involves a conserved and fundamental process, called the maternal-to-zygotic transition (MZT), which marks the switch from a maternal factors-dominated state to a zygotic factors-driven state. Yet the precise mechanism underlying MZT remains largely unknown. Here we report that the RNA helicase Ddx3xb in zebrafish undergoes liquid-liquid phase separation (LLPS) via its N-terminal intrinsically disordered region (IDR), and an increase in ATP content promotes the condensation of Ddx3xb during MZT. Mutant form of Ddx3xb losing LLPS ability fails to rescue the developmental defect of Ddx3xb-deficient embryos. Interestingly, the IDR of either FUS or hnRNPA1 can functionally replace the N-terminal IDR in Ddx3xb. Phase separation of Ddx3xb facilitates the unwinding of 5' UTR structures of maternal mRNAs to enhance their translation. Our study reveals an unprecedent mechanism whereby the Ddx3xb phase separation regulates MZT by promoting maternal mRNA translation.

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/β-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/β-catenin pathway. Nevertheless, Wnt/β-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 β-catenin, Lgr5, Bmi1, Ki67, KRT20, ZO-1, occludin, and claudin-1. Taken together, we conclude that heat exposure induces ERS and downregulates the Wnt/β-catenin signaling pathway to disrupt epithelial integrity by inhibiting the intestinal epithelial cell proliferation and stem cell expansion.

The intestinal epithelium is derived from intestinal stem cells (ISCs) and has direct contact with nutrients and toxins. However, whether methionine (Met) or a methionine hydroxyl analogue (2-hydroxy-4-(methylthio)butanoic acid (HMB)) can alleviate deoxynivalenol (DON)-induced intestinal injury remains unknown. Mice were treated orally with Met or HMB on days 1-11 and with DON on days 4-8. On day 12, the mice were sacrificed, and the jejunum was collected for crypt isolation and culture. Mouse enteroids were treated with DON and Met or HMB ex vivo. The results showed that Met and HMB increased the average daily feed intake and average daily gain of the mice. Met and HMB also improved the jejunal structure and barrier integrity and promoted ISC expansion, as indicated by the increased enteroid formation efficiency and area, under DON-induced injury conditions. In addition, DON-induced decreases in ISC activity were rescued Wnt/β-catenin signaling reactivation by Met or HMB in vivo and ex vivo. Collectively, our findings reveal that Met and HMB alleviated DON-induced intestinal injury by improving ISC expansion and reactivating Wnt/β-catenin signaling. Our study thus provides a nutritional intervention for intestinal diseases involving Wnt/β-catenin signaling.

Scope The intestinal epithelium is nourished by various nutrients and subjected to persistent and widespread feed-derived mycotoxin stress. l-Carnosine (LC) possesses robust antioxidant activity; however, its role in protecting intestinal mucosa against deoxynivalenol (DON) is still unclear. Methods and results In this study, 300 mg kg-1 BW LC and 3 mg kg-1 BW DON are orally administered to mice either alone or in combination for 10 days to investigate the role of LC in protecting the intestine against DON. This study found that LC alleviates the growth retardation of mice and repairs the damaged jejunal structure and barrier functions under DON exposure. LC rescues the intestinal stem cells (ISCs), increases the growth advantage in enteroids derived from jejunal crypts of mice in each group ex vivo, improves the proliferation and apoptosis of intestinal cells, and promotes ISC differentiation into absorptive cells, goblet cells, and Paneth cells. Furthermore, LC activates Nrf2 signaling by binding to Keap1 to reverse the striking DON-induced increase in ROS levels. Conclusion The study findings unveil that LC potentiates the antioxidant capacity of ISCs by regulating the Keap1/Nrf2 signaling pathway, which contributes to the intestinal epithelial regeneration response to DON insult.

Intestinal stem cell (ISC)-driven intestinal homeostasis is subjected to dual regulation by dietary nutrients and toxins. Our study investigated the use of lauric acid (LA) to alleviate deoxynivalenol (DON)-induced intestinal epithelial damage. C57BL/6 mice in the control, LA, DON, and LA + DON groups were orally administered PBS, 10 mg/kg BW LA, 2 mg/kg BW DON, and 10 mg/kg BW LA + 2 mg/kg BW DON for 10 days. The results showed that LA increased the average daily gain and average daily feed intake of the mice exposed to DON. Moreover, the DON-triggered impairment of jejunal morphology and barrier function was significantly improved after LA supplementation. Moreover, LA rescued ISC proliferation, inhibited intestinal cell apoptosis, and promoted ISC differentiation into absorptive cells, goblet cells, and Paneth cells. The jejunum crypt cells from the mice in the LA group expanded into enteroids, resulting in a significantly greater enteroid area than that in the DON group. Furthermore, LA reversed the DON-mediated inhibition of the Akt/mTORC1/S6K1 signaling axis in the jejunum. Our results indicated that LA accelerates ISC regeneration to repair intestinal epithelial damage after DON insult by reactivating the Akt/mTORC1/S6K1 signaling pathway, which provides new implications for the function of LA in ISCs.

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/β-catenin activity in the mice. Following a similar trend, the enteroid-budding efficiency and the expression of Active β-catenin, β-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/β-catenin activity by downregulating its membrane receptor Frizzled7 (FZD7). In contrast, Wnt/β-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/β-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/β-catenin signaling upregulation or reactivation. Our findings indicate Zn-Asp, especially Zn, enhances ISC activity to maintain the intestinal integrity by activating the Wnt/β-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.

The intake of food containing deoxynivalenol frequently causes damage to the intestine, the renewal of which is driven by intestinal stem cells (ISCs). Nevertheless, the toxicity of deoxynivalenol on ISCs and its underlying mechanisms remain to be elucidated. As pigs are the most sensitive animals to deoxynivalenol, we used piglets for investigation in this study. Here, we show that intestinal epithelial cell activity, B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1) protein level, and Wnt/β-catenin pathway activity were suppressed with acute expose to deoxynivalenol. We further established a novel system for porcine crypt isolation and ex vivo cultivation. Crypts and crypt cells expanded and budded with typical enteroid morphologies under this system. Our results show that both acute in vivo and in vitro administration of deoxynivalenol significantly decreased enteroid activity. Simultaneously, protein levels of β-catenin and leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) in enteroids were reduced by deoxynivalenol exposure. In conclusion, we established a reliable culture system for porcine enteroids and demonstrated for the first time that the activity of ISCs and the Wnt/β-catenin pathway is sensitively suppressed by acute deoxynivalenol exposure.

Disintegration of the intestine caused by deoxynivalenol (DON), which is a fungal metabolite found in cereal grain-based human and animal diets, triggers severe intestinal inflammatory disease. Hydrolyzed wheat gluten (HWG) can promote the development of intestine. Therefore, HWG was administered orally to male mice on 1-14 days, and DON was administered to them on 4-11 days. Feed, water intake and body weight were recorded all over the experimental period. Blood samples were collected then the mice were sacrificed to collect the jejunum for crypt isolation and culture. The intestinal morphology was observed by electron microscopy, and Western blotting was used to investigate intestinal stem cell (ISC) proliferation and differentiation, as well as the primary regulatory mechanism of the Wnt/β-catenin signaling. The results showed that HWG increased the average daily gain and average daily water intake of mice under DON-induced injury conditions, and increased the jejunum weight, villous height in the jejunum, and promoted jejunal crypt cell expansion. The DON-induced decrease in Wnt/β-catenin activity, the expression of Ki67, PCNA and KRT20 were rescued by HWG in the jejunum, crypt and enteroid, as well as the number of goblet cells and Paneth cells. Furthermore, HWG increased jejunum diamine oxidase (DAO) activity. In conclusion, HWG alleviates DON-induced intestinal injury by enhancing ISC proliferation and differentiation in a Wnt/β-catenin-dependent manner.