The sea cucumber Apostichopus japonicus can expel internal organs under stress and regenerate them subsequently. However, growth is delayed during regeneration, significantly impacting the industry. Circular RNAs (circRNAs) are single-stranded circular RNA molecules produced through alternative splicing of mRNA precursors. They play crucial roles in regulating gene expression via the ceRNA mechanism. In this study, circRNA profiles of control and regenerated intestines were constructed. A total of 15,874 circRNAs were identified, with a length of 300-350 nucleotides (nt) being the most abundant. Sanger sequencing confirmed the circular structure of circRNA398. Compared with the normal intestine, 50 and 83 differentially expressed circRNAs (DE-circRNAs) were identified in the regenerated intestine at 1 and 3 days post evisceration (dpe), respectively. Gene ontology (GO) terms for signal transduction and development regulation were most significantly enriched in 1dpeVScon and 3dpeVScon treatments, respectively. The dual-luciferase assay revealed that circRNA8388 functions as a sponge for miR-2392, participating in the remodeling of the extracellular matrix (ECM). In conclusion, these findings will contribute to the enhancement of the non-coding RNA database for echinoderms and lay the groundwork for future investigations into circRNA regulation during intestinal regeneration.

Ferroptosis plays important roles both in normal physiology and multiple human diseases. It is well known that selenoprotein named glutathione peroxidase 4 (GPX4) is a crucial regulator for ferroptosis. However, it remains unknown whether other selenoproteins responsible for the regulation of ferroptosis, particularly in gut diseases. In this study, it is observed that Selenoprotein I (Selenoi) prevents ferroptosis by maintaining ether lipids homeostasis. Specific deletion of Selenoi in intestinal epithelial cells induced the occurrence of ferroptosis, leading to impaired intestinal regeneration and compromised colonic tumor growth. Mechanistically, Selenoi deficiency causes a remarkable decrease in ether-linked phosphatidylethanolamine (ePE) and a marked increase in ether-linked phosphatidylcholine (ePC). The imbalance of ePE and ePC results in the upregulation of phospholipase A2, group IIA (Pla2g2a) and group V (Pla2g5), as well as arachidonate-15-lipoxygenase (Alox15), which give rise to excessive lipid peroxidation. Knockdown of PLA2G2A, PLA2G5, or ALOX15 can reverse the ferroptosis phenotypes, suggesting that they are downstream effectors of SELENOI. Strikingly, GPX4 overexpression cannot rescue the ferroptosis phenotypes of SELENOI-knockdown cells, while SELENOI overexpression can partially rescue GPX4-knockdown-induced ferroptosis. It suggests that SELENOI prevents ferroptosis independent of GPX4. Taken together, these findings strongly support the notion that SELENOI functions as a novel suppressor of ferroptosis during colitis and colon tumorigenesis.

Chemotherapy and radiotherapy frequently lead to intestinal damage. The mechanisms governing the repair or regeneration of intestinal damage are still not fully elucidated. Intraepithelial lymphocytes (IELs) are the primary immune cells residing in the intestinal epithelial layer. However, whether IELs are involved in intestinal epithelial injury repair remains unclear. Here, we found that IELs rapidly infiltrated the intestinal crypt region and are crucial for the recovery of the intestinal epithelium post-chemotherapy. Interestingly, IELs predominantly promoted intestinal regeneration by modulating the proliferation of transit-amplifying (TA) cells. Mechanistically, the expression of CD160 on IELs allows for interaction with herpes virus entry mediator (HVEM) on the intestinal epithelium, thereby activating downstream nuclear factor kappa (NF-κB) signaling and further promoting intestinal regeneration. Deficiency in either CD160 or HVEM resulted in reduced proliferation of intestinal progenitor cells, impaired intestinal damage repair, and increased mortality following chemotherapy. Remarkably, the adoptive transfer of CD160-sufficient IELs rescued the Rag1 deficient mice from chemotherapy-induced intestinal inflammation. Overall, our study underscores the critical role of IELs in intestinal regeneration and highlights the potential applications of targeting the CD160-HVEM axis for managing intestinal adverse events post-chemotherapy and radiotherapy.

Intestinal epithelium undergoes regeneration after injuries, and the disruption of this process can lead to inflammatory bowel disease and tumorigenesis. Intestinal stem cells (ISCs) residing in the crypts are crucial for maintaining the intestinal epithelium\'s homeostasis and promoting regeneration upon injury. However, the precise role of DGCR8, a critical component in microRNA (miRNA) biogenesis, in intestinal regeneration remains poorly understood. In this study, we provide compelling evidence demonstrating the indispensable role of epithelial miRNAs in the regeneration of the intestine in mice subjected to 5-FU or irradiation-induced injury. Through a comprehensive pooled screen of miRNA function in Dgcr8-deficient organoids, we observe that the loss of the miR-200 family leads to the hyperactivation of the p53 pathway, thereby reducing ISCs and impairing epithelial regeneration. Notably, downregulation of the miR-200 family and hyperactivation of the p53 pathway are verified in colonic tissues from patients with active ulcerative colitis (UC). Most importantly, the transient supply of miR-200 through the oral delivery of lipid nanoparticles (LNPs) carrying miR-200 restores ISCs and promotes intestinal regeneration in mice following acute injury. Our study implies the miR-200/p53 pathway as a promising therapeutic target for active UC patients with diminished levels of the miR-200 family. Furthermore, our findings suggest that the clinical application of LNP-miRNAs could enhance the efficacy, safety, and acceptability of existing therapeutic modalities for intestinal diseases.

Sea cucumbers have an extraordinary regenerative capability. Under stressful conditions, Holothuria glaberrima can eviscerate their internal organs, including the digestive tract. From the mesentery, a rudiment grows and gives rise to a new intestine within a few weeks. In the last decades, the cellular events that occur during intestinal regeneration have been characterized, including apoptosis, cell proliferation, and muscle cell dedifferentiation. Nevertheless, their contribution to the formation and early growth of the rudiment is still unknown. Furthermore, these cellular events\' relationship and potential interdependence remain a mystery. Using modulators to inhibit apoptosis and cell proliferation, we tested whether rudiment growth or other regenerative cellular events like muscle cell dedifferentiation were affected. We found that inhibition of apoptosis by zVAD and cell proliferation by aphidicolin and mitomycin did not affect the overall size of the rudiment seven days post-evisceration (7-dpe). Interestingly, animals treated with aphidicolin showed higher levels of muscle cell dedifferentiation in the distal mesentery, which could act as a compensatory mechanism. On the other hand, inhibition of apoptosis led to a decrease in cell proliferation in the rudiment and a delay in the spatiotemporal progression of muscle cell dedifferentiation throughout the rudiment-mesentery structure. Our findings suggest that neither apoptosis nor cell proliferation significantly contributes to early rudiment growth during intestinal regeneration in the sea cucumber. Nevertheless, apoptosis may play an essential role in modulating cell proliferation in the rudiment (a process known as apoptosis-induced proliferation) and the timing for the progression of muscle cell dedifferentiation. These findings provide new insights into the role and relationship of cellular events during intestinal regeneration in an emerging regeneration model.

Hepatocyte nuclear factor 4-alpha (HNF4α) drives a complex array of transcriptional programs across multiple organs. Beyond its previously documented function in the liver, HNF4α has crucial roles in the kidney, intestine, and pancreas. In the intestine, a multitude of functions have been attributed to HNF4 and its accessory transcription factors, including but not limited to, intestinal maturation, differentiation, regeneration, and stem cell renewal. Functional redundancy between HNF4α and its intestine-restricted paralog HNF4γ, and co-regulation with other transcription factors drive these functions. Dysregulated expression of HNF4 results in a wide range of disease manifestations, including the development of a chronic inflammatory state in the intestine. In this review, we focus on the multiple molecular mechanisms of HNF4 in the intestine and explore translational opportunities. We aim to introduce new perspectives in understanding intestinal genetics and the complexity of gastrointestinal disorders through the lens of HNF4 transcription factors.

The intestinal epithelium has high intrinsic turnover rate, and the precise renewal of the epithelium is dependent on the microenvironment. The intestine is innervated by a dense network of peripheral nerves that controls various aspects of intestinal physiology. However, the role of neurons in regulating epithelial cell regeneration remains largely unknown. Here, we investigated the effects of gut-innervating adrenergic nerves on epithelial cell repair following irradiation (IR)-induced injury. We observed that adrenergic nerve density in the small intestine increased post IR, while chemical adrenergic denervation impaired epithelial regeneration. Single-cell RNA sequencing experiments revealed a decrease in IL-22 signaling post IR in denervated animals. Combining pharmacologic and genetic tools, we demonstrate that β-adrenergic receptor signaling drives IL-22 production from type 3 innate lymphoid cells (ILC3s) post IR, which in turn promotes epithelial regeneration. These results define an adrenergic-ILC3 axis important for intestinal regeneration.

Cancer remains the second leading cause of mortality, with nearly 10 million deaths worldwide in 2020. In many cases, radiotherapy is used for its anticancer effects. However, radiation causes healthy tissue toxicity as a side effect. In intra-abdominal and pelvic malignancies, the healthy bowel is inevitably included in the radiation field, causing radiation-induced enteritis and dramatically affecting the gut microbiome. This condition is associated with significant morbidity and mortality that impairs cancer patients\' and survivors\' quality of life. This Review provides a critical overview of the main drivers in modulating the gut microenvironment in homeostasis, disease, and injury, focusing on gut microbial metabolites and microorganisms that influence epithelial regeneration upon radiation injury.

Intestinal ischemia underlies several clinical conditions and can result in the loss of the intestinal mucosal barrier. Ischemia-induced damage to the intestinal epithelium is repaired by stimulation of intestinal stem cells (ISCs), and paracrine signaling from the vascular niche regulates intestinal regeneration. Here, we identify FOXC1 and FOXC2 as essential regulators of paracrine signaling in intestinal regeneration after ischemia-reperfusion (I/R) injury. Vascular endothelial cell (EC)- and lymphatic EC (LEC)-specific deletions of Foxc1, Foxc2, or both in mice worsen I/R-induced intestinal damage by causing defects in vascular regrowth, expression of chemokine CXCL12 and Wnt activator R-spondin 3 (RSPO3) in blood ECs (BECs) and LECs, respectively, and activation of Wnt signaling in ISCs. Both FOXC1 and FOXC2 directly bind to regulatory elements of the CXCL12 and RSPO3 loci in BECs and LECs, respectively. Treatment with CXCL12 and RSPO3 rescues the I/R-induced intestinal damage in EC- and LEC-Foxc mutant mice, respectively. This study provides evidence that FOXC1 and FOXC2 are required for intestinal regeneration by stimulating paracrine CXCL12 and Wnt signaling.

The ability of stem cells to rapidly proliferate and differentiate is integral to the steady-state maintenance of tissues with high turnover such as the blood and intestine. Mutations that alter these processes can cause primary immunodeficiencies, malignancies and defects in barrier function. The Rho-kinases, Rock1 and Rock2, regulate cell shape and cytoskeletal rearrangement, activities essential to mitosis. Here, we use inducible gene targeting to ablate Rock1 and Rock2 in adult mice, and identify an obligate requirement for these enzymes in the preservation of the hematopoietic and gastrointestinal systems. Hematopoietic cell progenitors devoid of Rho-kinases display cell cycle arrest, blocking the differentiation to mature blood lineages. Similarly, these mice exhibit impaired epithelial cell renewal in the small intestine, which is ultimately fatal. Our data reveal a novel role for these kinases in the proliferation and viability of stem cells and their progenitors, which is vital to maintaining the steady-state integrity of these organ systems.