Zranb1-mutant mice display abnormal colonic mucus production and exacerbation of DSS-induced colitis.

Expression of mucin MUC2, a component of the colonic mucus layer, plays a crucial role in intestinal homeostasis. Here, we describe a new regulator of MUC2 expression, the deubiquitinase ZRANB1 (Trabid). A ZRANB1 mutation changing cysteine to serine in amino acid position 443, affects ubiquitination. To analyze ZRANB1 function in the intestine, we generated Zranb1 C443S mutant knock-in (Zranb1C443S/C443S) mice using the CRISPR/Cas9 system. Zranb1C443S/C443S mice exhibited decreased mRNA expression and MUC2 production. Colonic organoids from Zranb1C443S/C443S mice displayed decreased Muc2 mRNA expression following differentiation into goblet cells. Finally, we analyzed dextran sulfate sodium-induced colitis to understand ZRANB1's role in intestinal inflammation. Zranb1C443S/C443S mice with colitis exhibited significant weight loss, reduced colon length, and worsening clinical and pathological scores, indicating that ZRANB1 contributes to intestinal homeostasis. Together, these results suggest that ZRANB1 regulates MUC2 expression and intestinal inflammation, which may help elucidating the pathogenesis of inflammatory bowel disease and developing new therapeutics targeting ZRANB1.

Importance of Telomere Shortening in the Pathogenesis of Ulcerative Colitis: A New Treatment From the Aspect of Telomeres in Intestinal Epithelial Cells.

BACKGROUND AND AIMS: Ulcerative colitis [UC] is a chronic inflammatory disease of the colon with frequent relapses. Telomere shortening in intestinal epithelial cells has been reported in severe or longstanding cases. However, its influence on UC pathogenesis remains unelucidated. To this end, we evaluated telomere shortening using a long-term organoid inflammation model that we had originally established. METHODS: A UC model using human colon organoids was established to assess telomere changes chronologically. MST-312 was used for the telomerase inhibition assay. The potential of telomerase activators as a novel UC treatment was evaluated with an in vitro model, including microarray analysis, and histological changes were assessed using xenotransplantation into mouse colonic mucosa. RESULTS: Our UC model reproduced telomere shortening in vitro, which was induced by the continuous suppression of telomerase activity via P53. MST-312-based analysis revealed that telomere shortening was involved in the pathogenesis of UC. Madecassoside [MD] improved the telomere length of the UC model and UC patient-derived organoids, which further promoted cell proliferation in vitro and improved the graft take-rate of xenotransplantation. Moreover, histological analysis revealed that MD induced normal crypt structure with abundant goblet cells. CONCLUSIONS: This study is the first to reveal the mechanism and importance of telomere shortening in the pathogenesis of UC. MD could be a novel candidate for UC treatment beyond endoscopic mucosal healing.

Influence of chronic inflammation on the malignant phenotypes and the plasticity of colorectal cancer cells.

Sporadic adenoma or adenocarcinoma is often detected during endoscopic surveillance of patients with ulcerative colitis (UC). However, it is occasionally difficult to distinguish these neoplasms from dysplasia or colitis-associated cancers because of the influence of inflammation. However, the influence of inflammation on sporadic neoplasms is not well characterised. To assess this influence, we established a long-term inflammation model of colon cancer cells by inflammatory stimulation with tumour necrosis factor-α, flagellin and interleukin-1ß for 60 weeks. Then, the malignant phenotypes were evaluated using the MTS assay, Annexin V fluorescence assay, cell migration assay and sphere formation assay. The influence of P53 function on these phenotypes was assessed with a TP53 mutation model using the CRISPR/Cas9 system. A long-term inflammation model of LS174T cells was established for the first time with continuous inflammatory signalling. Chronic inflammation induced apoptosis and suppressed the proliferation and stemness of these cancer cells via the action of P53. It also enhanced the invasiveness of LS174T cells. Moreover, these phenotypic changes and changes in inflammatory signalling were recoverable after the removal of inflammatory stimuli, suggesting that colon cancer cells have higher plasticity than normal intestinal epithelial cells. In conclusion, our results suggest that sporadic neoplasms in patients with UC are affected by chronic inflammation but are not essentially altered.

Effect of oxytocin, prolactin-releasing peptide, or corticotropin-releasing hormone on feeding behavior in steers.

As a preliminary step to elucidate the involvement of central neurotransmitters in the dip in voluntary feed intake during the perinatal period in cows, we investigated the effect of intracerebroventricular (ICV) administration of oxytocin, prolactin-releasing peptide (PrRP), or corticotropin-releasing hormone (CRH), the central functions of all of which undergo drastic changes during the perinatal period, on feed intake in steers. Thirty minutes before the onset of feeding, the treatment solution was injected into the third ventricle through an implanted cannula, and feeding-related behaviors were observed for 1 h after the onset of feeding. Neither ICV oxytocin (5 and 50 µg) nor PrRP (2 and 20 nmol) reduced feed intake (n=6). Twenty nanomoles of bovine CRH noticeably inhibited feeding behavior compared with vehicle treatment (n=5, p<0.05). Fifty micrograms of oxytocin reduced latency to the first water access after feeding onset (p<0.1), which may be because of the stimulation of arginine vasopressin V1b receptor by the high dose of oxytocin. We conclude that CRH inhibits feeding behavior by its central action in this species, although this could also be an indirect effect due to the increased expression of abnormal behaviors caused by CRH. Central administration of neither oxytocin nor PrRP reduced feed intake in steers. Although the effects of sex steroids need to be examined, it appears that increased activity of oxytocin, and possibly PrRP, during the perinatal period does not contribute to the dip in voluntary feed intake in this species. On the other hand, it makes sense that suppressed central CRH activity during the perinatal period should act in the direction of maintaining or even increasing food intake to aid a steady supply of energy to the fetus or offspring. We thus speculate that CRH is not a prime candidate involved in the dip in voluntary feed intake during the perinatal period in cows.

Effect of intracerebroventricular injections of prolactin-releasing peptide on prolactin release and stress-related responses in steers.

Some evidence suggests that there might be a species difference in the effect of intracerebroventricularly administered (ICV) prolactin-releasing peptide (PrRP) between rodents and sheep. We compared the levels of cortisol (CORT) and prolactin (PRL), rectal temperature (RT) and behavioral responses to ICV bovine PrRP (bPrRP) in steers. ICV bPrRP (0.2, 2 and 20 nmol/200 µL) tended to evoke a dose-related increase in CORT concentrations and 0.2 nmol of bPrRP induced transient increase in PRL concentrations. A significant time-treatment interaction was observed for the percent change of CORT (P<0.05) and PRL (P<0.05) from pre-injection value. The time-treatment interaction for changes in RT was not significant (P=0.50). There tended to be a difference among the four treatments in terms of maximum change in RT from the pre-injection value between 0 and 90 min (P<0.1). Stress-related behavioral signs were not observed in the present experiment. These findings indicate that ICV bPrRP increased CORT and PRL levels, suggesting that central PrRP might participate in controlling the hypothalamo-pituitary-adrenal axis and PRL release in cattle, unlike sheep. In contrast, central PrRP is unlikely to be involved in controlling the behavior of this species because ICV bPrRP did not induce marked changes in their behavior.

A possible role of central serotonin in L-tryptophan-induced GH secretion in cattle.

To clarify the role of serotonin (5-HT) in the regulatory mechanism of L-tryptophan (TRP)--induced growth hormone (GH) secretion in cattle, changes in 5-HT concentrations in the cerebrospinal fluid (CSF) in the third ventricle (3V) and GH in plasma before and after the peripheral infusion of TRP were determined simultaneously. The direct effect of TRP on GH release from the dispersed anterior pituitary cells was also assessed. A chronic cannula was placed in 3V by stereotaxic surgery, then CSF and blood were withdrawn under physiological conditions. TRP (38.5 mg/kg BW) was infused through an intravenous catheter from 12.00 to 14.00 hours and CSF and blood sampling were performed from 11.00 to 18.00 hours at 1-h intervals. The concentration of 5-HT in CSF was determined by high-performance liquid chromatography with electrochemical detection. GH, melatonin (MEL), and cortisol (CORT) concentrations were measured by radio-immunoassay and enzyme-immunoassay. Concentrations of 5-HT were increased by TRP infusion. The TRP infusion significantly increased GH release. On the other hand, TRP did not stimulate GH release from the bovine pituitary cells. MEL and CORT concentrations were not altered by TRP infusion. These results suggest that TRP induced GH release via the activation of serotonergic neurons in cattle.

Relationships of stress responses with plasma oxytocin and prolactin in heifer calves.

Oxytocin and prolactin are potential candidates for the regulation of behavioral and physiological stress responses in the brain. To investigate the neurobiological basis of individual differences in stress responses in cattle, we examined the association of behavioral and hypothalamo-pituitary-adrenal axis reactivity to acute stressors and basal and stimulated levels of oxytocin and prolactin. Twenty Holstein heifer calves aged 2 weeks were subjected to a 10 min open-field test (OFT) followed by presentation of a feeding bucket for 15 min in the OF. If the calf contacted the bucket, a blast of air was applied to its muzzle (surprise test). Jugular blood samples collected before and after both tests were analyzed for oxytocin, prolactin, and cortisol. Relationships of basal and percent change in oxytocin or prolactin with behavioral responses in each test and percent change in cortisol were analyzed using principal components analysis and Spearman rank correlations. Plasma cortisol and prolactin concentrations were significantly elevated by the tests (p<0.005), but plasma oxytocin concentration did not significantly change (p>0.1). Four principal components explained 56.1% of the total variation: curiosity, general activity, fearfulness, and dependence on humans. Curiosity was inversely correlated with basal oxytocin level (rS=-0.683, p<0.05). General activity was positively correlated with prolactin reactivity (rS=0.448, p<0.05) and inversely with oxytocin reactivity to the novel environment (rS=-0.717, p<0.05). Fearfulness tended to correlate positively with basal oxytocin level (rS=0.583, p<0.1). Dependence on humans correlated with none of the hormonal parameters. The relationships of basal oxytocin level with curiosity and fearfulness for novel environments are of particular interest for future study.