Colon Homogenates Research Articles

Effects of Local Vasodilators and the Autonomic Nervous System on Microcirculation and Mitochondrial Function in Septic Rats.

Systemic vasodilating agents like nitroglycerin (NG) or iloprost (Ilo) show beneficial effects on intestinal microcirculation during sepsis, which could be attenuated by activation of the sympathetic nervous system or systemic side effects of vasodilating agents. This exploratory study aimed to investigate the effects of topically administered vasodilators and the parasympathetic drug carbachol on colonic microcirculatory oxygenation (µHbO2), blood flow (µFlow) and mitochondrial respiration. A total of 120 male Wistar rats were randomly assigned to twelve groups and underwent either colon ascendens stent peritonitis (CASP) or sham surgery. After 24 h, animals received the following therapeutic regimes: (1) balanced full electrolyte solution, (2) carbachol, (3) NG, (4) Ilo, (5) NG + carbachol, and (6) Ilo + carbachol. Mitochondrial respiration was measured in colon homogenates by respirometry. In sham animals, NG (-13.1%*) and Ilo (-10.5%*) led to a decrease in µHbO2. Additional application of carbachol abolished this effect (NG + carbachol: -4.0%, non-significant; Ilo + carbachol: -1.4%, non-significant). In sepsis, carbachol reduced µHbO2 when applied alone (-10.5%*) or in combination with NG (-17.6%*). Thus, the direction and degree of this effect depend on the initial pathophysiologic condition.

Hydroalcoholic extract of Araucaria sp. brown propolis alleviates ulcerative colitis induced by TNBS in rats by reducing inflammatory cell infiltration and oxidative damage.

To investigate the effects of Araucaria sp. brown propolis (ABP) against trinitrobenzenesulfonic acid (TNBS)-induced colitis in rats. Animals received vehicle (1% DMSO, 1 ml/kg) or hydroalcoholic extract of ABP (hydroalcoholic extract of Araucaria sp. brown propolis (HEABP), 30, 100, and 300 mg/kg) orally, or dexamethasone (25 mg/kg, s.c.) for 5 days. On day 4, the animals received intracolonic TNBS (150 mg/kg), on day 6 they were euthanized. The weight of the animals, the macroscopic and microscopic colonic damage, reduced glutathione (GSH) and malondialdehyde (MDA) levels, and the activity of glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and myeloperoxidase (MPO) were measured in colon homogenate. The action of HEABP and two isolated compounds in neutrophil migration was recorded. HEABP (100 and 300 mg/kg), but not dexamethasone, decreased colonic lesion, and increased colonic mucin staining. In parallel, HEABP decreased MDA and restored GSH levels and the activity of SOD, CAT, and GST in the colon. A dose-dependent inhibition of MPO activity was observed (LogIC50 = 1.9). Moreover, HEBPA and the junicedric and abietic acids inhibited the neutrophil chemotaxis in vitro and HEBPA reduced neutrophil migration in vivo. HEABP may be promising in the therapies for inflammatory bowel diseases, reducing oxidative and inflammatory damage, especially mediated by neutrophils.

Ameliorative effect of nodakenin in combating TNBS-induced ulcerative colitis by suppressing NFƙB-mediated NLRP3 inflammasome pathway.

Ulcerative colitis (UC) is an enduring and complex inflammatory bowel disease that is clinically prevalent, progressive, and debilitating. As of now, the few effective medical treatments for UC have unacceptably high side effects. It is crucial to find safer and more effective UC treatments. Nodakenin possesses anti-inflammatory and antioxidant activity by suppressing several pro-inflammatory mediators. In the present study, we aimed to evaluate the colonoprotective effect of nodakenin in combating colitis through the NFƙB-mediated NLRP3 inflammasome pathway. In mice, UC was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS). Nodakenin (10, 20, and 40mg/kg) was introduced intragastrically, and disease activity index (DAI) score was calculated. Malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD), nitric oxide (NO) levels, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) concentration were evaluated in colon homogenate. Colon samples were used for histopathological investigation and mRNA expression studies involving nuclear factor kappa B (NFƙB), cyclooxygenase-2 (COX-2), inducible nitric oxide (iNOS), nucleotide-binding receptor domain 3 (NLRP3), interleukin-1β (IL-1β), and interleukin-18 (IL-18). Nodakenin treatment was found effective in lowering the DAI score, histological score, MPO, MDA, and NO levels while elevating SOD levels as compared to the model control group, showcasing its anti-inflammatory and antioxidant properties. Nodakenin (40mg/kg) significantly downregulated the expression of TNF-α, IL-6, NFƙB (1.24-fold), iNOS (1.2-fold), COX-2 (1.98-fold), NLRP3 (1.78-fold), IL-1β (1.29-fold), and IL-18 (1.17-fold) conferring its great anti-inflammatory potential in combating colitis. Taking together, nodakenin presumably alleviated TNBS-induced colitis by NFƙB-mediated NLRP3 inflammasome pathway and reduced colon damage by downregulating various transcriptional genes and pro-inflammatory mediators.

The Effects of Sulfasalazine and Ezetimibe on Proinflammatory Cytokines in Male Rat with Induced Colitis: A Comparative Study

Abstract Background: Ulcerative colitis is a chronic debilitating disease. The existing treatment, including sulfasalazine, corticosteroids, azathioprine/6-mercaptopurine, cyclosporine, and antitumor necrosis factor therapy, frequently fails to cure the disease, necessitating the investigation of new drugs with less adverse effects. Objectives: The purpose of this study was to compare the anti-inflammatory effects of ezetimibe to those of sulfasalazine (salazosulfapyridine) in rats with experimentally induced colitis. Materials and Methods: A total of 40 adult males Albino-Wister rats were divided into four groups (each with 10 rats). Group I received no treatment and served as a negative control. Acetic acid4% (vol/vol) was used rectally to induce experimental colitis in the other three groups, where group II left without treatment. Sulfasalazine 100 mg/kg (group III) or ezetimibe 10 mg/kg (group IV) were used as a therapeutic dose orally for 1 week. The following parameter was estimated in the tissue homogenate of the colon: tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), nuclear factor kappa B-cell (NF-κB), and a histopathological score of the colonic tissue. Results: Both sulfasalazine and ezetimibe significantly reduced the level of TNF-α, IL-1β, and NF-κB compared with the induced colitis. Colon homogenate of TNF-α and IL-1β did not differ significantly between group III (197.25 ± 64.97 and 190.87 ± 36.86 pg/mL, respectively) and IV (223.72 ± 70.05 and 240.93 ± 61.56 pg/mL, respectively); however, ezetimibe-treated rats had significantly higher NF-κB than sulfasalazine-treated rats (3.35 ± 0.74 versus 2.11 ± 0.88 pg/mL). Both treatment modalities significantly ameliorated the histopathological score compared with induced colitis (3.0 ± 0.0), with the superiority of sulfasalazine over ezetimibe (0.57 ± 0.093 versus 1.39 ± 0.17). Conclusions: The results indicate that ezetimibe is an effective treatment (compared with sulfasalazine) for induced colitis by reducing the inflammatory response and ameliorating histopathological changes.

First report of isolation of Fusobacterium varium from liver abscesses and ruminal and colonic epithelial tissues of feedlot cattle*

ObjectiveOur objective was to isolate and determine prevalence of Fusobacterium varium in liver abscesses and the corresponding ruminal and colonic epithelial tissues and ruminal and colonic contents of feedlot cattle. Materials and MethodsA total of 96 intact liver abscess samples and matched ruminal and colonic tissues and contents from cattle, originating from feedlots that did not receive in-feed tylosin, were collected at slaughter. Liver abscesses and ruminal and colonic tissue were ho- mogenized and then plated, before and after enrichment in lactate or lysine medium with selective antibiotics, onto blood agar and selective lactate or lysine agar for isolation of Fusobacterium and to determine prevalence and concentration. Putative colonies were tested by a quantitative PCR assay targeting the hgdA gene for species confirma- tion. Results and DiscussionNone of the liver abscess samples yielded F. varium by direct plating; however, F. varium was isolated from 3 of 96 (3.1%) following enrich- ment of the homogenate in lactate or lysine medium. In contrast to liver abscesses, F. varium was isolated by direct plating from 27.1% (26/96) of ruminal epithelial and 3.1% (3/96) of colonic epithelial tissue homogenates. Overall, 10.1%, 77.1%, 44.8%, 86.5%, and 70.1% of liver abscess, ruminal, and colonic epithelial tissues and ruminal and colonic contents were positive for F. varium, respectively. Implications and ApplicationsThe increased fre- quency of isolation and high prevalence of F. varium in ruminal tissue and, to a lesser extent, in the colonic tissue confirms its ability to invade tissues and possibly cause bacterial ruminitis. However, the relatively low frequency of F. varium isolation and low prevalence in liver abscesses suggest that it is unlikely to be an etiologic agent. Inter- estingly, there is some evidence that ruminal strains of F. varium were resistant to tylosin; therefore, it would be of interest to determine the prevalence in cattle receiving in-feed tylosin.

Polysaccharides from an edible mushroom, Hericium erinaceus, alleviate ulcerative colitis in mice by inhibiting the NLRP3 inflammasomes and reestablish intestinal homeostasis

This study aimed to assess the effects of polysaccharides extracted from Hericium erinaceus fruiting bodies (HEFPs) on the inflammatory response to oxidative stress in a mouse model of ulcerative colitis (UC) induced by ingestion of dextran sodium sulfate. The results indicated reduced oxidative damage in the HEFPs groups, as evidenced by significantly decreased malondialdehyde levels and significantly increased levels of the antioxidant enzymes superoxide dismutase and catalase in colon homogenates, compared with those in the Model Control (MC) group. Additionally, compared with the levels in the MC group, the levels of the pro-inflammatory factors IL-6, IL-1β, and TNF-α in the positive-control (PC) and HEFPs groups were significantly lower, and that of the anti-inflammatory factor IL-10 was significantly higher. qRT-PCR analyses revealed that the colon expression patterns of IL-6, IL-1β, TNF-α, and IL-18 were consistent with the serum levels. Western-blotting results indicated significantly lower levels of NLRP3, ASC, and caspase 1 P20 in the HEFPs and PC groups than in the MC group. These findings suggest that HEFPs alleviate UC by suppressing the NLRP3 inflammasome/Caspase-1 pathway. Lachnospiraceae, Clostridiales, Parabacteroides, Oscillibacter, and Clostridium XlVa genera were more abundant in the gut microbiota of the HEFPs group than that of the MC group.

Modulatory effects of Moringa oleifera leaf extract on sodium nitrate-induced experimental colitis via regulation of P53, Ki-67 and PCNA biomarkers

BackgroundUlcerative colitis is a risk factor for colorectal carcinoma. Different mechanisms are related to colitis like apoptosis and hyperproliferation. Moringa oleifera leaves extract (MO) provides a promising option to overcome the risk. PurposeTo examine the colonic changes in a rat model of colitis induced by sodium nitrate (SN) and study the effects of MO. Study designEight adult male rats were allocated in each of the three group; control (distilled water), SN (100 mg/kg/day, orally via gastric gavage), and SN + MO (100 mg/kg/day, orally via gastric gavage). MethodsBody weight was measured after the end of the experiment. Colonic homogenates were tested for levels of oxidative stress indicators. Immunohistochemistry for P53, PCNA and Ki-67 was performed. Fresh colon specimens were used for quantitative real-time PCR for assessment of P53, PCNA and Ki-67 gene expression. ResultsSN group revealed a significant decreased weight (p = 0.002). MDA and NO levels were higher with SN administration than with MO co-administration (p= 0.04, 0.01 respectively). GSH level was reduced in SN group (p = 0.02) and significantly increased with MO intake (p = 0.04). SN-induced colonic destructive changes were reversed with MO. P53, PCNA and Ki-67 levels of gene expression were reduced in SN + MO group than SN group (P = 0.007, 0.02, 0.001 respectively). ConclusionMO protected the colonic mucosa against SN-induced changes regulating apoptosis, and cell proliferation.

Revisiting the Role of Valeric Acid in Manipulating Ulcerative Colitis.

Ulcerative colitis (UC) is characterized by a complicated interaction between mucosal inflammation, epithelial dysfunction, abnormal activation of innate immune responses, and gut microbiota dysbiosis. Though valeric acid (VA), one type of short-chain fatty acids (SCFAs), has been identified in other inflammatory disorders and cancer development, the pathological role of VA and underlying mechanism of VA in UC remain under further investigation. Studies of human clinical specimens and experimental colitis models were conducted to confirm the pathological manifestations of the level of SCFAs from human fecal samples and murine colonic homogenates. Valeric acid-intervened murine colitis and a macrophage adoptive transfer were applied to identify the underlying mechanisms. In line with gut microbiota dysfunction in UC, alteration of SCFAs from gut microbes were identified in human UC patients and dextran sodium sulfate -induced murine colitis models. Notably, VA was consistently negatively related to the disease severity of UC, the population of monocytes, and the level of interluekin-6. Moreover, VA treatment showed direct suppressive effects on lipopolysaccharides (LPS)-activated human peripheral blood mononuclear cells and murine macrophages in the dependent manner of upregulation of GPR41 and GPR43. Therapeutically, replenishment of VA or adoptive transfer with VA-modulated macrophages showed resistance to dextran sodium sulfate-driven murine colitis though modulating the production of inflammatory cytokine interleukin-6. In summary, the research uncovered the pathological role of VA in modulating the activation of macrophages in UC and suggested that VA might be a potential effective agent for UC patients.

Mangiferin (mango) attenuates AOM-induced colorectal cancer in rat’s colon by augmentation of apoptotic proteins and antioxidant mechanisms

Mangiferin (MF) is a natural C-glucosylxantone compound that has many substantial curative potentials against numerous illnesses including cancers. The present study's goal is to appraise the chemo preventive possessions of MF on azoxymethane (AOM)-mediated colonic aberrant crypt foci (ACF) in rats. Rats clustered into 5 groups, negative control (A), inoculated subcutaneously with normal saline twice and nourished on 0.5% CMC; groups B-E injected twice with 15 mg/kg azoxymethane followed by ingestion of 0.5% CMC (B, cancer control); intraperitoneal inoculation of 35 mg/kg 5-fluorouracil (C, reference rats) or nourished on 30 mg/kg (D) and 60 mg/kg (E) of MF. Results of gross morphology of colorectal specimens showed significantly lower total colonic ACF incidence in MF-treated rats than that of cancer controls. The colon tissue examination of cancer control rats showed increased ACF availability with bizarrely elongated nuclei, stratified cells, and higher depletion of the submucosal glands compared to MF-treated rats. Mangiferin treatment caused increased regulation of pro-apoptotic (increased Bax) proteins and reduced the β-catenin) proteins expression. Moreover, rats fed on MF had significantly higher glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), and lower malondialdehyde (MDA) concentrations in their colonic tissue homogenates. Mangiferin supplementation significantly down-shifted pro-inflammatory cytokines (transforming growth factor-α and interleukine-6) and up-shifted anti-inflammatory cytokines (interleukine-10) based on serum analysis. The chemo-protective mechanistic of MF against AOM-induced ACF, shown by lower ACF values and colon tissue penetration, could be correlated with its positive modulation of apoptotic cascade, antioxidant enzymes, and inflammatory cytokines originating from AOM oxidative stress insults.

Allicin ameliorates glucose and lipid metabolism via modulation of gut microbiota and bile acid profile in diabetic rats

Diabetes mellitus is a widely prevalent chronic disease, and effective treatments for it remain limited. Allicin has anti-diabetic effect, however, the detailed mechanism has not been well investigated. The diabetic rats were treated with allicin for 6 weeks, and then the fecal samples, serum and tissues were collected for measurement of gut microbiota (GM), bile acids (BAs) and other indexes. Allicin decreased the blood glucose levels, serum lipid levels, and alleviated hepatic lipid deposition of the diabetic rats. Simultaneously, allicin changed the composition of GM and increased secondary BAs in serum. Interestingly, allicin significantly increased the glucagon-like peptide-1 (GLP-1) content in colon homogenate, and reduced the protein expression of fibroblast growth factor 15 (FGF15) in ileum. Moreover, allicin increased 7α-hydroxylase 1 (CYP7A1) expression in liver. The study demonstrated that allicin ameliorates metabolisms in the diabetic rats though modulating gut microbiota, bile acids, and inhibiting enterohepatic FGF15-CYP7A1 signaling.

Preventive Anti-inflammatory Effects of Apocynin on Acetic Acid-Induced Colitis in Rats.

Ulcerative colitis is an inflammatory bowel disease with a complex aetiology characterised by abnormal immune responses and oxidative stress-induced tissue injury. Inflammatory cells play an important role in the progression of this pathology through the overproduction of reactive oxygen species (ROS) from various sources including the NADPH oxidases (NOXs). The aim of this study was to investigate the preventive effect of apocynin, a natural antioxidant molecule and a selective inhibitor of NOXs, on acetic acid (AA)-induced ulcerative colitis in rats. Our results first confirmed that apocynin has a high free radical scavenging capacity as well as a potent iron chelating ability. Oral pretreatment of rats with apocynin (200mg/kg and 400mg/kg) for 7days prior to AA-induced colitis suppressed the increase in pro-oxidant markers in colonic homogenates and preserved colonic cytoarchitecture from acetic acid-induced damage. Oral administration of apocynin (200mg/kg and 400mg/kg) also reduced several systemic inflammatory markers such as alkaline phosphatase, iron, pro-inflammatory cytokines, C-reactive protein and myeloperoxidase. This study shows that apocynin protects rats from acetic acid-induced colonic inflammation and suggests that apocynin may have a promising beneficial effect in the prevention of ulcerative colitis.

Antioxidant potential of ethanol extract of Annona muricata leaves and its inhibitory effect on lipid peroxidation in 1,2-dimethylhydrazine induced colon carcinogenesis

Annona muricata, as it is scientifically called but known in most parts of Nigeria as soursop. A fruit tree with various therapeutic uses, the plant belongs to the family of Annonaceae and has several medicinal properties. The medicinal properties of Annona muricata are well known, and they can be used to treat a variety of illnesses and conditions. Effects of ethanol extract of Annona muricata leaves (EEAML) on the enzymatic antioxidants of the kidney and colon in Wistar rats induced with 1, 2 Dimethylhydrazine (DMH) at 25 mg/kg bodyweight s.c. were investigated in this study. Thirty-six male Wistar rats weighing 110–170g were acclimated for two weeks and randomized into six groups (six per group). Group A (control), Group B (extract; 120 mg/kg body weight of ethanolic extract of A. muricata), Group C (DMH only), Group D (DMH + EXTRACT), Group E (pretreatment), and Group F (posttreatment). The extract was administered daily via oral gavage, while the DMH was given subcutaneously at a dosage of 25 mg/kg bodyweight. This study reveals the carcinogenic effect of DMH induced oxidative stress were ameliorated by the administration of this leaf extract of EEAML at a dose of 120mg/kg b.w. The colon and kidney homogenates of rats administered EEAML (co-treated, pretreatment and post- treatment) showed increased enzymatic antioxidant; glutathione peroxidase, catalase activities and SOD activities. But a reduction in malondialdehyde levels was observed. These findings validate the use of Annona muricata in traditional medicine by showing that the ethanolic extract of the leaves can reduce DMH-induced oxidative stress and alter the enzymatic antioxidant profile in colon carcinogenesis.

Moxifloxacin's Therapeutic Effects in AA-Induced Colitis: Anti-Inflammatory Action through NF-κB Pathway Inhibition, Including TNF-α Pathway and Downstream Inflammatory Processes

Objectives: The aim of the present study was to evaluate possible therapeutic effects of moxifloxacin against acetic acid-induced colitis in a rat model.
 Methods: Forty adult Wistar rats were separated into 4 groups, including the negative control group, acetic acid (AA) group, AA + sulfasalazine (100 mg/kg/day) group, and AA+ moxifloxacin (MFX) (8 mg/kg/day) group. Experimental colitis was induced in rats by rectal administration of (4%v/v) acetic acid. Rats with colitis were received either MFX 8mg/kg/day or sulfasalazine 100mg/kg orally for 7days.The parameters measured are macroscopical assessment, colon weight (indicator of edema) and the measurement of concentrations of the proinflammatory cytokine TNF-α, oxidative stress marker malondialdehyde (MDA), adhesion molecule selectin and the inflammatory mediator NF-κB in colonic tissue homogenate.
 Results: This study had shown that AA elevate macroscopical scores, colon weight and biochemical homogenate parameters and all measured parameters were significantly reduced by both moxifloxacin and sulfasalazine with nonsignificant difference between them.
 Conclusions: The study explain that moxifloxacin possesses therapeutic potential in in AA induced colitis and its anti-inflammatory actions may involve inhibition of NF-κB inflammatory pathways including TNF-α pathway with its downstream inflammatory process such as elevation of adhesion molecule synthesis and oxidative species overproduction in the colonic tissues.

Comparative treatment of Sulfasalazine+Ezetimibe combination and Sulfasalazine in a rat model with induced colitis.

Ulcerative colitis is a chronic inflammatory disease with high mortality and morbidity worldwide. It causes inflammation in the lining of the colon, resulting in several symptoms that negatively impact the quality of life. Unfortunately, there is currently no known cure for this condition. Therefore, it is crucial to explore alternative treatment approaches. This research aimed to investigate the anti-inflammatory and antioxidative effects of a combination therapy involving Sulfasalazine+Ezetimibe compared to Sulfasalazine alone in a rat model of ulcerative colitis. Forty adult rats were divided into four groups for this study. The groups consisted of a control group (negative control), an acetic acid group (positive control), an acetic acid+Sulfasalazine (100 mg/kg per day) group, and an acetic acid+Sulfasalazine (50 mg/kg)+Ezetimibe (5 mg/kg) group. Rats were treated for one week, and colitis was induced by administering 2 ml of 4% (v/v) acetic acid inter-rectally. After sacrifice, the colonic tissue homogenate was analyzed for several markers, including proinflammatory cytokines (TNF-α, IL-1β, NF-κB), oxidative stress markers (malondialdehyde, myeloperoxidase), and adhesive molecule markers (E-selectin, ICAM-1). Sulfasalazine and the combination of Sulfasalazine+Ezetimibe significantly reduced the colonic levels of TNF-α, IL-1β, NF-κB, MDA, and E-selectin in the homogenate. However, the combination therapy of Sulfasalazine and Ezetimibe demonstrated a superior effect.

Deletion of Endogenous Neuregulin-4 Limits Adaptive Immunity During Interleukin-10 Receptor-Neutralizing Colitis.

Growth factors are essential for maintenance of intestinal health. We previously showed that exogenous neuregulin-4 (NRG4) promotes colonocyte survival during cytokine challenge and is protective against acute models of intestinal inflammation. However, the function(s) of endogenous NRG4 are not well understood. Using NRG4-/- mice, we tested the role of endogenous NRG4 in models of colitis skewed toward either adaptive (interleukin-10 receptor [IL-10R] neutralization) or innate (dextran sulfate sodium [DSS]) immune responses. NRG4-/- and wild-type cage mate mice were subjected to chronic IL-10R neutralization colitis and acute DSS colitis. Disease was assessed by histological examination, inflammatory cytokine levels, fecal lipocalin-2 levels, and single cell mass cytometry immune cell profiling. Homeostatic gene alterations were evaluated by RNA sequencing analysis from colonic homogenates, with real-time quantitative polymerase chain reaction confirmation in both tissue and isolated epithelium. During IL-10R neutralization colitis, NRG4-/- mice had reduced colonic inflammatory cytokine expression, histological damage, and colonic CD8+ T cell numbers vs wild-type cage mates. Conversely, in DSS colitis, NRG4-/- mice had elevated cytokine expression, fecal lipocalin-2 levels, and impaired weight recovery. RNA sequencing showed a loss of St3gal4, a sialyltransferase involved in immune cell trafficking, in NRG4-null colons, which was verified in both tissue and isolated epithelium. The regulation of St3gal4 by NRG4 was confirmed with ex vivo epithelial colon organoid cultures from NRG4-/- mice and by induction of St3gal4 in vivo following NRG4 treatment. NRG4 regulates colonic epithelial ST3GAL4 and thus may allow for robust recruitment of CD8+ T cells during adaptive immune responses in colitis. On the other hand, NRG4 loss exacerbates injury driven by innate immune responses.

Experimental evidence of shikonin as a novel intervention for anti-inflammatory effects.

Shikonin is a natural product with antioxidant and anti-inflammatory activities. The biological activity of shikonin is still not fully understood, as well as its association with innate immunity and immune and inflammatory bowel disease (IBD) in humans. In this study, the toxicity of shikonin on Raw264.7 cells was assayed by MTT, and polarization of inflammatory macrophages was determined by flow cytometry. The results showed that shikonin can inhibit the polarization of macrophages towards M1 type and significantly inhibited the production of NO in the concentration range of 0.5-1μM. In addition, after treatment with shikonin, the production of IL-1β and TNF-α was significantly decreased. After shikonin administration, the body weight loss and decrease of colon length were significantly suppressed in DSS-treated colitis C57BL/6 mice. The pro-inflammatory cytokines TNF-α and IL-1β in colonic homogenate were significantly decreased. Shikonin treatment resulted in a notable improvement in the histopathological manifestations in DSS-treated animals at 25/50mg/kg. Meanwhile, we found that shikonin can regulate differentiation of T helper 17 cell (Th17)/regulatory T cell (Treg), thereby regulating the balance of Th17/Treg cells and exerting an anti-inflammatory effect in IBD animal models. In conclusion, we found that shikonin protects against DSS-induced acute colitis by, among other things, reducing immune cell infiltration, polarizing macrophages, and regulating Th17/Treg differentiation, as well as by downregulating the release of inflammatory cytokines. These findings showed that shikonin can improve inflammation by affecting macrophage polarization. Our experimental data provide experimental evidence and theory basis for research on anti-inflammatory effects for the shikonin as health or functional food.

THE ORAL GLUTMATE CARBOXYPEPTIDASE II INHIBITOR (S)-IBD3540 ROBUSTLY ATTENUATES INFLAMMATION IN SPONTANEOUS IL10 COLITIS

Abstract BACKGROUND & AIMS Glutamate carboxypeptidase II (GCPII) is elevated in active inflamed biopsies of both Crohn’s disease and ulcerative colitis patients and is a promising therapeutic target for the treatment of IBD. We have previously presented the development and characterization of a first-in-class, gut-restricted, and orally active GCPII inhibitor, (S)-IBD3540, and have shown that oral (S)-IBD3540 attenuates disease severity in multiple murine colitis models, including acute dextran sulfate sodium (DSS)-induced colitis. Here, we extend our knowledge regarding (S)-IBD3540’s anti-inflammatory effects in spontaneously occurring colitis of interleukin 10 (IL10-/-) mice by performing longitudinal measurement of inflammatory biomarker fecal lipocalin 2 (LCN2) and terminal colon cytokine and chemokine measurements. METHODS Female BALB/cAnNTac-Il10em7Tac mice (Taconic, Germantown, NY) were acquired at 4 weeks of age and monitored three times weekly for the onset of clinical signs, including alterations in stool consistency. Following onset of symptoms, groups were randomized and treatment was initiated with vehicle or 100 mg/kg eq. (S)-IBD3540 once daily by oral gavage. Prior to treatment, and biweekly thereafter, fecal LCN2 was quantified using a mouse Lipocalin-2/NGAL DuoSet ELISA (R&D Systems). Upon study termination, proximal colon homogenates were prepared and analyzed by a mouse Cytokine/Chemokine 31-Plex Discovery Assay Array (Eve Technologies). RESULTS Impressively, despite initiating treatment in highly symptomatic mice 100 mg/kg oral (S)-IBD3540 was found to reverse abnormalities in stool consistency. Additionally, (S)-IBD3540 had potent anti-inflammatory effects. As expected, fecal LCN2 was observed to increase throughout the study period in vehicle treated mice, however, fecal LCN2 was significantly decreased in (S)-IBD3540 treated mice (p<0.05). Similarly, colon multiplex analysis identified significant reductions in multiple cytokines and chemokines implicated in IL10 colitis pathobiology, with notable reductions in cytokines TNFa, IL-12p40, and IL-17, and chemokines IP-10 and MIP-2. CONCLUSION (S)-IBD3540 is a novel, gut-restricted, GCPII inhibitor that has robust anti-inflammatory activity in IL10-/- colitis. Encouragingly, we have now found that in this model (S)-IBD3540 reduces pro-inflammatory cytokines/chemokines that are the targets of existing IBD medications. Studies to compare efficacy to one of these agents, anti-IL-12p40, are in progress.

Knockdown of TRIM8 alleviates dextran sulfate sodium-induced colitis in mice by inhibiting the NF-κB signaling pathway.

Although TRIpartite Motif containing 8 (TRIM8) gene plays an important role in a number of biological processes, such as inflammation, its function and mechanism in ulcerative colitis (UC) remain unknown. The UC model was established by feeding mice with 3.5% dextran sulfate sodium (DSS). The animals were divided into the following four groups: control group, DSS group, DSS+short hairpin (sh)-NC group, and DSS+sh-TRIM8 group. Changes in body weight and disease activity index (DAI) score of mice in all the groups were recorded for 7 days. The animals were executed at the end of the experiment, and the expression of TRIM8 in colon tissue was detected by polymerase chain reaction and Western blot assays. The length of colon was measured, and the histopathological changes in mice colon were examined by hematoxylin and eosin staining. The expression of pro-inflammatory factors in mice serum and colonic tissue homogenate was detected by enzyme-linked-immunosorbent serologic assay. The expression of nuclear factor kappa B (NF-κB) pathway-related proteins in colonic tissues was detected by Western-blot analysis. TRIM8 was highly expressed in the colonic tissues of UC mice. Knockdown of TRIM8 improved DSS-induced weight loss, increased DAI score, shortened colon length, and alleviated colonic injury and inflammation in mice. Western-blot experiments showed that knockdown of TRIM8 inhibited DSS-induced phosphorylation of p65 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) protein but increased IκBα expression. Knockdown of TRIM8 inhibits UC injury and inflammatory response caused by DSS. This could be related to the regulation of NF-κB signaling pathway by TRIM8 protein.

The effect of olsalazine of chinese generic drugs on ulcerative colitis induced by dextran sulfate sodium salt in BALB/c mice.

To explore effect and mechanism of olsalazine of Chinese generic drugs on ulcerative colitis induced by dextran sulfate sodium salt (DSS) in BALB/c mice. The mouse model of ulcerative colitis was induced by free drinking of 3% (w/v) DSS aqueous solution for seven days. The mice were treated with olsalazine (0.6 g·kg-1) of Chinese generic drugs. The therapeutic effect of olsalazine on ulcerative colitis mice was evaluated by measuring disease activity index (DAI), colonic mucosal injury index (CMDI), histopathological score (HS), and detected the expression levels of interleukin (IL)-2, IL-10, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-1β in serum and IL-7, IL-17, IL-22, epidermal growth factor (EGF), transforming growth factor β1 (TGF-β1) in colonic homogenate of mice. Olsalazine significantly increased the contents of IL-2, IL-10, IL-22, TGF and EGF in ulcerative colitis rats, and significantly decreased the scores of DAI, CMDI, HS and the contents in IL-7, IL-17, TNF-α, IL-1β and IFN-γ when compared with the model group. It improved the degree of colonic lesion in ulcerative colitis mice. It was suggested that olsalazine has a therapeutic effect on ulcerative colitis induced by DSS in mice, and the mechanism may be related to the increase of IL-2, IL-10, IL-22, TGF, and EGF and the decrease of the expression of IL-7, IL-17, TNF-α, IL-1β, and IFN-γ.

Anti-inflammatory and antioxidant potential of Artemisia dracunculus L. aqueousextract against acetic acid induced ulcerative colitis in male Wistar rats

Ulcerative colitis (UC) is one of the two types of inflammatory bowel disease (IBD) which is increasing worldover due to modern life style. Patients with UC are prone to develop colorectal cancer. While the disease severity decides the treatment option, researchers look towards herbal medicines with anti-inflammatory properties for minimal or nil side effects. Artemisia dracunculus L., commonly called Tarragon, is a medicinal herb used in traditional Asian medicine mainly in Iran, India, Pakistan and Azerbaijan due to its special compounds. In this study, we tried to elucidate the effects of aqueous extract of tarragon on acetic acid induced ulcerative colitis (UC) in rats. Male Wistar rats were grouped into four groups of eight each viz., control; experimental control (UC was induced via luminal instillation of 4% acetic acid); and UC induced + aqueous tarragon extract (100 mg/kg) or prednisolone (2 mg/kg) orally for ten consecutive days. Tissue specimens were collected after the experimental period for evaluation of caspase-3 and cyclooxygenase-2 (COX-2) expression by immunohistochemistry. Real-time PCR was used to monitor the levels of IL-1, IL-6 and TNF-α in colonic homogenates. Moreover, the levels of myeloperoxidase, nitric oxide and total antioxidant capacity were measured in colonic homogenates. The results showed that both treatment regimens could similarly reduce the severity of disease symptoms. Treatment with aqueous extract of tarragon caused a better improvement (P <0.05) in the levels of myeloperoxidase enzyme, and total antioxidant capacity of colonic homogenates compared to prednisolone. Nevertheless, the levels of the expression of caspase-3, and COX-2 and TNF-α were reduced in UC rats received prednisolone more than UC rats received aqueous extract of tarragon. The was no statistical difference in the levels of nitric oxide, IL-1 and IL-6 between UC rats received tarragon extract or prednisolone. Overall, these findings suggest that the aqueous extract of tarragon is a promising strategy to control ulcerative colitis. Aqueous extract can also be used as an anti-inflammatory and immune system stimulant in conditions where the immune system is damaged.