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Trefoil factor 3 (intestinal trefoil factor) is a cytoprotective factor in the gut. Herein we compared the effect of trefoil factor 3 with tumor necrosis factor-[alpha] on 1) activation of NF-[kappa]B in intestinal epithelial cells; 2) expression of Twist protein (a molecule essential for downregulation of nuclear factor-[kappa]B activity in vivo); and 3) production of interleukin-8. We showed that Twist protein is constitutively expressed in intestinal epithelial cells. Tumor necrosis factor-[alpha] induced persistent degradation of Twist protein in intestinal epithelial cells via a signaling pathway linked to proteasome, which was associated with prolonged activation of NF-[kappa]B. In contrast to tumor necrosis factor, trefoil factor 3 triggered transient activation of NF-[kappa]B and prolonged upregulation of Twist protein in intestinal epithelial cells via an ERK kinase-mediated pathway. Unlike tumor necrosis factor-[alpha], transient activation of NF-[kappa]B by trefoil factor 3 is not associated with induction of IL-8 in cells. To examine the role of Twist protein in intestinal epithelial cells, we silenced the Twist expression by siRNA. Our data showed that trefoil factor 3 induced interleukin-8 production after silencing Twist in intestinal epithelial cells. Together, these observations indicated that 1) trefoil factor 3 triggers a diverse signal from tumor necrosis factor-[alpha] on the activation of NF-[kappa]B and its associated molecules in intestinal epithelial cells; and 2) trefoil factor 3-induced Twist protein plays an important role in the modulation of inflammatory cytokine production in intestinal epithelial cells. trefoil factor 3; signal transduction

Zhu, Ya-Qin, Yu Lu, and Xiao-Di Tan. Monochloramine induces reorganization of nuclear speckles and phosphorylation of SRp30 in human colonic epithelial cells: role of protein kinase C. Am J Physiol Cell Physiol 285: C1294-C1303, 2003. First published June 25, 2003; 10.1152/ajpcell.00090.2003.--Intestinal epithelial cells are constantly stimulated by reactive oxidant metabolites (ROMs) in inflamed mucosa. Monochloramine (N[H.sub.2]Cl), a cell-permeant ROM, is particularly relevant to the pathogenesis of inflammation in the gastrointestinal tract. Nuclear speckles, a unique nuclear subcompartment, accumulate a family of proteins, namely, serine- and arginine-rich (SR) proteins. They play important roles in regulation of pre-mRNA splicing. Currently, little is known about the link between inflammatory stimulation and the pre-mRNA splicing process, although gene expression is changed in inflamed tissues. The present study was designed to investigate whether stimulation of human colonic epithelial cells (HT-29 and Caco-2 cell lines) with (N[H.sub.2]Cl) affects nuclear speckles and their components. By indirect immunofluorescence, nuclear speckles have been shown to undergo rapid aggregation after (N[H.sub.2]Cl) stimulation. By utilizing Western blotting, SRp30 (a subset of SR proteins) in intestinal epithelial cells was found to be phosphorylated after (N[H.sub.2]Cl) treatment, whereas other SR proteins were not responsive to (N[H.sub.2]Cl) stimulation. The cytotoxic effect of (N[H.sub.2]Cl) was excluded by both negative lactate dehydrogenase assay and propidium iodide staining. Therefore, (N[H.sub.2]Cl)-induced morphological changes on nuclear speckles and phosphorylated SRp30 do not result from intestinal epithelial injury. Furthermore, the effect of (N[H.sub.2]Cl) on nuclear speckles and SRp30 was blocked by bisindolylmaleimide I, a selective PKC inhibitor. Together, the available data suggest that stimulation of intestinal epithelial cells with (N[H.sub.2]Cl) results in a consequent change on pre-mRNA splicing machinery via a distinctive signal pathway involving activation of PKC. This effect may contribute to oxidant-induced pathophysiological changes in the gastrointestinal tract. intestinal epithelial cell; reactive oxygen metabolites; pre-mRNA splicing machinery; SR proteins; signal transduction

Apoptosis, inflammation, and wound healing are critical pathophysiological events associated with various liver diseases. Currently, there is a lack of in vivo approaches to study hepatocyte apoptosis-induced liver injury and repair. To address this critical knowledge gap, we developed a unique genetically modified mouse model, namely, 3xTg-iHAP ( 3 - T rans g ene with i nducible H epatocyte A poptosis P henotype) in this study. The 3xTg-iHAP mice possess three transgenes including Alb-Cre , Rosa26- rtTA , and tetO-Fasl on a B6 background. These mice are phenotypically normal, viable, and fertile. After subcutaneous administration of a single dose of doxycycline (5 mg/kg, Dox) to 3xTg-iHAP mice, we observed a complete histological spectrum of sterile liver wound-healing responses: asymptomatic hepatocyte apoptosis at 8 h, necrotic liver injury and sterile inflammation at 48 h, followed by hepatocyte mitosis and regeneration within 7 days. During the injury phase, the mice exhibited an increase in biomarkers of ALT, CXCL1, and IL-6 in peripheral blood and α-SMA protein in liver tissues. Conversely, the mice displayed a decrease in these markers in the recovery phase. Remarkably, this model shows that the sterile liver injury following elevated hepatocyte apoptosis is associated with an increase in myeloid cells in the liver. Within 7 days post-Dox administration, the liver of Dox-treated 3xTg-iHAP mice displays a normal histological structure, indicating completion of wound-healing. Together, we established a novel mouse model of injury and regeneration induced by hepatocyte apoptosis. This tool provides a robust in vivo platform for studying the pathophysiology of sterile liver inflammation, regeneration, and new therapeutic interventions for liver diseases.

Feeding modes influence the gut microbiome, immune system, and intestinal barrier homeostasis in neonates; how feeding modes impact susceptibility to neonatal gastrointestinal (GI) diseases is still uncertain. Here, we investigated the impact of dam feeding (DF) and formula feeding (FF) on features of the gut microbiome and physiological inflammation during the first 2 days of postnatal development and on the susceptibility to intestinal injury related to the inflammatory state in neonatal mouse pups. 16S rRNA sequencing data revealed microbiome changes, lower α-diversity, and a distinct pattern of β-diversity including expansion of f_Enterobacteriaceae and f_Enterococcaceae in the ileum of FF pups compared with DF pups by postnatal day (P)2. Together with gut dysbiosis, the FF cohort also had greater ileal mucosa physiological inflammatory activity compared with DF pups by P2 but maintained normal histological features. Interestingly, FF but not DF mouse pups developed necrotizing enterocolitis (NEC)-like intestinal injury within 24 h after anti-CD3 mAb treatment, suggesting that FF influences the susceptibility to intestinal injury in neonates. We further found that NEC-like incidence in anti-CD3 mAb-treated FF neonatal pups was attenuated by antibiotic treatment. Collectively, our data suggest that FF predisposes mouse pups to anti-CD3 mAb-induced intestinal injury due to abnormal f_Enterobacteriaceae and f_Enterococcaceae colonization. These findings advance our understanding of FF-associated microbial colonization and intestinal inflammation, which may help inform the development of new therapeutic approaches to GI diseases like NEC in infants. NEW & NOTEWORTHY This report shows that a feeding mode profoundly affects gut colonization in neonatal mice. Furthermore, our results demonstrate that formula feeding predisposes mouse pups to anti-CD3 mAb-induced necrotizing enterocolitis (NEC)-like intestinal injury upon inadequate microbial colonization. The study suggests the role of the combined presence of formula feeding-associated dysbiosis and mucosal inflammation in the pathogenesis of NEC and provides a new mouse model to study this disease.

Sirtuin 6 (Sirt6) is predominantly expressed in epithelial cells in intestinal crypts. It plays an important role in protecting intestinal epithelial cells against inflammatory injury. Previously, we found that colitis is associated with the downregulation of Sirt6 protein in the intestines. Here, we report that murine interferon-γ (Ifnγ) inhibits Sirt6 protein but not mRNA expression in young adult mouse colonocytes (YAMC, a mouse colonic epithelial cell line) in a dose- and time-dependent manner. Using microRNA array analysis, we showed that Ifnγ induces expression of miR-92b in YAMC cells. With in silico analysis, we found that the Sirt6 3'-untranslated region (UTR) contains a putative binding site for miR-92b . Luciferase assay showed that Ifnγ inhibited Sirt6 3'-UTR activity and this effect was mimicked by miR-92b via directly targeting the miR-92b seed site in the 3'-UTR of Sirt6 mRNA. Furthermore, Western blot demonstrated that miR-92b downregulated Sirt6 protein expression in YAMC cells. Blocking miR-92b with a specific inhibitor attenuated the inhibitory effect of Ifnγ on Sirt6 protein expression in the cells. Collectively, our data suggest that Ifnγ inhibits Sirt6 protein expression in intestinal epithelial cells via a miR-92b -mediated mechanism. miR-92b may be a novel therapeutic target for rescuing Sirt6 protein levels in intestinal epithelial cells, thereby protecting against intestinal mucosal injury caused by inflammation.

Prenatal inflammation is a risk factor for necrotizing enterocolitis (NEC), and it increases intestinal injury in a rat NEC model. We previously showed that maldevelopment of the intestinal microvasculature and lack of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) signaling play a role in experimental NEC. However, whether prenatal inflammation affects the intestinal microvasculature remains unknown. In this study, mouse dams were injected intraperitoneally with lipopolysaccharide (LPS) or saline at embryonic day 17 . Neonatal intestinal microvasculature density, endothelial cell proliferation, and intestinal VEGF-A and VEGFR2 proteins were assessed in vivo. Maternal and fetal serum TNF concentrations were measured by ELISA. The impact of TNF on the neonatal intestinal microvasculature was examined in vitro and in vivo, and we determined whether prenatal LPS injection exacerbates experimental NEC via TNF. Here we found that prenatal LPS injection significantly decreased intestinal microvascular density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression in neonatal mice. Prenatal LPS injection increased maternal and fetal serum levels of TNF. TNF decreased VEGFR2 protein in vitro in neonatal endothelial cells. Postnatal TNF administration in vivo decreased intestinal microvasculature density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression and increased the incidence of severe NEC. These effects were ameliorated by stabilizing hypoxia-inducible factor-1α, the master regulator of VEGF. Furthermore, prenatal LPS injection significantly increased the incidence of severe NEC in our model, and the effect was dependent on endogenous TNF. Our study suggests that prenatal inflammation increases the susceptibility to NEC, downregulates intestinal VEGFR2 signaling, and affects perinatal intestinal microvascular development via a TNF mechanism. NEW & NOTEWORTHY This report provides new evidence that maternal inflammation decreases neonatal intestinal VEGF receptor 2 signaling and endothelial cell proliferation, impairs intestinal microvascular development, and predisposes neonatal mouse pups to necrotizing enterocolitis (NEC) through inflammatory cytokines such as TNF. Our data suggest that alteration of intestinal microvascular development may be a key mechanism by which premature infants exposed to prenatal inflammation are at risk for NEC and preserving the VEGF/VEGF receptor 2 signaling pathway may help prevent NEC development.

The pathogenesis of necrotizing enterocolitis (NEC), a common gastrointestinal disease affecting premature infants, remains poorly understood. We previously found that intestinal VEGF-A expression is decreased in human NEC samples and in a neonatal mouse NEC model prior to detectable histological injury. Therefore, we hypothesized that lack of VEGF receptor 2 (VEGFR2) signaling facilitates neonatal intestinal injury by impairing intestinal microvasculature development. Here, we found that intestinal VEGF-A and its receptor, VEGFR2, were highly expressed at the end of fetal life and significantly decreased after birth in mice. Furthermore, selective inhibition of VEGFR2 kinase activity and exposure to a neonatal NEC protocol significantly decreased the density of the intestinal microvascular network, which was further reduced when both interventions were provided together. Furthermore, VEGFR2 inhibition resulted in greater mortality and incidence of severe injury in pups submitted to the NEC model. The percentage of lamina propria endothelial cells was decreased during NEC induction, and further decreased when VEGFR2 signaling was inhibited. This was associated with decreased endothelial cell proliferation rather than apoptosis. In conclusion, we found that VEGF-A and VEGFR2 proteins are highly expressed in the intestine before birth, and are significantly downregulated in the immediate neonatal period. Furthermore, VEGFR2 signaling is necessary to maintain the integrity of the intestinal mucosal microvasculature during the postnatal period and lack of VEGFR2 signaling predisposes to NEC in neonatal mice., (Copyright © 2016 the American Physiological Society.)

Liver steatosis in nonalcoholic fatty liver disease is affected by genetics and diet. It is associated with insulin resistance (IR) in hepatic and peripheral tissues. Here, we aimed to characterize the severity of diet-induced steatosis, obesity, and IR in two phylogenetically distant mouse strains, C57BL/6J and DBA/2J. To this end, mice (male, 8 wk old) were fed a high-fat and high-carbohydrate (HFHC) or control diet for 16 wk followed by the application of a combination of classic physiological, biochemical, and pathological studies to determine obesity and hepatic steatosis. Peripheral IR was characterized by measuring blood glucose level, serum insulin level, homeostasis model assessment of IR, glucose intolerance, insulin intolerance, and AKT phosphorylation in adipose tissues, whereas the level of hepatic IR was determined by measuring insulin-triggered hepatic AKT phosphorylation. We discovered that both C57BL/6J and DBA/2J mice developed obesity to a similar degree without the feature of liver inflammation after being fed an HFHC diet for 16 wk. C57BL/6J mice in the HFHC diet group exhibited severe pan-lobular steatosis, a marked increase in hepatic triglyceride levels, and profound peripheral IR. In contrast, DBA/2J mice in the HFHC diet group developed only a mild degree of pericentrilobular hepatic steatosis that was associated with moderate changes in peripheral IR. Interestingly, both C57BL/6J and DBA/2J developed severe hepatic IR after HFHC diet treatment. Collectively, these data suggest that the severity of diet-induced hepatic steatosis is correlated to the level of peripheral IR, not with the severity of obesity and hepatic IR. Peripheral rather than hepatic IR is a dominant factor of pathophysiology in nonalcoholic fatty liver disease.