Matrine reduced intestinal stem cell damage in eimeria necatrix-infected chicks via blocking hyperactivation of Wnt signaling.

BACKGROUND: Coccidiosis is a rapidly spreading and acute parasitic disease that seriously threatening the intestinal health of poultry. Matrine from leguminous plants has anthelmintic and anti-inflammatory properties. PURPOSE: This assay was conducted to explore the protective effects of Matrine and the AntiC (a Matrine compound) on Eimeria necatrix (EN)-infected chick small intestines and to provide a nutritional intervention strategy for EN injury. STUDY DESIGN: The in vivo (chick) experiment: A total of 392 one-day-old yellow-feathered broilers were randomly assigned to six groups in a 21-day study: control group, 350 mg/kg Matrine group, 500 mg/kg AntiC group, EN group, and EN + 350 mg/kg Matrine group, EN + 500 mg/kg AntiC group. The in vitro (chick intestinal organoids, IOs): The IOs were treated with PBS, Matrine, AntiC, 3 µM CHIR99021, EN (15,000 EN sporozoites), EN + Matrine, EN + AntiC, EN + Matrine + CHIR99021, EN + AntiC + CHIR99021. METHODS: The structural integrity of chicks jejunal crypt-villus axis was evaluated by hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM). And the activity of intestinal stem cells (ISCs) located in crypts was assessed by in vitro expansion advantages of a primary in IOs model. Then, the changes of Wnt/ß-catenin signaling in jejunal tissues and IOs were detected by Real-Time qPCR,Western blotting and immunohistochemistry. RESULTS: The results showed that dietary supplementation with Matrine or AntiC rescued the jejunal injury caused by EN, as indicated by increased villus height, reduced crypt hyperplasia, and enhanced expression of tight junction proteins. Moreover, there was less budding efficiency of the IOs expanded from jejunal crypts of chicks in the EN group than that in the Matrine and AntiC group, respectively. Further investigation showed that AntiC and Matrine inhibited EN-stimulated Wnt/ß-catenin signaling. The fact that Wnt/ß-catenin activation via CHIR99021 led to the failure of Matrine and AntiC to rescue damaged ISCs confirmed the dominance of this signaling. CONCLUSION: Our results suggest that Matrine and AntiC inhibit ISC proliferation and promote ISC differentiation into absorptive cells by preventing the hyperactivation of Wnt/ß-catenin signaling, thereby standardizing the function of ISC proliferation and differentiation, which provides new insights into mitigating EN injury by Matrine and AntiC.

Heat stress disrupts intestinal stem cell migration and differentiation along the crypt-villus axis through FAK signaling.

During heat stress (HS), the intestinal epithelium suffers damage due to imbalance of tissue homeostasis. However, the specific mechanism by which intestinal stem cells (ISCs) migrate and differentiate along the crypt-villus axis to heal lesions upon insult is unclear. In our study, C57BL/6 mice and IPEC-J2 cells were subjected to normal ambient conditions (25 °C for 7 days in vivo and 37 °C for 18 h in vitro) or 41 °C. The results showed that HS impaired intestinal morphology and barrier function. The numbers of ISCs (SOX9+ cells), mitotic cells (PCNA+ cells), and differentiated cells (Paneth cells marked by lysozyme, absorptive cells marked by Villin, goblet cells marked by Mucin2, enteroendocrine cells marked by Chromogranin A, and tuft cells marked by DCAMKL1) were reduced under high temperature. Importantly, BrdU incorporation confirmed the decreased migration ability of jejunal epithelial cells exposed to 41 °C. Furthermore, intestinal organoids (IOs) expanded from jejunal crypt cells in the HS group exhibited greater growth disadvantages. Mechanistically, the occurrence of these phenotypes was accompanied by FAK/paxillin/F-actin signaling disruption in the jejunum. The fact that the FAK agonist ZINC40099027 reversed the HS-triggered inhibition of IPEC-J2 cell differentiation and migration further confirmed the dominant role of FAK in response to high-temperature conditions. Overall, the present investigation is the first to reveal a major role of FAK/paxillin/F-actin signaling in HS-induced ISC migration and differentiation along the crypt-villus axis, which indicates a new therapeutic target for intestinal epithelial regeneration after heat injuries.

Selenomethionine Alleviates DON-Induced Oxidative Stress via Modulating Keap1/Nrf2 Signaling in the Small Intestinal Epithelium.

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.

L-glutamate requires ß-catenin signalling through Frizzled7 to stimulate porcine intestinal stem cell expansion.

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 ß-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.

Recombinant Porcine R-Spondin 1 Facilitates Intestinal Stem Cell Expansion along the Crypt-Villus Axis through Potentiating Wnt/ß-Catenin Signaling in Homeostasis and Deoxynivalenol Injury.

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/ß-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/ß-catenin activity to increase ISC activity. Our work systematically demonstrates that rpRSPO1 facilitates ISC expansion by potentiating Wnt/ß-catenin signaling during homeostasis and responding to deoxynivalenol perturbations.

Lauric acid alleviates deoxynivalenol-induced intestinal stem cell damage by potentiating the Akt/mTORC1/S6K1 signaling axis.

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.