Response Variability to Drug Testing in Two Models of Chemically Induced Colitis.

The lack of knowledge regarding the pathogenesis of IBD is a challenge for the development of more effective and safer therapies. Although in vivo preclinical approaches are critical for drug testing, none of the existing models accurately reproduce human IBD. Factors that influence the intra-individual response to drugs have barely been described. With this in mind, our aim was to compare the anti-inflammatory efficacy of a new molecule (MTADV) to that of corticosteroids in TNBS and DSS-induced colitis mice of both sexes in order to clarify further the response mechanism involved and the variability between sexes. The drugs were administered preventively and therapeutically, and real-time bioluminescence was performed for the in vivo time-course colitis monitoring. Morphometric data were also collected, and colonic cytokines and acute plasma phase proteins were analyzed by qRT-PCR and ELISA, respectively-bioluminescence images correlated with inflammatory markers. In the TNBS model, dexamethasone worked better in females, while MTADV improved inflammation in males. In DSS-colitis, both therapies worked similarly. Based on the molecular profiles, interaction networks were constructed to pinpoint the drivers of therapeutic response that were highly dependent on the sex. In conclusion, our results suggest the importance of considering sex in IBD preclinical drug screening.

Cornus mas L. Extract Targets the Specific Molecules of the Th17/Treg Developmental Pathway in TNBS-Induced Experimental Colitis in Rats.

Given that one of the crucial events in the pathogenesis of inflammatory bowel disease is the loss of homeostasis between Th17 and Treg cells, targeting the specific molecules of the Th17/Treg axis developmental pathway is a promising strategy for inflammatory bowel disease prevention and treatment. The current study aimed to assess the impact of cornelian cherry (Cornus mas L.) extract, rich in iridoids and polyphenols known for their potential anti-inflammatory activity, at two doses (20 or 100 mg/kg) on the crucial factors for Th17/Treg cell differentiation in the course of experimental colitis and compare this action with that of sulfasalazine. This study was conducted on the biobank colon tissue samples collected during the previous original experiment, in which colitis in rats was induced by trinitrobenzenesulfonic acid (TNBS). The levels of IL-6, RORγt, total STAT3, p-STAT3, and Foxp3 were determined by ELISA. The expression of PIAS3 mRNA was quantified by qPCR. Cornelian cherry extract at a dose of 100 mg/kg counteracted the TNBS-induced elevation of IL-6, RORγt, and p-STAT3 levels and a decrease in Foxp3 level and PIAS3 mRNA expression, while given concomitantly with sulfasalazine was more effective than sulfasalazine alone in reversing the TNBS-induced changes in IL-6, RORγt, total STAT3, p-STAT3, Foxp3 levels, and PIAS3 mRNA expression. The beneficial effect of cornelian cherry extract on experimental colitis may be due to its immunomodulatory activity reflected by the influence on factors regulating the Th17/Treg axis.

Effect of Aqueous Extract of Phenolic Compounds Obtained from Red Wine in Experimental Model of Colitis in Mice.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder represented by Crohn's disease and ulcerative colitis. Currently, there is no cure and pharmacological treatment aims to induce and maintain remission on patients. Because the therapy reveals a relatively high toxicity, during a long-term utilization, it is essential to investigate new pharmacological approaches. Polyphenols, commonly present on red wine, have shown health-beneficial effects related to their antioxidant and anti-inflammatory effects through the inhibition of NF-kB activation, COX-2 and iNOS induction. In this sense, it would be interesting to study their effects in an IBD context. Therefore, this study aims to evaluate the effects of an aqueous extract of phenolic compounds in a 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced model of colitis. METHOD: Experimental colitis was induced in mice through an intrarectal administration of TNBS and then the mice were treated with an aqueous extract of phenolic compounds intraperitoneally for four days. RESULTS AND DISCUSSION: The extract demonstrated an anti-inflammatory effect, reducing TNF-α levels in the colon, and had a beneficial effect on the extraintestinal manifestations related to IBD, without any significant side effects. The extract of phenolic compounds demonstrated to be a valuable object of study for the management of IBD in the future.

Inhibition of TNBS-induced intestinal inflammation in crucian carp (Carassius carassius) by oral administration of bioactive Bioactive food derived peptides.

Intestinal enteritis is a main issue in crucian carp production which results in massive economic loss. Traditional antibiotics used for disease prevention of crucian carp (Carassius carassius) have been banned, thus an alternative approach needs to be identified. In this study, the bioactive peptide was evaluated as a diet supplement for preventing intestinal inflammation in crucian carp. Intestinal inflammation was induced by intrarectal administration of a 2,4,6-trinitrobenzene sulfonic acid (TNBS) solution. The fish samples were fed with different diets for 14 days. The disease activity index (DAI), which included, fish swimming, food intake, anal inflammation, body surface, and ascites was determined daily. Intestine segments were stained with haematoxylin and eosin (H.E.) for histopathological analysis. The expression of cytokines, including interleukin-1ß (IL-1ß), interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and myeloperoxidase (MPO) in crucian carp were determined. In TNBS-induced groups, the DAI scores were dramatically increased compared to the control group. The histopathological analysis showed that the damage of the fish intestine after the injection of TNBS. The relative expression levels of pro-inflammation cytokines (TNF-α, IL-1ß, IL-8, MPO) were significantly increased compared to the control group on day 1. In the TNBS-induced group feed with a diet supplemented with bioactive peptide, the symptoms of intestinal inflammation were relieved on day 3 and the mRNA expression levels of pro-inflammation cytokines (TNF-α, IL-1ß, IL-8, MPO) were reduced compared to day 1. On day 7, the fish samples enrofloxacin group and bioactive peptide group were recovered from TNBS-induced intestinal inflammation. This study showed that the fish diet supplemented with bioactive peptide could help to prevent and recover from intestinal inflammation. Thus, the bioactive peptide can be used as a replacement for antibiotics to prevent disease in aquaculture production.

Protective effect of naringin ameliorates TNBS-induced colitis in rats via improving antioxidant status and pro-inflammatory cytokines.

Aim: Ulcerative colitis (UC) is a chronic inflammatory bowel disease that disturbs the colon mucosal lining and is characterized by oxido-nitrosative stress and the release of pro-inflammatory cytokines. Naringin (NG) belongs to a group of chemicals called bioflavonoids derived from grapefruit and related citrus species. NG has been widely used as folk medicine in many countries, due to its several health benefits.Method: This study examined the effect of NG on 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. Forty-two male Wistar rats were divided into seven groups like Normal Control (NC), Ethanol Control (EC), Disease Control (DC), NG 20 (20 mg/kg, p.o.), NG 40 (40 mg/kg, p.o.), NG 80 (80 mg/kg, p.o.), and Dexamethasone (DEX) (2 mg/kg, p.o.). Colitis was induced in Wistar albino rats by administering TNBS intra-rectally (in 50% ethanol). The rats were then given 14 days of NG (20, 40, and 80 mg/kg) and DEX (2 mg/kg) treatment. Several behavioral, biochemical, molecular, and histological analyses were performed.Result: The treatment of rats with NG significantly increased the body weight (p < .05, p < .01), hematological parameters like hemoglobin (p < .05, p < .01, p < .001), red blood cells (p < .01, p < .001), and platelets count (p < .01, p < .001) and decreased in spleen weight (p < .01, p < .001), colon weight (p < .01, p < .001), colon weight to length ratio (p < .05, p < .01, p < .001), macroscopic score (p < .01, p < .001), adhesion score (p < .01, p < .001), diarrhea score (p < .05, p < .001), stool consistency (p < .01, p < .001), rectal bleeding score (p < .05, p < .01, p < .001), white blood cells count (p < .01, p < .001). NG significantly (p < .01, p < .001) increased colonic superoxide, glutathione, and catalase levels and decreased malondialdehyde and myeloperoxidase levels. It also significantly (p < .01, p < .001) decreased the biochemical parameters, proinflammatory cytokines and reduced the histological damage in the colon tissue caused by TNBS.Conclusion: Our results demonstrated that NG treatment attenuated pathologic changes of TNBS-induced colitis in rats through restoring colonic damage and reducing inflammatory response in the colon tissue. Thus, NG might be considered as an effective candidate for the treatment of UC patients.

Anti-Inflammatory Effect of Topiramate in a Chronic Model of TNBS-Induced Colitis.

Inflammatory bowel disease (IBD) is characterized by a chronic and relapsing inflammatory response in the gastrointestinal tract, resulting in severe symptoms such as abdominal pain, vomiting, diarrhea, bloody stools, and weight loss. Currently, there is no cure, and the pharmacological treatment includes drugs that induce and keep the patient in remission, not reversing the underlying pathogenic mechanism. These therapies, in the long term, may cause various side effects and complications, which has increased the need to investigate new, more effective, and safer pharmacological approaches. In preclinical studies, topiramate has demonstrated a potential anti-inflammatory effect by inhibiting the production of several pro-inflammatory cytokines. This study aimed to investigate the effect of topiramate in a chronic TNBS-induced colitis model in rodents. Experimental colitis was induced by four intrarectal administrations of 1% TNBS in female CD-1 mice. Topiramate 10 and 20 mg were administered intraperitoneally for 14 days. Several parameters were evaluated, such as bodyweight, alkaline phosphatase (ALP), fecal hemoglobin, fecal calprotectin, tumor necrosis factor (TNF)-α, and interleukin (IL)-10. Topiramate reduces TNBS-induced colonic damage in a model of chronic experimental colitis and normalizes the stool consistency and anus appearance. Additionally, topiramate significantly reduced the concentration of ALP, fecal hemoglobin, fecal calprotectin, TNF-α, and IL-10, demonstrating it to be a promising pharmacological approach for the treatment of IBD in the future.

Characteristics of an In Vitro Mesenteric Lymph Node Cell Suspension Model and Its Possible Association with In Vivo Functional Evaluation.

In a previous study, we uncovered three immune-responsive patterns of gut microbes using an in vitro mesenteric lymph node cell suspension model, abbreviated as the MLN model hereafter. We used Akkermansia muciniphila and Clostridium butyricum as the first group directly inducing an immune response, Bifidobacterium sp. and Bacteroides sp. as the second group evoking an immune response with the help of stimuli (anti-CD3 and anti-CD28 antibodies), and Lactobacillus sp. as the third group blunting the immune response with or without stimuli. Our group previously clarified the immune-activation characteristics of A. muciniphila and linked its in vivo immune induction effect in GF and SPF mice under homeostasis. In the present study, we supplemented the characteristics of C. butyricum and B. bifidum in the in vitro MLN model and addressed the specific elements of the model. Finally, we used an in vivo TNBS-challenge model to show the functional differences between these species with different response patterns in vitro. The results showed that C. butyricum and B. bifidum evoked an immune response in vitro in a dose-dependent and strain-unique manner. Although TLR2, rather than TLR4, is indispensable for immune activation in the present in vitro model, it may not involve interaction between TLR2 and bacterial ligands. Like the PBMC model, the present in vitro MLN model is highly dependent on cell resources and should be given more attention when used to conduct a quantitative comparison. Finally, a mixture of two strong immunogenic strains, A. muciniphila and C. butyricum, significantly increased the mortality of TNBS-challenged (2,4,6-trinitrobenzene sulfonic acid, TNBS) mice, indicating a possible link between the in vitro MLN model and in vivo functional evaluation. However, more evidence is needed to clarify the associations and underlying mechanisms.

Chronic Experimental Model of TNBS-Induced Colitis to Study Inflammatory Bowel Disease.

Background: Inflammatory bowel disease (IBD) is a world healthcare problem. In order to evaluate the effect of new pharmacological approaches for IBD, we aim to develop and validate chronic trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. Methods: Experimental colitis was induced by the rectal administration of multiple doses of TNBS in female CD-1 mice. The protocol was performed with six experimental groups, depending on the TNBS administration frequency, and two control groups (sham and ethanol groups). Results: The survival rate was 73.3% in the first three weeks and, from week 4 until the end of the experimental protocol, the mice's survival remained unaltered at 70.9%. Fecal hemoglobin presented a progressive increase until week 4 (5.8 ± 0.3 µmol Hg/g feces, p < 0.0001) compared with the ethanol group, with no statistical differences to week 6. The highest level of tumor necrosis factor-α was observed on week 3; however, after week 4, a slight decrease in tumor necrosis factor-α concentration was verified, and the level was maintained until week 6 (71.3 ± 3.3 pg/mL and 72.7 ± 3.6 pg/mL, respectively). Conclusions: These findings allowed the verification of a stable pattern of clinical and inflammation signs after week 4, suggesting that the chronic model of TNBS-induced colitis develops in 4 weeks.

In Silico Study and Effects of BDMC33 on TNBS-Induced BMP Gene Expressions in Zebrafish Gut Inflammation-Associated Arthritis.

The bone morphogenic protein (BMP) family is a member of the TGF-beta superfamily and plays a crucial role during the onset of gut inflammation and arthritis diseases. Recent studies have reported a connection with the gut-joint axis; however, the genetic players are still less explored. Meanwhile, BDMC33 is a newly synthesized anti-inflammatory drug candidate. Therefore, in our present study, we analysed the genome-wide features of the BMP family as well as the role of BMP members in gut-associated arthritis in an inflammatory state and the ability of BDMC33 to attenuate this inflammation. Firstly, genome-wide analyses were performed on the BMP family in the zebrafish genome, employing several in silico techniques. Afterwards, the effects of curcumin analogues on BMP gene expression in zebrafish larvae induced with TNBS (0.78 mg/mL) were determined using real time-qPCR. A total of 38 identified BMP proteins were revealed to be clustered in five major clades and contain TGF beta and TGF beta pro peptide domains. Furthermore, BDMC33 suppressed the expression of four selected BMP genes in the TNBS-induced larvae, where the highest gene suppression was in the BMP2a gene (an eight-fold decrement), followed by BMP7b (four-fold decrement), BMP4 (four-fold decrement), and BMP6 (three-fold decrement). Therefore, this study reveals the role of BMPs in gut-associated arthritis and proves the ability of BDMC33 to act as a potential anti-inflammatory drug for suppressing TNBS-induced BMP genes in zebrafish larvae.

Inhibitor of apoptosis-stimulating p53 protein protects against inflammatory bowel disease in mice models by inhibiting the nuclear factor kappa B signaling.

Drugs and therapies available for the treatment of inflammatory bowel disease (IBD) are not satisfactory. Our previous study has established the inhibitor of apoptosis-stimulating p53 protein (iASPP) as an oncogenic regulator in colorectal cancer by forming a regulatory axis or feedback loop with miR-124, p53, or p63. As iASPP could target and inhibit nuclear factor kappa B (NF-κB) activation, in this study the role and mechanism of iASPP in IBD was investigated. The aberrant up-regulation of iASPP in IBD was subsequently confirmed, based on online data sets, clinical sample examinations and 2,4,6-trinitrobenzene sulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced colitis mice models. TNBS or DSS stimulation successfully induced colon shortness, body weight loss, mice colon oxidative stress and inflammation. In both types of colitis mice models, iASPP over-expression improved, whereas iASPP knockdown aggravated TNBS or DSS stimulation-caused colon shortness, body weight loss and mice colon oxidative stress and inflammation. Meanwhile, in both types of colitis mice models, iASPP over-expression inhibited p65 phosphorylation and decreased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, C-X-C motif chemokine ligand (CXCL)1 and CXCL2 in mice colons, whereas iASPP knockdown exerted opposite effects.

Rectal administration of carbon monoxide inhibits the development of intestinal inflammation and promotes intestinal wound healing via the activation of the Rho-kinase pathway in rats.

The inhalation of carbon monoxide (CO) gas and the administration of CO-releasing molecules were shown to inhibit the development of intestinal inflammation in a murine colitis model. However, it remains unclear whether CO promotes intestinal wound healing. Herein, we aimed to evaluate the therapeutic effects of the topical application of CO-saturated saline enemas on intestinal inflammation and elucidate the underlying mechanism. Acute colitis was induced with trinitrobenzene sulfonic acid (TNBS) in male Wistar rats. A CO-saturated solution was prepared via bubbling 50% CO gas into saline and was rectally administrated twice a day after colitis induction; rats were sacrificed 3 or 7 days after induction for the study of the acute or healing phases, respectively. The distal colon was isolated, and ulcerated lesions were measured. In vitro wound healing assays were also employed to determine the mechanism underlying rat intestinal epithelial cell restitution after CO treatment. CO solution rectal administration ameliorated acute TNBS-induced colonic ulceration and accelerated ulcer healing without elevating serum CO levels. The increase in thiobarbituric acid-reactive substances and myeloperoxidase activity after induction of acute TNBS colitis was also significantly inhibited after CO treatment. Moreover, the wound healing assays revealed that the CO-saturated medium enhanced rat intestinal epithelial cell migration via the activation of Rho-kinase. In addition, the activation of Rho-kinase in response to CO treatment was confirmed in the inflamed colonic tissue. Therefore, the rectal administration of a CO-saturated solution protects the intestinal mucosa from inflammation and accelerates colonic ulcer healing through enhanced epithelial cell restitution. CO may thus represent a novel therapeutic agent for the treatment of inflammatory bowel disease.

Synthesis and activity of N-(5-hydroxy-3,4,6-trimethylpyridin-2-yl)acetamide analogues as anticolitis agents via dual inhibition of TNF-α- and IL-6-induced cell adhesions.

Tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) are the critical pro-inflammatory cytokines involved in the pathogenesis of inflammatory bowel disease (IBD). Inhibition of these cytokines and related signaling pathways has been a target for the development of IBD therapeutics. In the current study, 6-acetamido-2,4,5-trimethylpyridin-3-ol (1) and various analogues with the amido scaffold were synthesized and examined for their inhibitory activities in in vitro and in vivo IBD models. The parent compound 1 (1 µM) showed an inhibitory activity against TNF-α- and IL-6-induced adhesion of monocytes to colon epithelial cells, which was similar to tofacitinib (1 µM), a JAK inhibitor, but much better than mesalazine (1,000 µM). All the analogues showed a positive relationship (R2 = 0.8943 in a linear regression model) between the inhibitory activities against TNF-α-induced and those against IL-6-induced adhesion. Compound 2-19 turned out to be the best analogue and showed much better inhibitory activity against TNF-α- and IL-6-induced adhesion of the cells than tofacitinib. In addition, oral administration of compound 1 and 2-19 resulted in a significant suppression of clinical signs of TNBS-induced rat colitis, including weight loss, colon tissue edema, and myeloperoxidase activity, a marker for inflammatory cell infiltration in colon tissues. More importantly, compound 2-19 (1 mg/kg) was more efficacious in ameliorating colitis than compound 1 and sulfasalazine (300 mg/kg), the commonly prescribed oral IBD drug. Taken together, the results suggest that compound 2-19 can be a novel platform for dual-acting IBD drug discovery targeting both TNF-α and IL-6 signaling.

Cinnamaldehyde and hesperetin attenuate TNBS-induced ulcerative colitis in rats through modulation of the JAk2/STAT3/SOCS3 pathway.

Ulcerative colitis is an autoimmune inflammatory disorder with a negative impact on the life quality of patients. Cinnamaldehyde and hesperetin were chosen due to their antioxidants and anti-inflammatory effects. This study explored the protective effects of cinnamaldehyde (40 and 90 mg/kg, po) and hesperetin (50 and 100 mg/kg, po) on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced ulcerative colitis in rats. Cinnamaldehyde and hesperetin significantly improved macroscopic and histopathological examinations with a significant reduction in myeloperoxidase and intracellular adhesion molecule-1 expression. They significantly reduced colon oxidative stress by a significant elevation in both reduced glutathione content and superoxide dismutase activity with a significant reduction of NO content. Furthermore, cinnamaldehyde and hesperetin alleviated the inflammatory injury by a significant reduction in interleukin-6 along with suppression of nuclear factor-κB, receptor for advanced glycation end products, and tumor necrosis factor-α expression. Moreover, cinnamaldehyde and hesperetin significantly decreased p-JAK2 and p-STAT3 while significantly increased suppressors of cytokine signaling 3 (SOCS3) protein expression. In conclusion, cinnamaldehyde and hesperetin counteracted TNBS-induced ulcerative colitis through antioxidant, anti-inflammatory properties as well as modulation of the JAk2/STAT3/SOCS3 pathway.

Betulinic acid hydroxamate prevents colonic inflammation and fibrosis in murine models of inflammatory bowel disease.

Intestinal fibrosis is a common complication of inflammatory bowel disease (IBD) and is defined as an excessive accumulation of scar tissue in the intestinal wall. Intestinal fibrosis occurs in both forms of IBD: ulcerative colitis and Crohn's disease. Small-molecule inhibitors targeting hypoxia-inducing factor (HIF) prolyl-hydroxylases are promising for the development of novel antifibrotic therapies in IBD. Herein, we evaluated the therapeutic efficacy of hydroxamate of betulinic acid (BHA), a hypoxia mimetic derivative of betulinic acid, against IBD in vitro and in vivo. We showed that BAH (5-20 µM) dose-dependently enhanced collagen gel contraction and activated the HIF pathway in NIH-3T3 fibroblasts; BAH treatment also prevented the loss of trans-epithelial electrical resistance induced by proinflammatory cytokines in Caco-2 cells. In two different murine models (TNBS- and DSS-induced IBD) that cause colon fibrosis, oral administration of BAH (20, 50 mg/kg·d, for 17 days) prevented colon inflammation and fibrosis, as detected using immunohistochemistry and qPCR assays. BAH-treated animals showed a significant reduction of fibrotic markers (Tnc, Col1a2, Col3a1, Timp-1, α-SMA) and inflammatory markers (F4/80+, CD3+, Il-1ß, Ccl3) in colon tissue, as well as an improvement in epithelial barrier integrity and wound healing. BHA displayed promising oral bioavailability, no significant activity against a panel of 68 potential pharmacological targets and was devoid of genotoxicity and cardiotoxicity. Taken together, our results provide evidence that oral administration of BAH can alleviate colon inflammation and colitis-associated fibrosis, identifying the enhancement of colon barrier integrity as a possible mechanism of action, and providing a solid rationale for additional clinical studies.

Ginsenoside Rb1 alleviates colitis in mice via activation of endoplasmic reticulum-resident E3 ubiquitin ligase Hrd1 signaling pathway.

Endoplasmic reticulum (ER) homeostasis is regulated by ER-resident E3 ubiquitin ligase Hrd1, which has been implicated in inflammatory bowel disease (IBD). Ginsenoside Rb1 (GRb1) is the major ginsenoside in ginseng with multiple pharmacological activities. In this study we investigated the role of Hrd1 in IBD and its regulation by GRb1. Two mouse colitis models were established to mimic human IBD: drinking water containing dextran sodium sulfate (DSS) as well as intra-colonic infusion of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS). Colitis mice were treated with GRb1 (20, 40 mg·kg-1·d-1, ig) or a positive control drug sulfasalazine (500 mg·kg-1·d-1, ig) for 7 days. The model mice showed typical colitis symptoms and pathological changes in colon tissue. In addition to significant inflammatory responses and cell apoptosis in colon tissue, colon epithelial expression of Hrd1 was significantly decreased, the expression of ER stress markers GRP78, PERK, CHOP, and caspase 12 was increased, and the expression of Fas was increased (Fas was removed by Hrd1-induced ubiquitination). These changes were partially, or completely, reversed by GRb1 administration, whereas injection of Hrd1 inhibitor LS102 (50 mg·kg-1· d-1, ip, for 6 days) exacerbated colitis symptoms in colitis mice. GRb1 administration not only normalized Hrd1 expression at both the mRNA and protein levels, but also alleviated the ER stress response, Fas-related apoptosis, and other colitis symptoms. In intestinal cell line IEC-6, the expression of Hrd1 was significantly decreased by LPS treatment, but was normalized by GRb1 (200 µM). GRb1 alleviated LPS-induced ER stress and cell apoptosis in IEC-6 cells, and GRb1 action was inhibited by knockdown of Hrd1 using small interfering RNA. In summary, these results reveal a pathological role of Hrd1 in colitis, and provide a novel insight into alternative treatment of colitis using GRb1 activating Hrd1 signaling pathway.

Vitamin D Deficiency Exacerbates Colonic Inflammation Due to Activation of the Local Renin-Angiotensin System in the Colon.

BACKGROUND: The renin-angiotensin system (RAS) is activated in inflammatory bowel disease (IBD), and vitamin D deficiency aggravates the development of colitis, but the relationship between the local colonic RAS and vitamin D is unclear with regard to the pathogenesis of IBD. AIMS: To investigate whether vitamin D suppresses the local colonic RAS to prevent colonic mucosal inflammation in a mouse model of experimental colitis. METHODS: C57BL/6 mice fed vitamin D-deficient (VDD) diet for 8 weeks were induced to colitis by 2,4,6-trinitrobenzenesulfonic acid (TNBS), with mice fed vitamin D-sufficient (VDS) diet as controls. Colitis severity was assessed by histology, and pro-inflammatory cytokines, RAS components, and signaling pathways were quantified by real-time RT-PCR and Western blotting. RESULTS: C57BL/6 mice fed the VDD diet for 8 weeks exhibited significantly lower serum 25(OH)D3 concentrations compared to mice fed the VDS diet. When these VDD mice were induced to colitis by TNBS, they exhibited more severe colonic inflammation and developed more severe colitis compared to the VDS counterparts. VDD diet feeding resulted in higher production of mucosal pro-inflammatory cytokines, higher activation of the myosin light chain kinase-tight junction regulatory pathway, and greater increases in mucosal permeability. VDD diet feeding also enhanced colonic RAS activation. Treatment with angiotensin II receptor blocker losartan markedly alleviated colitis in TNBS-induced VDD mice. CONCLUSION: Vitamin D deficiency promotes colonic inflammation at least in part due to over activation of the local RAS in the colon.

Hecogenin and fluticasone combination attenuates TNBS-induced ulcerative colitis in rats via downregulation of pro-inflammatory mediators and oxidative stress.

OBJECTIVE: Ulcerative colitis is common types of severe, progressive, idiopathic inflammatory bowel disease that involves the mucosal lining of the large intestine. The purpose of the study is to explore the effects of hecogenin in TNBS (2, 4, 6- trinitrobenzene sulfonic acid) induced ulcerative colitis model in rats. MATERIAL AND METHODS: Thirty Wistar rats were randomized into five groups: (i) Normal Control (NC), (ii) Disease Control (DC), (iii) Hecogenin (HG) (50 µg/rat), (iv) Fluticasone (FC) (50 µg/rat), (v) Hecogenin + Fluticasone (HG + FC) combination (25 µg/rat). Colitis was induced by trans-rectal administration of TNBS using a catheter inserted 8 cm into the rectal portion of the rat. Colitis was evaluated by an independent observer who was blinded to the treatment. All treatment group results were compared to the TNBS group results. RESULTS: The study results revealed that treatment of rats with HG and HG + FC significantly improved the body weight and colon length whereas; decreased the spleen weight, colon weight/length ratio, macroscopic lesions score, diarrhea score and adhesion score. The drug treatment in rats substantially decreased the development of inflammatory cytokines, levels of serum immunoglobulin E, colonic nitric oxide contents and restoration of antioxidant stress markers. Histopathological colon sample study significantly reduced colonic inflammation with a substantial decrease in inflammation score. CONCLUSION: Thus, HG and HG + FC combination could change the pathogenesis of the disease and may be a potential therapeutic target for the treatment of ulcerative colitis by a reduction in dose in conjunction with FC to prevent the persistent adverse effects associated with FC.

Interaction between the Renin-Angiotensin System and Enteric Neurotransmission Contributes to Colonic Dysmotility in the TNBS-Induced Model of Colitis.

Angiotensin II (Ang II) regulates colon contraction, acting not only directly on smooth muscle but also indirectly, interfering with myenteric neuromodulation mediated by the activation of AT1 /AT2 receptors. In this article, we aimed to explore which mediators and cells were involved in Ang II-mediated colonic contraction in the TNBS-induced rat model of colitis. The contractile responses to Ang II were evaluated in distinct regions of the colon of control animals or animals with colitis in the absence and presence of different antagonists/inhibitors. Endogenous levels of Ang II in the colon were assessed by ELISA and the number of AT1/AT2 receptors by qPCR. Ang II caused AT1 receptor-mediated colonic contraction that was markedly decreased along the colons of TNBS-induced rats, consistent with reduced AT1 mRNA expression. However, the effect mediated by Ang II is much more intricate, involving (in addition to smooth muscle cells and nerve terminals) ICC and EGC, which communicate by releasing ACh and NO in a complex mechanism that changes colitis, unveiling new therapeutic targets.

Euterpe oleracea Mart. (Açaí) attenuates experimental colitis in rats: involvement of TLR4/COX-2/NF-ĸB.

Euterpe oleracea Mart., commonly known as açaí, has been demonstrated to exhibit significantly antioxidant and inflammatory activities in experimental models. These effects of the hydroalcoholic extract from the açaí seed (ASE) were investigated in TNBS-induced (2,4,6-trinitrobenzenesulfonic acid) acute colitis model in rats. Wistar rats (180-220 g) were orally pretreated with saline (0.3 mL), ASE (10, 30 and 100 mg/kg) and dexamethasone (control group, 1 mg/kg) once daily for 3 days starting before TNBS instillation. On day 3 after TNBS, the animals were euthanized, the portion of distal colon was collected and washed with 0.9% saline for macroscopy and histological evaluation, glutathione (GSH) and malonyldialdehyde (MDA) levels, myeloperoxidase (MPO) and catalase (CAT) activity, nitrate and nitrite (NO3/NO2) concentration, pro-inflammatory cytokines levels and intestinal barrier integrity. We also evaluated Toll-like Receptor 4/cyclooxygenase-2/nuclear factor kappa B expression as a possible mechanism related to the ASE effects. Treatment with ASE 100 mg/kg decreased significantly macroscopic and microscopic damage induced by TNBS. In addition, MPO activity, TNF-α (tumor necrosis factor-alpha) and IL-1ß (interleukin 1) levels were reduced in rats with colitis. ASE 100 mg/kg restored GSH and MDA levels, CAT activity, NO3/NO2 concentration and improved the intestinal barrier integrity in the TNBS group. ASE 100 mg/kg significantly reduced TNBS-induced expression of the TLR4, COX-2 and NF-κB p65. ASE 100 mg/kg improved macroscopy and histological parameters, inflammation, intestinal barrier integrity and nitric and oxidative stress through the TLR-4/COX-2/NF-κB pathway.

Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane.

Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol-auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that <20% of DHE fluorescence was quenched when the DHE-containing cells were exposed to membrane-impermeant collisional quenchers (spin-labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass-bead lysis or repeated freeze-thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer.