Nowadays, the pharmacological management of visceral hypersensitivity associated with colitis is ineffective. In this context, targeting purinergic P2X4 receptor (P2X4R), which can modulate visceral pain transmission, could represent a promising therapeutic strategy. Herein, we tested the pain-relieving effect of two novel and selective P2X4R antagonists (NC-2600 and NP-1815-PX) in a murine model of DNBS-induced colitis and investigated the mechanisms underlying their effect. Tested drugs and dexamethasone (DEX) were administered orally, two days after colitis induction. Treatment with tested drugs and DEX improved tissue inflammatory parameters (body weight, spleen weight, macroscopic damage, TNF and IL-1β levels) in DNBS-rats. In addition, NC-2600 and NP-1815-PX attenuated visceral pain better than DEX and prevented the reduction of occludin expression. In in vitro studies, treatment of CaCo2 cells with supernatant from THP-1 cells, previously treated with LPS plus ATP, reduced the expression of tight junctions protein. By contrast, CaCo2 cells treated with supernatant from THP-1 cells, previously incubated with tested drugs, counteracted the reduction of tight junctions due to the inhibition of P2X4R/NLRP3/IL-1β axis. In conclusion, these results suggest that the direct and selective inhibition of P2X4R represents a viable approach for the management of visceral pain associated with colitis via NLRP3/IL-1β axis inhibition.

Improving clinical outcomes and delaying disease recrudescence in Ulcerative Colitis (UC) patients is crucial for clinicians. In addition to traditional and new pharmacological therapies that utilize biological drugs, the development of medical devices that can ameliorate UC and facilitate the remission phase should not be overlooked. Drug-based therapy requires time to be personalized and to evaluate the benefit/risk ratio. However, the increasing number of diagnosed UC cases worldwide necessitates the exploration of new strategies to enhance clinical outcomes. By incorporating medical devices alongside pharmacological treatments, clinicians can provide additional support to UC patients, potentially improving their condition and slowing down the recurrence of symptoms. Chemically identified as an azelaic acid derivative and palmitoylethanolamide (PEA) analog, adelmidrol is a potent anti-inflammatory and antioxidant compound. In this study, we aimed to evaluate the effect of an intrarectal administration of 2% adelmidrol (Ade) and 0.1% hyaluronic acid (HA) gel formulation in both the acute and resolution phase of a mouse model of colitis induced via DNBS enema. We also investigated its activity in cultured human colon biopsies isolated from UC patients in the remission phase at follow-up when exposed in vitro to a cytomix challenge. Simultaneously, with its capacity to effectively alleviate chronic painful inflammatory cystitis when administered intravesically to urological patients such as Vessilen, the intrarectal administration of Ade/HA gel has shown remarkable potential in improving the course of colitis. This treatment approach has demonstrated a reduction in the histological damage score and an increase in the expression of ZO-1 and occludin tight junctions in both in vivo studies and human specimens. By acting independently on endogenous PEA levels and without any noticeable systemic absorption, the effectiveness of Ade/HA gel is reliant on a local antioxidant mechanism that functions as a \"barrier effect\" in the inflamed gut. Building on the findings of this preliminary study, we are confident that the Ade/HA gel medical device holds promise as a valuable adjunct in supporting traditional anti-UC therapies.

As a dynamical system, complex disease always has a sudden state transition at the tipping point, which is the result of the long-term accumulation of abnormal regulations. This paper proposes a novel approach to detect the early-warning signals of influenza A (H3N2 and H1N1) outbreaks by dysregulated dynamic network biomarkers (dysregulated DNBs) for individuals. The results of cross-validation show that our approach can detect early-warning signals before the symptom appears successfully. Unlike the traditional DNBs, our dysregulated DNBs are anchored and very few, which is essential for disease early diagnosis in clinical practice. Moreover, the genes of dysregulated DNBs are significantly enriched in the influenza-related pathways. The source code of this paper can be freely downloaded from https://github.com/YanhaoHuo/dysregulated-DNBs.git.

BACKGROUND: IBD is a spectrum of pathologies characterized by dysregulated immune activation leading to uncontrolled response against the intestine, thus resulting in chronic gut inflammation and tissue damage. Due to its complexity, the molecular mechanisms responsible for disease onset and progression are still elusive, thus requiring intense research effort. In this context, the development of models replicating the etiopathology of IBD and allowing the testing of new potential therapies is critical.
METHODS: Colon from C57BL/6 or BALB/c mice was cultivated in a Gut-Ex-Vivo System (GEVS), exposed for 5 h to DNBS 1.5 or 2.5 mg/mL, in presence or absence of two probiotic formulations (P1 = Bifidobacterium breve BR03 (DSM16604) and B632 (DSM24706); P2 = Lacticaseibacillus rhamnosus LR04 (DSM16605), Lactiplantibacillus plantarum LP14 (DSM33401) and Lacticaseibacillus paracasei LPC09), and the main hallmarks of IBD were evaluated.
RESULTS: Gene expression analysis revealed the following DNBS-induced effects: (i) compromised tight junction organization, responsible for tissue permeability dysregulation; (ii) induction of ER stress, and (iii) tissue inflammation in colon of C57BL/6 mice. Moreover, the concomitant DNBS-induced apoptosis and ferroptosis pathways were evident in colon from both BALB/c and C57BL/6 mice. Finally, the co-administration of probiotics completely prevented the detrimental effects of DNBS.
CONCLUSIONS: Overall, we have provided results demonstrating that GEVS is a consistent, reliable, and cost-effective system for modeling DNBS-induced IBD, useful for studying the onset and progression of human disease at the molecular level, while also reducing animal suffering. Moreover, we have confirmed the beneficial effect of probiotics administration in promoting the remission of IBD.

The pharmacological blockade of P2X4 receptors has shown potential benefits in the management of several immune/inflammatory diseases. However, data regarding the involvement of P2X4 receptors in the pathophysiological mechanisms of action in intestinal inflammation are not well defined. We aimed to evaluate the anti-inflammatory effects of two novel and selective P2X4 receptor antagonists, NC-2600 and NP-1815-PX, and characterize the molecular mechanisms of their action in a murine model of 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis. These two drugs and dexamethasone (DEX) were administered orally for 6 days, immediately after the manifestation of DNBS. The body weight decrease, resulting from colitis, was attenuated by NC-2600 and NP-1815-PX, but not DEX. However, all three drugs attenuated the increase in spleen weight and ameliorated macroscopic and microscopic colonic tissue damage. Furthermore, all three compounds decreased tissue IL-1β levels and caspase-1 expression and activity. Colonic tissue increase of tumor necrosis factor was downregulated by DEX, while both NC-2600 and NP-1815-PX were ineffective. The reduction of occludin associated with colitis was ameliorated by NC-2600 and NP-1815-PX, but not DEX. In THP-1 cells, lipopolysaccharide and ATP upregulated IL-1β release and NLRP3, caspase-1, caspase-5, and caspase-8 activity, but not of caspase-4. These changes were prevented by NC-2600 and NP-1815-PX treatment. For the first time, the above findings show that the selective inhibition of P2X4 receptors represents a viable approach to manage bowel inflammation via the inhibition of NLRP3 inflammasome signaling pathways.

Inflammatory bowel disease (IBD) is a complex, chronic, and dysregulated inflammatory condition which etiology is still largely unknown. Its prognosis and disease progression are highly variable and unpredictable. IBD comprises several heterogeneous inflammatory conditions ranging from Ulcerative Colitis (UC) to Crohn\'s Disease (CD). Importantly, a definite, well-established, and effective clinical treatment for these pathologies is still lacking. The urgent need for treatment is further supported by the notion that patients affected by UC or CD are also at risk of developing cancer. Therefore, a deeper understanding of the molecular mechanisms at the basis of IBD development and progression is strictly required to design new and efficient therapeutic regimens. Although the development of animal models has undoubtedly facilitated the study of IBD, such in vivo approaches are often expensive and time-consuming. Here we propose an organ ex vivo culture (Gut-Ex-Vivo system, GEVS) based on colon from Balb/c mice cultivated in a dynamic condition, able to model the biochemical and morphological features of the mouse models exposed to DNBS (5-12 days), in 5 h. Indeed, upon DNBS exposure, we observed a dose-dependent: (i) up-regulation of the stress-related protein transglutaminase 2 (TG2); (ii) increased intestinal permeability associated with deregulated tight junction protein expression; (iii) increased expression of pro-inflammatory cytokines, such as TNFα, IFNγ, IL1β, IL6, IL17A, and IL15; (iv) down-regulation of the anti-inflammatory IL10; and (v) induction of Endoplasmic Reticulum stress (ER stress), all markers of IBD. Altogether, these data indicate that the proposed model can be efficiently used to study the pathogenesis of IBD, in a time- and cost-effective manner.

The pharmacological activation of A3 receptors has shown potential usefulness in the management of bowel inflammation. However, the role of these receptors in the control of visceral hypersensitivity in the presence of intestinal inflammation has not been investigated. The effects of AR170, a potent and selective A3 receptor agonist, and dexamethasone (DEX) were tested in rats with 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis to assess their tissue inflammatory parameters. The animals received AR170, DEX, or a vehicle intraperitoneally for 6 days, starting 1 day before the induction of colitis. Visceral pain was assessed by recording the abdominal responses to colorectal distension in animals with colitis. Colitis was associated with a decrease in body weight and an increase in spleen weight. The macroscopic damage score and tissue tumor necrosis factor (TNF), interleukin 1β (IL-1β), and myeloperoxidase (MPO) levels were also enhanced. AR170, but not DEX, improved body weight. Both drugs counteracted the increase in spleen weight, ameliorated macroscopic colonic damage, and decreased TNF, IL-1β, and MPO tissue levels. The enhanced visceromotor response (VMR) in rats with colitis was decreased via AR170 administration. In rats with colitis, AR170 counteracted colonic inflammatory cell infiltration and decreased pro-inflammatory cytokine levels, thereby relieving visceral hypersensitivity.

The management of chronic visceral pain related to Inflammatory Bowel Diseases or Irritable Bowel Syndrome is still a clinical problem and new therapeutic strategies continue to be investigated. In the present study, the efficacy of a pomegranate decoction and of its polysaccharide and ellagitannin components in preventing the development of colitis-induced abdominal pain in rats was evaluated. After colitis induction by 2,4-dinitrobenzenesulfonic acid (DNBS), the pomegranate decoction (300 mg kg-1), polysaccharides (300 mg kg-1), and ellagitannins (45 mg kg-1) were orally administered for 14 days. Repeated treatment with decoction reduced visceral hypersensitivity in the colitic animals both at 7 and 14 days. Similar efficacy was shown by polysaccharides, but with lower potency. Ellagitannins administered at dose equivalent to decoction content showed higher efficacy in reducing the development of visceral pain. Macroscopic and microscopic evaluations performed on the colon 14 days after the damage showed that all three preparations reduced the overall amount of mast cells, the number of degranulated mast cells, and the density of collagen fibers in the mucosal stroma. Although ellagitannins seem to be responsible for most of the beneficial effects of pomegranate on DNBS-induced colitis, the polysaccharides support and enhance its effect. Therefore, pomegranate mesocarp preparations could represent a complementary approach to conventional therapies for promoting abdominal pain relief.

Mitochondria exist in a constantly remodelling network, and excessive fragmentation can be pathophysiological. Mitochondrial dysfunction can accompany enteric inflammation, but any contribution of altered mitochondrial dynamics (ie, fission/fusion) to gut inflammation is unknown. We hypothesized that perturbed mitochondrial dynamics would contribute to colitis.
Quantitative polymerase chain reaction for markers of mitochondrial fission and fusion was applied to tissue from dextran sodium sulfate (DSS)-treated mice. An inhibitor of mitochondrial fission, P110 (prevents dynamin related protein [Drp]-1 binding to mitochondrial fission 1 protein [Fis1]) was tested in the DSS and di-nitrobenzene sulfonic acid (DNBS) models of murine colitis, and the impact of DSS ± P110 on intestinal epithelial and macrophage mitochondria was assessed in vitro.
Analysis of colonic tissue from mice with DSS-colitis revealed increased mRNA for molecules associated with mitochondrial fission (ie, Drp1, Fis1) and fusion (optic atrophy factor 1) and increased phospho-Drp1 compared with control. Systemic delivery of P110 in prophylactic or treatment regimens reduced the severity of DSS- or DNBS-colitis and the subsequent hyperalgesia in DNBS-mice. Application of DSS to epithelial cells or macrophages caused mitochondrial fragmentation. DSS-evoked perturbation of epithelial cell energetics and mitochondrial fragmentation, but not cell death, were ameliorated by in vitro co-treatment with P110.
We speculate that the anti-colitic effect of systemic delivery of the anti-fission drug, P110, works at least partially by maintaining enterocyte and macrophage mitochondrial networks. Perturbed mitochondrial dynamics can be a feature of intestinal inflammation, the suppression of which is a potential novel therapeutic direction in inflammatory bowel disease.

Abdominal pain is a frequent symptom of irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBDs). Although the knowledge of these pathologies is progressing, new therapeutic strategies continue to be investigated. In the present study, the effect of a system of molecules of natural origin (a medical device according to EU Directive 93/42/EC, engineered starting from Boswellia serrata resins, Aloe vera polysaccharides and Matricaria chamomilla and Melissa officinalis polyphenols) was evaluated against the intestinal damage and visceral pain development in DNBS-induced colitis model in rats. The system (250 and 500 mg kg-1) was orally administered once daily, starting three days before the injection of 2,4-dinitrobenzenesulfonic acid (DNBS) and for 14 days thereafter. The viscero-motor response (VMR) to colon-rectal balloon distension (CRD) was used as measure of visceral sensitivity. The product significantly reduced the VMR of DNBS-treated animals. Its effect on pain threshold was better than dexamethasone and mesalazine, and not lower than amitriptyline and otilonium bromide. At microscopic and macroscopic level, the tested system was more effective in protecting the intestinal mucosa than dexamethasone and mesalazine, promoting the healing of tissue lesions. Therefore, we suggest that the described system of molecules of natural origin may represent a therapeutic option to manage painful bowel diseases.