Macroscopic Damage Score Research Articles

DOP035 Anti-inflammatory potential of novel tethered agonists of the adhesion G protein-coupled receptor F5

Abstract Background An accumulating data suggest that the adhesion G protein-coupled receptor F5 (ADGRF5) is fundamental for human health and diseases1-3. It was noted that ADGRF5 mediates immune response in cancers but its impact on colitis development is still unknown. Moreover, recent findings identified mature tethered agonist of ADGRF5 which corresponds to Stachel sequence of this receptor4. The purpose of this work was to design peptides corresponding to Stachel sequence able to activate ADGRF5 with higher activity than the mature ADGRF5 tethered agonist and evaluate them in colitis model. Methods Single amino acid substitutions in the mature ADGRF5 tethered agonist sequence combined with calcium accumulation and inositol phosphate conversion assays as well as ADGRF5 overexpressing cells were used to identify novel ADGRF5 tethered agonists. The downstream signalling pathway induced by ADGRF5 tethered agonists was estimated using Western blot technique. In vivo studies employing murine model of colitis induced by dextran sulphate sodium salt (DSS) administration were performed. Anti-inflammatory potential of novel ADGRF5 tethered agonists was assessed using macroscopic and microscopic damage score as well as total length of inflamed colon. Results The comparison of mature and modified ADGRF5 tethered agonist activity documented that substitution of the amino acids in position 11 and 13 improves their activity in ADGRF5 overexpressing cells when compared to wild-type cells. In vitro approach using calcium accumulation and inositol phosphate conversion assays revealed that novel ADGRF5 tethered agonists are characterized by 6-fold lower EC50 value than parent ADGRF5 tethered agonist. Moreover, our novel ADGRF5 tethered agonists are able to induce phosphorylation of protein kinase A and B as well as extracellular signal-regulated kinase signalling pathways in time and dose-dependent manner. Finally, administration of novel ADGRF5 tethered agonists in the murine model of colitis led to a significant improvement of macroscopic and microscopic damage score compared to animals treated with mature ADGRF5 tethered agonist. Additionally, the colons of mice treated with novel ADGRF5 tethered agonists but not with mature ADGRF5 tethered agonist were characterized by reduction of total length of inflammation and improvement of colon architecture. Conclusion We found critical positions in the Stachel sequence of ADGRF5 agonists which are required for their activity. New ADGRF5 tethered agonists displayed anti-inflammatory action highlighting that modulation of ADGRF5 activity may serve as an attractive target in colitis treatment.

Protective effect of Tripterygium glycosides on ulcerative colitis rats based on ERK/p38 MAPK signaling pathway

This study explored the effect of Tripterygium glycosides(TG) on ulcerative colitis in rats and examined the regulatory role of the extracellular signal-regulated kinase/p38 mitogen-activated protein kinase(ERK/p38 MAPK) signaling pathway. Seventy male Wistar rats were selected and randomly divided into control, model, low-dose TG, medium-dose TG, high-dose TG, positive control, and pathway inhibitor groups. The disease activity index(DAI) score, macroscopic damage score, and microscopic colonic injury score were observed in each group. HE staining was used to detect pathological changes in colonic tissues. TUNEL assay was employed to detect the apoptosis rate of intestinal mucosal cells. ELISA was used to measure serum levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-alpha(TNF-α). Western blot and PCR methods were used to detect the expression of ERK/p38 MAPK pathway-related proteins and mRNA in tissues. The results showed that TG could effectively improve the histopathological condition; compared with the control group, DAI score, morphological injury score, colon injury score, apoptosis rate of colonic mucosal cells, serum levels of IL-1β, IL-6 and TNF-α, protein levels of p-ERK/ERK and p-p38 MAPK/p38 MAPK, mRNA levels of ERK and p38 MAPK were significantly elevated and significantly increased in the model group(P<0.05). Compared with the model group, DAI scores of rats in the high dose group of TG, the positive control group and the pathway inhibitor group were reduced, and the pathway inhibitor group was the lowest(P<0.05). Compared with the model group, morphological injury score and colonic injury score were reduced in the remaining groups, and lowest in the pathway inhibitor group(P<0.05). Compared with the model group, the apoptosis rate of rat intestinal mucosa cells was decreased in the high-dose TG, the positive control group, and the pathway inhibitor group, and the levels of IL-1β, IL-6, and TNF-α in serum, and the expression of p-ERK/ERK, p-p38 MAPK/p38 MAPK, ERK mRNA, p38 MAPK mRNA in the colonic tissues was reduced(P<0.05), and the improvement effect of the above indexes in the pathway inhibitor group was most significant. In conclusion, TG can effectively improve inflammatory responses in ulcerative colitis, promote colonic mucosal recovery, and enhance immune function. Its mechanism may be related to the regulation of the ERK/p38 MAPK signaling pathway.

Eosinophil Depletion as a Potential Therapeutic Strategy in Acute and Chronic Intestinal Inflammation Based on a Dextran Sulfate Sodium Colitis Model.

A role for eosinophils in intestinal inflammation and fibrosis in the context of inflammatory bowel disease has been suggested, yet the precise nature, whether causal or secondary remains debated. Hence, it remains unclear whether targeting eosinophils should be further explored as a treatment option in inflammatory bowel disease. Acute and chronic dextran sulfate sodium colitis was induced in wild-type C57BL/6 mice. Eosinophils were depleted by anti-CCR3 injections before colitis induction in a chronic model and after colitis onset in an acute model in order to investigate the impact of eosinophil depletion on pre-existing colitis. Inflammation was assessed using the disease activity index, macroscopic damage, and histological disease activity score. In the chronic model, fibrosis was assessed by examining colon weight/length ratio, collagen deposition through Martius Scarlet Blue staining, hydroxyproline assay, and COL1A1 expression. Protein and gene expression were assessed using the Meso Scale Discovery platform and real-time quantitative polymerase chain reaction. In the acute and chronic colitis model, eosinophil depletion resulted in reduced disease activity and faster recovery, as observed via the total area under the curve of the disease activity index (P = .004 and P = .02, respectively), macroscopic damage score (P = .009 and P = .08, respectively), and histological disease activity score (P = .09 and P = .002, respectively). In the acute model, the accelerated recovery was accompanied by an increase in interleukin (IL)-10 (P = .03) and a decrease in IL-4 (P = .03) and IL-6 (P = .009). Colon weight/length ratio and collagen deposition were not affected by eosinophil depletion. Eosinophil depletion prevents and decreases intestinal inflammation in a preclinical dextran sulfate sodium model without affecting fibrosis. These results pave the way for exploring eosinophil depletion as a novel treatment modality in addressing intestinal inflammation.

Docosahexaenoic acid (DHA) alleviates inflammation and damage induced by experimental colitis.

Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are chronic gastrointestinal disorders associated with significant morbidity and complications. This study investigates the therapeutic potential of docosahexaenoic acid (DHA) in a trinitrobenzene sulfonic acid (TNBS) induced colitis model, focusing on inflammation, oxidative stress, and intestinal membrane permeability. Wistar albino rats were divided into Control, Colitis, and Colitis + DHA groups (n = 8-10/group). The Colitis and Colitis + DHA groups received TNBS intrarectally, while the Control group received saline. DHA (600 mg/kg/day) or saline was administered via gavage for six weeks. Macroscopic and microscopic evaluations of colon tissues were conducted. Parameters including occludin and ZO-1 expressions, myeloperoxidase (MPO) activity, malondialdehyde (MDA), glutathione (GSH), total antioxidant status (TAS), total oxidant status (TOS), Interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) levels were measured in colon tissues. Colitis induction led to significantly higher macroscopic and microscopic damage scores, elevated TOS levels, reduced occludin and ZO-1 intensity, decreased mucosal thickness, and TAS levels compared to the Control group (p < 0.001). DHA administration significantly ameliorated these parameters (p < 0.001). MPO, MDA, TNF-α, and IL-6 levels were elevated in the Colitis group but significantly reduced in the DHA-treated group (p < 0.001 for MPO, MDA; p < 0.05 for TNF-α and IL-6). DHA demonstrated antioxidant and anti-inflammatory effects by reducing reactive oxygen species production, enhancing TAS capacity, preserving GSH content, decreasing proinflammatory cytokine levels, preventing neutrophil infiltration, reducing shedding in colon epithelium, and improving gland structure and mucosal membrane integrity. DHA also upregulated the expressions of occludin and ZO-1, critical for barrier function. Thus, DHA administration may offer a therapeutic strategy or supplement to mitigate colitis-induced adverse effects.

Remdesivir ameliorates ulcerative colitis-propelled cell inflammation and pyroptosis in acetic acid rats by restoring SIRT6/FoxC1 pathway

IntroductionUlcerative colitis (UC) is a primary culprit of inflammatory bowel disease that entails prompt and effective clinical intervention. Remdesivir (RDV), a broad-spectrum antiviral nucleotide, has been found to exert anti-inflammatory effects in experimental animals. AimThis study investigates the prospective anti-inflammatory merit of RDV on an experimental model of UC. The role of SIRT6/FoxC1 in regulating colonic cell inflammation and pyroptosis is delineated. MethodRats were challenged with a single intrarectal dose of acetic acid (AA) solution (2 ml; 4 % v/v) to induce colitis. RDV (20 mg/kg, ip) and sulfasalazine (100 mg/kg, po) were administered to rats 14 days before the injection of AA. ResultsAdministration of RDV ameliorated colonic cell injury and loss as manifested by improvement of severe colon histopathological mutilation and macroscopic damage and disease activity index scores together with restoration of normal colon weight/length ratio. In addition, RDV alleviated colonic inflammatory reactions, thereby curtailing NF-κB activation and the inflammatory cytokines, TNF-α, IL-18, and IL-1β. Mitigation of colonic oxidative stress and apoptotic reactions were also evident in the setting of RDV treatment. Mechanistically, RDV enhanced the anti-inflammatory cascade, SIRT6/FoxC1, together with curbing the pyroptotic signal, NLRP3/cleaved caspase-1/Gasdermin D-elicited colonic inflammatory cell death. ConclusionThis study reveals, for the first time, the anti-inflammatory effect of RDV against experimental UC. Augmenting SIRT6/FoxC1-mediated repression of colonic inflammation and pyroptosis might advocate the colo-protective potential of RDV.

Exploring the synergistic potential of probiotics and vitamin D to ameliorate acute TNBS-induced colitis in mice

Inflammatory Bowel Disease (IBD), either Crohn’s disease or Ulcerative Colitis, are diseases characterized by chronic inflammation of the gastrointestinal tract, disruption of mucosal barrier, gut dysbiosis and vitamin D deficiency. The use of probiotics or vitamin D supplements have been studied individually in different colitis models to determine their beneficial effects by increasing tight junction proteins such as occludin. However, the synergistic effects of combining these treatments to target tight junction expression are still unknown. Therefore, the objective of this research was to demonstrate the effcacy of probiotics and high dietary vitamin D to ameliorate colonic inflammation by increasing mucosal barrier integrity. We hypothesized that co-administration of a probiotic mixture with vitamin D would reduce colitis-induced inflammation by increasing tight junction expression. For the methodology, male and female C57BL/6 mice were divided into four treatment groups: Colitis+Vehicle (n=10), Colitis+Probiotic (n=10), Colitis+Vitamin D (n=11), Colitis+Probiotic+Vitamin D (n=7). Animals received seven days of pretreatment with Vivomixx probiotics in water, high vitamin D (5IU/g) in food, or both. On day 7, animals were anesthetized lightly, and colitis induction was done by intracolonic administration of 0.1mL of 4mg TNBS in 30% ethanol. Water, food, and body weight were monitored daily. Animals were sacrificed on day ten, the colonic tissue was removed and assessed for inflammation then stained for occludin expression by immunofluorescence, and probiotic colonization was confirmed within fecal samples by RT-PCR amplification of S. Thermophilus gene. Results indicate that all animals decreased weight after colitis induction, but only animals receiving probiotic had a slight recovery by day 10. Animals receiving the probiotic mixture had higher S. Thermophilus expression in fecal samples compared to vehicle (p&lt;0.001) indicating appropriate colonization. A significant reduction in macroscopic damage score (3.92 vs. 7.42, p&lt;0.05) and microscopic score (3.42 vs 7.20, p&lt;0.01) was found in animals with Probiotic+Vitamin D compared to vehicle. Although an increase in occludin expression was observed with probiotic administration it did not reach statistical significance. In conclusion, findings indicate a beneficial effect of combining probiotics and vitamin D on colonic tissue damage in an animal model of acute colitis but this did not impact the tight junction protein occludin. This work was supported by RISE (R25GM082406), G-RISE (T32GM144896), PRI MD Summer Program and Porter Physiology Development Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Potential mitigating impact of a dipeptidyl peptidase-IV inhibitor, vildagliptin, on oxazolone-induced ulcerative colitis: Targeting the role of PI3K/AKT/mTOR and AMPK/Nrf2 signaling pathways

Growing evidence suggests that phosphoinositide 3-kinase (PI3K) and adenosine monophosphate-activated protein kinase (AMPK) signaling cascades are critical in ulcerative colitis (UC) pathophysiology by influencing gut mucosal inflammation. Recently, the coloprotective properties of dipeptidyl peptidase-IV (DPP-IV) inhibitors have emerged. Thus, this study assessed for the first time the potential mitigating impact of a DPP-IV inhibitor, vildagliptin (Vilda), on oxazolone (OXZ)-induced colitis in rats, targeting the role of PI3K/AKT/mTOR and AMPK/Nrf2 pathways. Thirty-two adult Albino rats were divided into four groups: control, Vilda (10 mg/kg/day orally), OXZ (300 µL of 5 % OXZ in 50 % aqueous ethanol solution introduced once into the colon via catheter), and Vilda+OXZ. Inflammatory cytokines (interleukin 13, tumor necrosis factor-α, interleukin 10), oxidative/endoplasmic reticulum stress markers (myeloperoxidase, reduced glutathione, catalase, CHOP), mitochondrial reactive oxygen species, adenosine triphosphate levels, and mitochondrial transmembrane potential were estimated. p-AMPK, p-AKT, beclin-1, and SQSTM1 levels were immunoassayed. Nrf2, PI3K, and mTOR expression levels were quantified using the real-time polymerase chain reaction. Furthermore, p-NF-ĸBp65 and LC3II immunoreactivity were evaluated. Vilda administration effectively ameliorated OXZ-induced colitis, as evidenced by the reduced Disease Activity Index, macroscopic colon damage score, colon weight/length ratio, ulcer index, and histopathological and electron microscopic changes in the colon tissues. Vilda treatment also counteracted OXZ-triggered inflammation, oxidative/endoplasmic reticulum stress, mitochondrial dysfunction, and enhanced autophagy in the colon. Vilda substantially suppressed PI3K/AKT/mTOR and activated the AMPK/Nrf2 pathway. Vilda has potent coloprotective and anti-ulcerogenic properties, primarily attributed to its antiinflammatory, antioxidant, and modulatory impact on mitochondrial dysfunction and autophagy activity. These effects were mostly mediated by suppressing PI3K/AKT/mTOR and activating AMPK/Nrf2 signaling cascades, suggesting a potential role of Vilda in UC therapy.

DOP43 Histologic improvement, attenuation of inflammation and microbiome modulation by engineered high acetate producing Saccharomyces boulardii in DSS-induced colitis

Abstract Background The probiotic Saccharomyces cerevisiae var. boulardii (Sb) may be optimized for therapeutic use in ulcerative colitis by enhancing its acetate production, as acetate is a key factor for its probiotic efficacy and was shown to have anti-inflammatory and barrier-protective effects in vitro (Deleu, 2023). Therefore, higher acetate producing Sb strains have been engineered which were preclinically evaluated in an acute DSS mice model of colitis. Methods Nine-week-old female C57/Bl6 mice (N=120) were allocated to 12 treatment groups receiving drinking water or 2.75% DSS in combination with PBS (control), Baker’s yeast (non-probiotic control), SDH1 (no-acetate Sb), ENT (transient acetate strain-probiotic Enterol control), SbP (high acetate Sb) and ENT3 (extra high acetate Sb). Disease activity including weight loss, diarrhoea and the presence of occult blood were scored daily. On day 7, the DSS groups were transferred to regular drinking water and on day 14 mice were sacrificed. Colonic tissue and faeces (0, 9, 14d) were collected for resp. histology, RNA, and 16S rRNA sequencing. Results Disease activity in DSS subgroups (Fig.A), determined by the area under the curve, was lower for SbP compared to PBS and Baker’s yeast (both p&amp;lt;0.05). Sb SDH1 showed higher disease activity compared to the Sb strains ENT, SbP and ENT3 (all p&amp;lt;0.05). At sacrifice, the macroscopic damage score in DSS was lower for SbP and ENT3 (both p&amp;lt;0.05) compared to Sb SDH1, and the colon weight/length-ratio was decreased for ENT and SbP compared to Sb SDH1 (resp. p=0.06 and p=0.08). Higher histologic inflammation was noted in the non- or transient-acetate producing strains on DSS compared to healthy PBS control (all p&amp;lt;0.05), whereas this increase was not observed for both high-acetate producing strains SbP and ENT3 on DSS (p=NS) indicating a production dependent response. Anti-inflammatory findings were confirmed by transcriptomic analysis of differentially expressed genes e.g. decreased expression of Tnf, Il1b and S100a8/9, predominantly expressed as calprotectin, upon stimulation with the highest acetate producing strain ENT3 (all adj.p&amp;lt;0.1; except PBS adj.p=0.16). Subsequent, microbial analysis showed a superior effect of ENT3 in maintaining a stable alpha diversity (p=0.58, Fig.E), which was not observed for the other strains. Conclusion Engineered high acetate producing Sb strains attenuated DSS-induced colitis, with its anti-inflammatory effects further supported by transcriptomic analysis and superiority in maintaining a stable microbiome composition. Together with our previous in vitro work in patient-derived organoids, these data indicate a role for high-acetate producing Sb- in attenuating intestinal inflammation.

Gilaburu (Viburnum opulus L.) fruit extract has potential therapeutic and prophylactic role in a rat model of acetic acid-induced oxidant colonic damage

Ethnopharmacological relevanceUlcerative colitis (UC) which has a global impact on the health care system with its recurrent and incompletely curable characteristics, affects the patients’ quality of life. Gilaburu (GB; Viburnum opulus L.) is a fruit with rich polyphenol ingredient which is used ethnobotanically in Türkiye for medicinal purposes (for example, to pass kidney stones, to treat stomach, heart, and liver diseases, hemorrhages, hypertension, ulcers, common cold, tuberculosis, rheumatic and menstrual pain, and diabetes). On the other hand, the effects of GB in the experimental UC model have not been studied. Aim of the studyThis study aimed to explore the potential antioxidant and anti-inflammatory effects of GB fruit extract in improving acetic acid (AA)-induced UC. Materials and methodsStarting immediately after (AA + GB group) or 1 week before (GB + AA + GB group) the colitis induced by intrarectal AA (5%; v/v) administration, the rats orally received GB (100 mg/kg) once per day for 3 days. The control and AA groups were administered orally saline (1 ml), while the AA + SS group were administered sulfasalazine (SS; 100 mg/kg; orally) as a positive control once per day for 3 days. Distal colonic tissue specimens were obtained for the histological and biochemical [myeloperoxidase (MPO), malondialdehyde (MDA), glutathione (GSH), chemiluminescence (CL), caspase-3, 8-hydroxy-2’-deoxyguanosine (8-OHdG), matrix metalloproteinase (MMP)-9, transforming growth factor (TGF)-β1, smad-3 and cytokine (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, interferon (IFN)-γ), measurements] evaluations on the 3rd day. ResultsElevated macroscopic and microscopic damage scores, high tissue wet weight values, increased tissue-associated MPO, MDA, CL, caspase-3, 8-OHdG, cytokines (TNF-α, IL-1β, IL-6, IL-8), MMP-9, TGF-β1, smad-3 levels, and decreased GSH values of the AA group were all reversed by GB treatments (AA + GB and GB + AA + GB groups) (p < 0.05–0.001). However, sulfasalazine treatment (AA + SS group) did not change the IL-8, 8-OHdG, MMP-9, and TGF-β1 measurements significantly. ConclusionsGilaburu shows both anti-inflammatory and antioxidant effects against AA-induced colonic damage by suppressing neutrophil infiltration, regulating inflammatory mediators, inhibiting reactive species production, lipid peroxidation, and apoptosis, conserving endogenous antioxidant glutathione, and ameliorating oxidative DNA damage. Since the current ulcerative colitis drugs display limited benefits and adverse side effects, potential therapeutic and/or prophylactic role of gilaburu can be evaluated in ulcerative colitis.

Evaluation of role of Jasminum sambac against ulcerative colitis in rats

Current investigation is designed to find out the role of Jasminum sambac (JS) against Acetic Acid (AA)-induced ulcerative colitis in rats. 30 male albino Wistar rats were randomly separated into 5 batches (n=6): normal: admitted with distilled water for 7 days; negative: received the distilled water for 1- 3 days and from 4-7 days received the 2 mL of AA (4% v/v) rectally; test 1, 2 and standard group: orally received the ethanolic extract of leaves of Jasminum sambac (EEJS) at doses 250, 500 mg/kg and prednisolone 2 mg/kg from 1-3 days without AA and from 4-7 days received the 2 mL of 4% v/v AA rectally after 2 h administration of EEJS and prednisolone. On 8th day rats were sacrificed and colon tissues were examined. Rats with acetic acid-induced colitis exhibited significant decrease in body weight, colon length, superoxide dismutase, catalase, reduced glutathione, significant rise in colon weight, oedema, disease activity index, macroscopic damage score, occult blood in stool, stool consistency and lipid peroxidation. Pre-treatment with EEJS at doses 250, 500 mg/kg orally significantly regularized the above-mentioned parameters in dose dependent manner. Ethanolic extract of Jasminum sambac could protect the AA-produced ulcerative colitis by attenuation of macroscopic damage, microscopic damage, and oxidative biomarkers.

Impact of Eugenol on Acetic Acid-induced Colitis in Rats

Background: Inflammatory bowel diseases (IBD) including ulcerative colitis and Crohn's disease, are immune-mediated chronic relapsing intestinal disorders characterized by the presence of an acute or chronic inflammatory process in the bowel wall. Eugenol essential oil is used in traditional medicine to treat digestive disorders such as gastrointestinal ulcers, indigestion, and inflammation of the intestines. Aim: To investigate the effects of Eugenol on colitis in rat models. Methods: Eugenol was administered per rectum (5 and 10 mg/kg) and intraperitoneally (0.25 and 0.5 mg/kg) for 6 days after induction of colitis by acetic acid. The changes were examined macroscopically, histologically, and biochemically and compared with Dexamethasone. Results: Results showed a significant decrease in the macroscopic damage score (P &lt; 0.05), and reduction in the weight ratio of the colon (P &lt; 0.01), the histological signs of inflammation (P &lt; 0.01) such as infiltration of lymphocytes and macrophages into the mucosa, mucin depletion, crypt abscess, edema, and tissue damage, as well as leukocyte accumulation and myeloperoxidase level in compare with the colitis control group. Conclusion: This animal trial demonstrates the beneficial anti-inflammatory and wound-healing effects of Eugenol in the treatment of acetic acid-induced colitis in rats. Eugenol can potentially be advantageous as a supplemental remedy for the treatment of IBD.

Ameliorating effects of Acacia arabica and Ocimum basilicum on acetic acid-induced ulcerative colitis model through mitigation of inflammation and oxidative stress

IntroductionUlcerative colitis (UC) is a chronic recurrent inflammatory disease of the large intestine and rectum. The disease is characterized by oxidative stress and severe inflammation. Research has shown the anti-oxidative and anti-inflammatory effects induced by consuming the Acacia arabia and Ocimum basilicum. The present study aimed to evaluate the effect of treatment with O. basilicum together with A. arabica on healing, inflammation, and oxidative stress in the course of experimental colitis in rats. MethodsA total number of 50 male rats were selected and randomly assigned to five groups of 10 rats each. Colitis was induced in rats by enemas with a 4 % acetic acid solution. Four days after the colitis induction, the rats were orally treated for the next 4 days with saline or a combination of A. arabica and O. basilicum (1000 mg/kg) or sulfasalazine (100 mg/kg). ResultsAcetic acid-induced colitis increased the colon's macroscopic and histopathological damage scores; increased colon levels of MDA (Malondialdehyde), MPO (Myeloperoxidase), TNF-α (Tissue necrosis factor α), IL6 (Interleukin 6), and IL17 (Interleukin 17); and decreased SOD (Superoxide Dismutase), GPx (Glutathione Peroxidase), and IL10 (Interleukin 10) levels in the treated rats compared with the control group (P < 0.001). Overall, a combination of A. arabica and O. basilicum reduced macroscopic and histopathological damage scores (P < 0.01) of the colon, and MDA, MPO, TNF-α, IL6 (P < 0.001), and IL17 (P < 0.01) levels of the colon. Furthermore, it increased SOD, GPx, and IL10 levels compared to the colitis group (P < 0.01). ConclusionA. arabica and O. basilicum have improving effects on UC by reducing inflammation and oxidative stress.

The use of a multi-strain probiotic and high dietary vitamin D to ameliorate acute TNBS-induced colitis in mice

Inflammatory Bowel Diseases (IBD) either Crohn’s disease or Ulcerative colitis are debilitating disorders that affect the gastrointestinal tract with chronic inflammation. Patients with IBD are often characterized by having a change in the gut microbiome composition and decreased levels of vitamin D, which contribute to disease development. Because of this, the separate use of probiotics and vitamin D has gained attention as complementary treatments. However, the beneficial effects of combining these treatments to target colitis inflammation by modulating gut microbiome and immune cells are still unknown. Therefore, the objective of this research was to demonstrate the efficacy of probiotics and high dietary vitamin D to ameliorate colonic inflammation. We hypothesized that co-administration of probiotics and vitamin D will synergistically reduce colitis induced inflammation. For the methodology, male and female C57bl/6 mice were divided into 4 groups: Colitis (n=6), Colitis + Probiotic (n=6), Colitis +Vitamin D (n=6), Colitis + Probiotic + Vitamin D (n=6). Animals received 7 days of pretreatment with Vivomixx probiotic in drinking water, high vitamin D (5IU/g) in food, or both treatments combined. At day 7, animals were lightly anesthetized with ether and colitis induction was done by intracolonic administration of 0.1mL of 4mg trinitrobenzenesulfonic acid in 30% ethanol. Water, food, and body weight were monitored daily. Animals were sacrificed 3 days post colitis induction and colonic tissue was collected to assess colonic inflammation by macroscopic score, colon length, and myeloperoxidase activity (MPO). Results obtained indicate that animals had similar food and water consumption during the 7 days pretreatment of probiotic and vitamin D.After 48 hours of colitis induction, all animals lost an average of 15% of their weight. At day of sacrifice, colitis untreated animals continued to lose weight, while administration of probiotic and probiotic +Vitamin D significantly recovered body weight (7.55%, p&lt;0.05 and 9.55%, p&lt;0.01, respectively) compared to colitis untreated. Similarly, animals treated with probiotic (4.11, p=0.0017) and probiotic + Vitamin D (4.02, p=0.0012) had significantly decreased macroscopic damage score compared to colitis (10.9). Treatment with vitamin D alone did not improve weight loss or macroscopic score. In conclusion, results obtained indicate that probiotic administration may be responsible for colitis recovery by decreasing weight loss and colonic inflammation. Future studies will increase the number of animals to determine if synergistic effects occur between probiotic and vitamin D to ameliorate colitis by modulating the gut microbiome and increasing anti-inflammatory cytokines. Supported in part by 5R25GM082406 and 1T32GM144896 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lafutidine Ameliorates Indomethacin-Induced Small Intestinal Damage in Rats by Modifying the Intestinal Mucosal Barrier, Inflammation, and Microbiota

Introduction: Nonsteroidal anti-inflammatory drug (NSAID)-induced small intestinal damage is a serious and escalating clinical problem without effective treatment. Lafutidine (LAF) is a novel histamine H2 receptor antagonist with a mucosal protective action. This study aimed to investigate the protective effect of LAF on indomethacin (IND)-induced enteropathy in rats. Methods: Rats were treated with LAF for 10 days with concomitant IND treatment on the final 5 days. Changes in metabolism and hematological and biochemical parameters were measured, and intestinal damage was blindly scored. Intestinal mucosal tissue and luminal contents were collected for transcriptome and microbiota sequencing. Intestinal inflammation and barrier function were also evaluated. Results: LAF treatment prevented anorexia and weight loss in rats and ameliorated reductions in hemoglobin, hematocrit, total protein, and albumin levels. LAF reduced the severity of IND-induced intestinal damage including macroscopic and histopathological damage score. Transcriptome sequencing results indicated that LAF might have positive effects on intestinal inflammation and the intestinal mucosal barrier. Further research revealed that LAF decreased neutrophil infiltration, and IL-1β and TNF-α expression in intestinal tissue. Besides, the treatment increased mucus secretion, MUC2, Occludin, and ZO-1 expression, and decreased serum D-lactate levels. LAF treatment also ameliorates microbial dysbiosis in small intestine induced by IND and increased the abundance of Lactobacillus acidophilus. Conclusion: LAF may protect against NSAID enteropathy via enhancing the intestinal mucosal barrier, inhibiting inflammation, and regulating microbiota.

Budesonide-liposomes inclusion complex in thermosensitive gel alleviates DSS-induced ulcerative colitis in mice

Purpose A novel formulation for Ulcerative Colitis (UC) treatment by rectal administration with budesonide liposomes (Bud Lip) and thermosensitive gel (Gel) was developed for future clinical use. To evaluate the anti-inflammatory activity and colon mucosal protection of this novel formulation compared with the other three in mice. Methods Bud Lip was prepared by reverse evaporation method and then dispersed in solutions with PL407 and PL188 by a cold method. Male mice were induced to UC by dextran sulfate sodium (DSS) and were treated for 14 days by rectal administration, as follows: Bud enema (a conventional suspension formulation); Bud Lip; Bud Gel; Bud Lip-Gel; saline. And a negative control without colitis was also used. Disease activity index (DAI), and macroscopic and microscopic damage scores in colon tissues were used to evaluate the effect of therapy. The levels of IL-6 and IL-10 in serum and the concentrations of TNF-α and IL-10 and myeloperoxidase (MPO) activity in colon tissue were also introduced. Results In UC mice model, Bud Lip-Gel showed inflammation was alleviated significantly, and the treatment was highly associated with lower DAI, less macroscopic and microscopic colonic damage and downregulation of pro-inflammatory cytokines TNF-α, IL-6 and MPO. Bud Lip-Gel had advantages over Bud, Bud Lip, Bud Gel in the treatment of active UC. Conclusion Novel Bud liposomes complex in thermosensitive Gel effectively mitigated symptoms, alleviated macroscopic and microscopic colon damage, and reduced inflammatory reaction in UC mice, which might be a potential strategy for UC treatment.

Imatinib mitigates experimentally-induced ulcerative colitis: Possible contribution of NF-kB/JAK2/STAT3/COX2 signaling pathway

RationaleUlcerative colitis (UC) is a chronic immune-mediated disease characterized by recurrent inflammation, damage, and alteration of the large intestine's mucosal and submucosal surfaces. The purpose of this research was to evaluate the impact of tyrosine kinase inhibitor (imatinib) on experimentally induced UC in rats via acetic acid (AA). MethodsMale rats were randomly assigned to four groups: control, AA, AA + imatinib (10 mg/kg), and AA + imatinib (20 mg/kg). Imatinib (10 and 20 mg/kg/day) was orally supplied by oral syringe for one week before induction of UC. On the eighth day, Rats received enemas containing a 4 % solution of acetic acid to induce colitis. One day after inducing colitis, rats were euthanized and their colons were subjected to morphological, biochemical, histological, and immunohistochemical analysis. ResultsImatinib pretreatment significantly decreased macroscopic and histological damage scores, decreased disease activity index as well as colon mass index. In addition, imatinib successfully lowered the levels of malondialdehyde (MDA) in colonic tissues and enhanced superoxide dismutase activity (SOD) and glutathione content (GSH). Imatinib also reduced colonic levels of inflammatory interleukins (IL-23, IL-17, IL-6), JAK2 and STAT3. Furthermore, imatinib suppressed nuclear transcription factor kappa B (NF-kB/p65) level, and COX2 expression in colonic tissues. SignificanceImatinib may be a viable therapy option for UC as it halts the interaction network of NF-kB/JAK2/STAT3/COX2 signaling pathway.

A novel oral formulation of the melanocortin-1 receptor agonist PL8177 resolves inflammation in preclinical studies of inflammatory bowel disease and is gut restricted in rats, dogs, and humans.

PL8177 is a potent and selective agonist of the melanocortin 1 receptor (MC1R). PL8177 has shown efficacy in reversing intestinal inflammation in a cannulated rat ulcerative colitis model. To facilitate oral delivery, a novel, polymer-encapsulated formulation of PL8177 was developed. This formulation was tested in 2 rat ulcerative colitis models and evaluated for distribution, in vivo, in rats, dogs, and humans. The rat models of colitis were induced by treatment with 2,4-dinitrobenzenesulfonic acid or dextran sulfate sodium. Single nuclei RNA sequencing of colon tissues was performed to characterize the mechanism of action. The distribution and concentration of PL8177 and the main metabolite within the GI tract after a single oral dose of PL8177 was investigated in rats and dogs. A phase 0 clinical study using a single microdose (70 µg) of [14C]-labeled PL8177 investigated the release of PL8177 in the colon of healthy men after oral administration. Rats treated with 50 µg oral PL8177 demonstrated significantly lower macroscopic colon damage scores and improvement in colon weight, stool consistency, and fecal occult blood vs the vehicle without active drug. Histopathology analysis resulted in the maintenance of intact colon structure and barrier, reduced immune cell infiltration, and increased enterocytes with PL8177 treatment. Transcriptome data show that oral PL8177 50 µg treatment causes relative cell populations and key gene expressions levels to move closer to healthy controls. Compared with vehicle, treated colon samples show negative enrichment of immune marker genes and diverse immune-related pathways. In rats and dogs, orally administered PL8177 was detected at higher amounts in the colon vs upper GI tract. [14C]-PL8177 and the main metabolite were detected in the feces but not in the plasma and urine in humans. This suggests that the parent drug [14C]-PL8177 was released from the polymer formulation and metabolized within the GI tract, where it would be expected to exert its effect. Collectively, these findings support further research into the oral formulation of PL8177 as a possible therapeutic for GI inflammatory diseases in humans.

P079 Attenuation of inflammation and histologic production-dependent improvement by engineered high acetate producing Saccharomyces boulardii in DSS-induced colitis

Abstract Background It has been hypothesized that the probiotic potential of the yeast Saccharomyces boulardii (Sb) is associated with its acetate production. This SCFA is of interest in IBD due to its cross-feeding potential with beneficial butyrate-producing bacteria and low toxicity to epithelial cells . Our previous in vitro work supports this hypothesis (Deleu et al, 2022) but still requires in vivo validation. Therefore, we evaluated the effect of different engineered Sb strains producing variable amounts of acetate in DSS-induced colitis in mice. Methods Nine week old female C57/Bl6 mice (N=120) were allocated to 12 treatment groups receiving drinking water or 2.75% DSS in combination with PBS (control), Baker’s yeast (non-probiotic control), SDH1 (non-acetate producing Sb), ENT (transient acetate producing Enterol strain-probiotic control), SbP (high acetate producing Sb) and ENT3 (extra high acetate producing Sb). Disease activity including weight loss, diarrhoea and the presence of occult blood was scored daily. On day 7, the DSS groups were transferred to regular drinking water and on day 14 mice were sacrificed. Colonic tissue and blood were collected for resp. histologic and cytokine analysis. Results Disease activity, determined by the area under the curve, in DSS subgroups was lower for SbP compared to PBS and Baker’s yeast (both p&amp;lt;0.05). Remarkably, Sb SDH1 showed even higher disease activity compared to the Sb strains ENT, SbP and ENT3 (all p&amp;lt;0.05). At sacrifice, macroscopic damage score in DSS subgroups was lower for SbP and ENT3 (both p&amp;lt;0.05) compared to Sb SDH1 and the colon weight/length-ratio was decreased for ENT and SbP compared to Sb SDH1 (resp. p=0.06 and p=0.08). Higher histologic inflammation was noted in the non- or only transient-acetate producing strains on DSS compared to healthy PBS control (all p&amp;lt;0.05), whereas this increase was not observed for both high-acetate producing strains SbP and ENT3 on DSS (p=NS). Lower IL1β, IL2 and IL4 concentrations for DSS groups on SbP and ENT3 compared to DSS groups on Sb SDH1 and ENT were observed (all p&amp;lt;0.05). In contrast, IL10, TNFα and KC/GRO were lower for the DSS groups on Sb SDH1 and ENT compared to DSS groups on SbP, ENT3, and even PBS for IL2 and 4 (all p&amp;lt;0.05). Conclusion Engineered high acetate producing Sb strains show a significant trend towards improved attenuation of DSS-induced colitis compared to the parent Sb strain on disease activity, macroscopic damage score and show production-dependent response on histology. Mixed pro-inflammatory serum profiles were observed potentially pointing in the direction of other effects of acetate accumulation. Together with our previous in vitro work, these data indicate a role for Sb-produced acetate in attenuating inflammation.

DOP09 Eosinophils exert a pro-inflammatory role in a chronic DSS colitis model without an impact on fibrosis

Abstract Background Eosinophils might play a pro-inflammatory role in inflammatory bowel disease (IBD). However, the role of eosinophils in intestinal fibrosis remains poorly understood, although a pro-fibrotic function has been hypothesized based on data outside the gastrointestinal tract. Therefore, we aimed to unravel the role of eosinophils in chronic intestinal inflammation and fibrosis and to explore eosinophil depletion as a potential therapeutic target. Methods A 3-cycles chronic dextran sodium sulphate (DSS) model was induced in 6-8-week-old C57BL/6 wild type mice. Mice received 3 DSS cycles (1.5% - 2.00% - 2.00%) in which 1 DSS cycle consisted of 1 week of DSS administration followed by 2 weeks of recovery. During the 3 cycles, and starting 3 days prior to colitis induction, anti-CCR3 antibody or isotype injections were given twice weekly to study the effect of the eosinophil depletion (total of 18 injections). The disease activity index (DAI; weight loss, stool consistency and presence of blood) was determined twice weekly. At sacrifice, colonic damage was scored macroscopically (presence of hyperaemia, adhesions and length and degree of colon affected by inflammation) and colonic single cells were isolated and fluorescently stained for flow cytometry. Eosinophils were thereby identified as CD45+ CD11b+ Siglec-F+ CD117- cells. Lastly, a histological active disease score was determined comprising of the sum of neutrophil infiltration, mononuclear cell infiltration, changes in mucosal architecture, goblet cell loss and epithelial defects (Creyns et al., 2019). Intestinal fibrosis was assessed via colon weight/length ratio, COL1A1 gene expression and collagen deposition with a Martius Scarlet Blue (MSB) staining and a colorimetric hydroxyproline assay. Results Blood and colonic eosinophil depletion via anti-CCR3 injections was confirmed via flow cytometry (figure 1). The DAI in the eosinophil depleted group was decreased compared to the isotype injected group (area under the curve 115.4 ± 29 vs 160.6 ± 28; p=0.02). A similar trend was seen in the macroscopic damage score (2.0 ± 1.3 vs 3.9 ± 2.1; p=0.08). Furthermore, a lower histological active disease score was found in the mice in which the eosinophils were depleted compared to the isotype injected mice (8.8 ± 0.8 vs 12.8 ± 1.5; p=0.002) (figure 2). Lastly, no differences in the parameters for fibrosis between the anti-CCR3 injected and isotype injected groups were observed (figure 3). Conclusion Eosinophil depletion via intraperitoneal anti-CCR3 injections resulted in partial protection from intestinal inflammation in chronic DSS, and could therefore be further explored as a potential therapeutic agent. In contrast, eosinophil depletion does not seem to have any anti-fibrotic effect

Role of cyclooxygenases 1 and 2 in the maintenance of colonic mucosal integrity in an experimental colitis model.

The role of cyclooxygenase (COXs) isoforms in maintaining colonic mucosal integrity is not fully understood. This study aimed to evaluate the role of COX-1 and -2 on colonic mucosal integrity in an experimental colitis model. Colitis was induced in Wistar rats by intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (20 mg + 50% ethanol). The control group (sham group) received saline only. After 7, 14, or 28 days, colonic samples were removed, and macroscopic lesion scores, wet weight, myeloperoxidase activity, and transepithelial electrical resistance (TER) were determined. In other rat groups, colonic samples from the sham group and a 7th day post-colitis group were mounted in Üssing chambers with the luminal side exposed to a buffer solution (control), acetylsalicylic acid (ASA), SC-560 (COX-1 inhibitor), or celecoxib (COX-2 inhibitor). TER and epithelial permeability to fluorescein were measured. The 7th day colitis group had higher macroscopic damage scores, wet weight, and myeloperoxidase activity and lower basal TER than the sham, 14th day colitis, and 28th day colitis groups. Inhibition of COX-1 but not COX-2 significantly decreased TER and increased permeability to fluorescein in the 7th day post-colitis group compared to the sham group. Additionally, ASA decreased the colonic mucosal integrity on day seven post-colitis compared to the sham group. A decrease in the colonic mucosa integrity in the experimental colitis model can be aggravated only by the inhibition of COX-1, which demonstrated the importance of this enzyme in the maintenance of colonic mucosal integrity.