Cannabidiol triggers fatty acids β-oxidation mediated by Stat2 to facilitate intestinal stem cells regeneration post radiation.

Background: Acute gastrointestinal syndrome (AGS) is one of the most severe clinical manifestations after exposure to high doses of radiation, and is life-threatening in radiological emergency scenarios. However, an unmet challenge is lacking of an FDA-approved drug that can ameliorate the damage of radiation-exposed intestinal tissues and accelerate the regeneration of injured epithelia. In this study, we investigated whether the small molecule Me6TREN (Me6) can regulate intestinal stem cell (ISC) proliferation and promote crypt regeneration after irradiation.Methods: Lethally irradiated mice were administered with Me6 or PBS to study the survival rate, and sections of their small intestine were subjected to immunostaining to evaluate epithelial regeneration. An intestinal organoid culture system was employed to detect the role of Me6 in organoid growth and ISC proliferation. We further investigated the key signaling pathways associated with Me6 using microarray, western blotting, and RNA interference techniques.Results: We identified the small molecule Me6 as a potent intestinal radiation countermeasure. Systemic administration of Me6 significantly improved ISC and crypt cell regeneration and enhanced the survival of mice after high doses of radiation. Using an in vitro intestinal organoid culture system, we found that Me6 not only induced ISC proliferation but also increased the budding rate of intestinal organoids under unirradiated and irradiated conditions. Me6 remarkably activated the expression of ISC-associated and proliferation-promoting genes, such as Ascl2, Lgr5, Myc, and CyclinD1. Mechanistically, Me6 strongly stimulated the phosphorylation of β-catenin at the S552 site and increased the transcriptional activity of β-catenin, a key signaling pathway for ISC self-renewal and proliferation. This is further evidenced by the fact that knockdown of β-catenin abolished the effect of Me6 on intestinal organoid growth in vitro and crypt regeneration in irradiated mice.Conclusion: The small molecule Me6TREN induced ISC proliferation, enhanced intestinal organoid growth in vitro, and promoted intestinal tissue regeneration after radiation injury by activating β-catenin signaling.

PERK is essential for proliferation of intestinal stem cells in mice

Sphingolipids, including ceramides, are an important component of high-fat diets. These molecules can regulate fatty acid oxidation and intestinal stem cell proliferation, predisposing the gut to tumorigenesis. However, the molecular mechanisms involved in ceramide metabolism-mediated intestinal stem cell (ISC) proliferation and tumorigenesis are poorly understood. To understand how changes in sphingolipid metabolite flux affect intestinal stem cells, we manipulated the activities of each of the enzymes of the ceramide synthetic pathway using cell type-specific over-expression or depletion of the corresponding mRNAs in each intestinal cell type of the Drosophila midgut. We documented cell-autonomous and non-cell-autonomous effects, including alterations in cell size, number, differentiation, and proliferation. In our screen, the altered expression of several ceramide metabolism enzymes led to changes in ISC proliferation, cell sizes, and overall cellularity. Among other genes, over-expression of ceramidase homolog, Brain washing (bwa) in gut enteroblasts (EB) increased EB cell size and caused a non-cell-autonomous, 7-8-fold increase in ISC proliferation. Our analysis confirmed previous reports that bwa does not have ceramidase activity, and lipidomic studies indicated that bwa increases the saturation status of sphingolipids, free fatty acids, and other lipids. The pro-proliferative effects of bwa could be counter-acted by depleting a serine palmitoyltransferase, Lace , or a sphingosine acyltransferase, Schlank , which are needed for ceramide synthesis, or by co-expressing a ceramide desaturase enzyme, ifc , indicating that increased saturated ceramides were causal for ISC proliferation and the disruption of gut homeostasis. Accumulating saturated sphingolipids and fatty acids induced inflammatory signaling in the gut, and activated ISC proliferation through the pro-inflammatory cytokines, Upd3 and Upd2. We propose that saturated sphingolipids promote ISC proliferation through pro-inflammatory pathways.

Grape Exosome-like Nanoparticles Induce Intestinal Stem Cells and Protect Mice From DSS-Induced Colitis

INTRODUCTION: Increased serotonin activity enhances small intestinal (SI) mucosal growth and nutrient absorption, but the mechanism behind this observation is unknown. This study evaluated the effect of serotonin signaling on intestinal stem cell (ISC) number and proliferation in mouse SI crypts. We hypothesized that alterations in serotonin activity correspond to changes in ISC proliferation. METHODS: Lgr5-EGFP-reporter mice were administered intraperitoneal citalopram, a selective serotonin reuptake inhibitor (SSRI), to potentiate serotonin activity, or para-chlorophenylalanine (PCPA), to inhibit serotonin synthesis. Control animals received saline alone. After 14 days, mice were injected intraperitoneally with bromodeoxyuridine (BrdU) to label actively dividing cells, followed by SI harvest 1 hour later. Immunofluorescence staining and microscopy identified Lgr5-positive ISC and BrdU-positive proliferating cells in epithelial crypts. ISC proliferation was quantified by calculating the proportion of crypts per animal with ≥1 cell expressing Lgr5 and labeled with BrdU. RESULTS: Compared with controls, SSRI treatment was associated with more Lgr5-positive cells per crypt (4.8 ± 0.9 vs 3.1 ± 0.6 cells/crypt; p < 0.01), and PCPA treatment resulted in no difference (3.5 ± 1.3 vs 3.1 ± 0.6 cells/crypt; p = 0.11); 66.6% of crypts in control animals did not contain proliferating stem cells. SSRI treatment was associated with a greater number of crypts exhibiting ISC proliferation (60.0% vs 33.3%; p < 0.01), and PCPA treatment was associated with fewer crypts containing proliferating ISCs (16.7% vs 33.3%; p < 0.01) compared with controls (Figure).FigureCONCLUSION: Serotonin activity is directly correlated with ISC proliferation in SI crypts. Modulation of stem cell activity with SSRIs may have therapeutic potential in patients who undergo SI resection.

Intestinal stem cells (ISCs) in the Drosophila adult midgut are essential for maintaining tissue homeostasis, and their proliferation and differentiation speed up in order to meet the demand for replenishing the lost cells in response to injury. Several signaling pathways including JAK-STAT, EGFR and Hippo (Hpo) pathways have been implicated in damage-induced ISC proliferation, but the mechanisms that integrate these pathways have remained elusive. Here, we demonstrate that the Drosophila homolog of the oncoprotein Myc (dMyc) functions downstream of these signaling pathways to mediate their effects on ISC proliferation. dMyc expression in precursor cells is stimulated in response to tissue damage, and dMyc is essential for accelerated ISC proliferation and midgut regeneration. We show that tissue damage caused by dextran sulfate sodium feeding stimulates dMyc expression via the Hpo pathway, whereas bleomycin feeding activates dMyc through the JAK-STAT and EGFR pathways. We provide evidence that dMyc expression is transcriptionally upregulated by multiple signaling pathways, which is required for optimal ISC proliferation in response to tissue damage. We have also obtained evidence that tissue damage can upregulate dMyc expression post-transcriptionally. Finally, we show that a basal level of dMyc expression is required for ISC maintenance, proliferation and lineage differentiation during normal tissue homeostasis.

Many adult tissues are maintained by resident stem cells that elevate their proliferation in response to injury. The regulatory mechanisms underlying regenerative proliferation are still poorly understood. Here we show that injury induces Hedgehog (Hh) signaling in enteroblasts (EBs) to promote intestinal stem cell (ISC) proliferation in Drosophila melanogaster adult midgut. Elevated Hh signaling by patched (ptc) mutations drove ISC proliferation noncell autonomously. Inhibition of Hh signaling in the ISC lineage compromised injury-induced ISC proliferation but had little if any effect on homeostatic proliferation. Hh signaling acted in EBs to regulate the production of Upd2, which activated the JAK-STAT pathway to promote ISC proliferation. Furthermore, we show that Hh signaling is stimulated by DSS through the JNK pathway and that inhibition of Hh signaling in EBs prevented DSS-stimulated ISC proliferation. Hence, our study uncovers a JNK-Hh-JAK-STAT signaling axis in the regulation of regenerative stem cell proliferation.

The insect midgut epithelium is remodeled during larval-pupal metamorphosis when larval polyploid cells (LPCs) are replaced by the daughters of intestinal stem cells (ISCs). We characterized the proliferation of ISCs during midgut remodeling in the red flour beetle, Tribolium castaneum. Midgut remodeling is initiated at 96 hr after ecdysis into the final instar larval stage. Immunocytochemistry with bromodeoxyuridine and phospho-histone H3 antibodies showed that the ISCs are the progenitors of the pupal/adult midgut epithelium and they undergo proliferation and differentiation to form new midgut epithelium. In vitro midgut culture experiments revealed that 20-hydroxyecdysone (20E) in the absence of juvenile hormone induces proliferation of ISCs. RNA interference (RNAi) mediated silencing of ecdysone receptors (EcRA and EcRB) and ultraspiracle (USP) identified EcRA and USP but not EcRB as the proteins involved in 20E regulation of ISCs proliferation. These data show that the proliferation of ISCs is under both developmental and endocrine regulation.

Long noncoding RNAs (lncRNAs) play important regulatory roles in stem cell self-renewal, pluripotency maintenance, and differentiation. Till now, there is very limited knowledge about how lncRNAs regulate intestinal stem cells (ISCs), and lncRNAs mediating ISC regeneration in Drosophila have yet been characterized. Here, we identify a lncRNA, CR46040, that is essential for the injury-induced ISC regeneration in Drosophila. Loss of CR46040 greatly impairs ISC proliferation in response to tissue damage caused by dextran sulfate sodium (DSS) treatment. We demonstrate that CR46040 is a genuine lncRNA that has two isoforms transcribed from the same transcription start site and works in trans to regulate intestinal stem cells. Mechanistically, CR46040 knock-out flies failed to fully activate JNK, JAK/STAT, and HIPPO signaling pathways after tissue damage, which are required for ISC proliferation after intestinal injury. Moreover, CR46040 knock-out flies are highly susceptible to DSS treatment and enteropathogenic bacteria Erwinia carotovora ssp. carotovora 15 (Ecc15) infection. Our findings characterize, for the first time, a lncRNA that mediates damage-induced ISC proliferation in Drosophila and provide new insights into the functional links among the long noncoding RNAs, ISC proliferation, and tissue homeostasis.

Growth hormone (GH) and glutamine (Gln) stimulate the growth of the intestinal mucosa. GH activates the proliferation of intestinal stem cells (ISCs), enhances the formation of crypt organoids, increases ISC stemness markers in the intestinal organoids, and drives the differentiation of ISCs into Paneth cells and enterocytes. Gln enhances the proliferation of ISCs and increases crypt organoid formation; however, it mainly acts on the post-proliferation activity of ISCs to maintain the stability of crypt organoids and the intestinal mucosa, as well as to stimulate the differentiation of ISCs into goblet cells and possibly Paneth cells and enteroendocrine cells. Since GH and Gln have differential effects on ISCs. Their use in combination may have synergistic effects on ISCs. In this review, we summarize the evidence of the actions of GH and/or Gln on crypt cells and ISCs in the literature. Overall, most studies demonstrated that GH and Gln in combination exerted synergistic effects to activate the proliferation of crypt cells and ISCs and enhance crypt organoid formation and mucosal growth. This treatment influenced the proliferation of ISCs to a similar degree as GH treatment alone and the differentiation of ISCs to a similar degree as Gln treatment alone.

Bacillus amyloliquefaciens is a well-accepted probiotic, with many benefits for both humans and animals. The ability of intestinal stem cells (ISCs) to develop into several intestinal epithelial cell types helps accelerate intestinal epithelial regeneration. Limited knowledge exists on how bacteria regulated ISCs proliferation and regeneration. Our study investigated the effects of Bacillus amyloliquefaciens supplementation on ISC proliferation and regeneration and intestinal mucosal barrier functions in piglets exposed to lipopolysaccharide (LPS). Eighteen piglets (male, 21 days old) were randomly split into 3 clusters: CON cluster, LPS cluster, and SC06+LPS cluster. On day 21, 100 μg/kg body weight of LPS was intraperitoneally administered to the SC06+LPS and LPS groups. We found SC06 supplementation maintained the intestinal barrier integrity, enhanced intestinal antioxidant capacity, reduced generation of inflammatory response, and suppressed enterocyte apoptosis against the deleterious effects triggered by LPS. In addition, our research indicated that the SC06 supplementation not only improved the ISC regeneration, but also resulted in upregulation of aryl hydrocarbon receptor (AhR) in LPS-challenge piglets. Further studies showed that SC06 also induced ISC differentiation toward goblet cells and inhibited their differentiation to intestinal absorptive cells and enterocytes. The coculture system of SC06 and ileum organoids revealed that SC06 increased the growth of ISCs and repaired LPS-induced organoid damage through activating the AhR/STAT3 signaling pathway. These findings showed that SC06, possibly through the AhR/STAT3 pathway, accelerated ISC proliferation and promoted epithelial barrier healing, providing a potential clinical treatment for IBD. Our research demonstrated that SC06 is effective in preventing intestinal epithelial damage after pathological injury, restoring intestinal homeostasis, and maintaining intestinal epithelial regeneration.

Krüppel-like factor 5 is essential for proliferation and survival of mouse intestinal epithelial stem cells

Regulation of small intestinal epithelial growth by endogenous and environmental factors is critical for intestinal homeostasis and recovery from insults. Depletion of the intestinal microbiome increases epithelial proliferation in small intestinal crypts, similar to the effects observed in animal models of serotonin potentiation. Based on prior evidence that the microbiome modulates serotonin activity, we hypothesized that microbial depletion-induced epithelial proliferation is dependent on host serotonin activity. A mouse model of antibiotic-induced microbial depletion (AIMD) was employed. Serotonin potentiation was achieved through either genetic knockout of the serotonin transporter (SERT) or pharmacologic SERT inhibition, and inhibition of serotonin synthesis was achieved with para-chlorophenylalanine. AIMD and serotonin potentiation increased intestinal villus height and crypt proliferation in an additive manner, but the epithelial proliferation observed after AIMD was blocked in the absence of endogenous serotonin. Using Lgr5-EGFP-reporter mice, we evaluated intestinal stem cell (ISC) quantity and proliferation. AIMD increased the number of ISCs per crypt and ISC proliferation compared to controls, and changes in ISC number and proliferation were dependent on the presence of host serotonin. Furthermore, western blotting demonstrated that AIMD reduced epithelial SERT protein expression compared to controls. In conclusion, host serotonin activity is necessary for microbial depletion-associated changes in villus height and ISC proliferation in crypts, and microbial depletion produces a functional serotonin-potentiated state through reduced SERT protein expression. These findings provide an understanding of how changes to the microbiome contribute to intestinal pathology and can be applied therapeutically.

Objectives: Iron deficiency impairs intestinal mucosal structure and function, yet its impact on intestinal stem cells (ISCs) remains unclear. This study was therefore designed to examine how iron deficiency affects the proliferation and differentiation of ISCs. Methods: Iron-deficient mouse and enteroid models were established. Expression of key cell markers was analyzed using Western blot, qPCR, and immunofluorescence. Results: Iron deficiency led to structural impairment of the intestinal mucosa, characterized by decreased small intestinal villus height. In iron-deficient mice, expression of ChrA (enteroendocrine cell marker), Lyz (Paneth cell marker), and Muc2 (goblet cell marker) was significantly downregulated across duodenum, jejunum and ileum, whereas Vil1 (enterocyte marker) expression increased. Moreover, both Lgr5 (an ISC marker) expression and the number of Ki67-positive proliferating cells were significantly reduced, along with a decrease in Ki67 transcriptional levels under iron-deficient conditions. Similarly, deferoxamine (DFO)-treated enteroids showed fewer Lgr5-positive ISCs, downregulation of Lgr5, Lyz and Muc2, and upregulation of Vil1. RNA-seq further confirmed that iron deficiency skews ISC differentiation toward absorptive lineage. This shift was associated with modulation of the Notch signaling pathway: upregulation of the ligand Dll1, receptors Notch2 and Notch3, and the protease ADAM10, alongside downregulation of the negative regulator Atoh1. These findings indicate that Notch pathway activation promotes enterocyte differentiation under iron deprivation. Conclusions: Iron deficiency suppressed the proliferation of ISCs and induced their differentiation toward enterocytes, which is associated with the modulation of the Notch signaling pathway, providing a mechanistic insights for impaired intestinal repair and the potential for nutrient-targeted therapies.

The proliferation and differentiation of intestinal stem cells (ISCs) are the basis of intestinal renewal and regeneration, and gut microbiota plays an important role in it. Dietary nutrition has the effect of regulating the activity of ISCs; however, the regulation effect of α-linolenic acid (ALA) has seldom been reported. After intervening mice with different doses of ALA for 30 days, it is found that ALA (0.5g kg-1 ) promotes small intestinal and villus growth by activating the Wnt/β-catenin signaling pathway to stimulate the proliferation of ISCs. Furthermore, ALA administration increases the abundance of the Ruminococcaceae and Prevotellaceae, and promotes the production of short-chain fatty acids (SCFAs). Subsequent fecal transplantation and antibiotic experiments demonstrate that ALA on the proliferation of ISCs are gut microbiota dependent, among them, the functional microorganism may be derived from Ruminococcaceae. Administration of isobutyrate shows a similar effect to ALA in terms of promoting ISCs proliferation. Furthermore, ALA mitigates 5-fluorouracil-induced intestinal mucosal damage by promoting ISCs proliferation. These results indicate that SCFAs produced by Ruminococcaceae mediate ALA promote ISCs proliferation by activating the Wnt/β-catenin signaling pathway, and suggest the possibility of ALA as a prebiotic agent for the prevention and treatment of intestinal mucositis.