Gadolinium Chloride Improves The Course Of TNBS And Dss-induced Colitis Through Protecting Against Colonic Mucosal Inflammation

BackgroundInflammatory bowel disease (IBD) comprises Crohn’s disease and ulcerative colitis that cause chronic and remittent-relapsing intestinal inflammation of all or part of the intestinal tract. Although the aetiology of IBD remains unclear, it is thought to result from dysregulation of the mucosal immune responses to intestinal bacterial antigens in genetically predisposed individuals. Currently, treatment options for IBD are mainly focused on suppressing mucosal immune responses, including the use of5-aminosalicylic acid (5-ASA) agents, steroids, antimicrobials, and some immunomodulators. However, although they are reasonably successful in many patients, still a great number cannot achieve remission, highlighting the need for novel therapeutic targets.Infiltration and activation of macrophages in colon are central features of IBD, and inflammatory macrophages in mucosa are thought to play an essential role in the pathogenesis of IBD. In IBD and experimental colitis, monocytes in blood are recruited to the mucosa and differentiate into activated macrophages that produce pro-inflammatory cytokines, such as tumor necrosis factor a (TNFa), interleukin (IL)-1and IL-6. Activation of NF-κB is thought as a strong inducer of these proinflammatory cytokine expressions, and activated NF-κB has been demonstrated in colonic macrophages of patients with IBD. Alteration in cytokine production by macrophages is one major component of the pathology of IBD. Therefore, strategies for targeting inflammatory mucosal macrophages may be important for developing new therapeutics. However, studies on intervention with mucosal macrophages in colon are really limited, which might mainly due to the limited intervention methods.Gadolinium chloride (GdCl3), a rare earth metal, is widely used experimentally. The role of GdCl3in macrophage elimination has been widely proven in liver of experimental animals, indicating a preventive or therapeutic effect in liver injury. GdCl3is also employed as a macrophage selective inhibitor in vivo. It has been reported that GdCl3has no depletion effect in tissue macrophages in lung, but it decreases the expression of TNFa and IL-6after LPS stimulation in rat. However, the effect of GdCl3on mucosal macrophages in colon remains largely unknown.Objectives1. To study the effect of GdCl3on mucosal inflammation and disease activity in colitis mice.2. To investigate the potential mechanisms of GdCl3in improving mucosalinflammation in colitis mice.3. To study the inhibiotory role of GdCl3in RAW264.7cells in vitro.Methods1. Animal treatmentMale C57BL/6J mice weighing20-25g (aged8-10weeks) were used in this study. GdCl3was administrated to mice via the tail vein. The PBS vehicle (0.1mL) was administrated as a control. Mice were sacrificed for detection of the amount of mucosal macrophages in colon after GdCl3treatment for1day,2days and3days.2. Colitis induction and design of treatmentThe TNBS colitis was induced by rectal administration of TNBS (2mg in50%ethanol,0.1mL in total) via a polyethylene catheter inserted2to3cm from the anus. An equivalent volume of PBS was instilled into control mice. The DSS colitis was induced by an intake of3%(w/v) DSS (40,000MW) dissolved in drinking water for7consecutive days. Control mice received only drinking water. GdCl3(10mg/kg body weight) was given to mice through intravenous route or intrarectal route after colitis induction. Control mice received equivalent administration of PBS. 3. Evaluation of colitis progressionBody weights were recorded daily. Severity of colitis was assessed by the disease activity index (DAI). Mice were killed on day7or day14with colons removed. Colons were measured and cut into sections. Histopathological studies were performed, and histology was scored microscopically in a blinded fashion.4. Flow cytometric analysis of mucosal macrophages in colonColonic mucosal cells were harvested. The cells were incubated with the antibodies, and then were detected using a flow cytometer. The following antibody clones were used:F4/80-FITC, and CD3-FITC.5. Measurement of cytokine levels in serum and colonic mucosaAfter blood collection, serum was collected. Colonic mucosa protein was extracted. Levels of TNFα, IL-1β and IL-6in serum and colonic mucosa were measured by ELISA kits.6. Cell culture and treatmentRAW264.7cells were seeded onto the96-well plate with5.0×103cells per well. After12hours, cells were treated with gadolinium chloride in various concentrations (0-200μM) for required incubation times (24hours,48hours and72hours).7. Cell viabilities and apoptosis of RAW264.7cells with GdCl3treatmentAt the end of incubation of GdCl3in RAW264.7cells,3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazo-lium bromide (MTT) in PBS was added. Then the supernatant was removed, and DMSO was added to dissolve the formazan. Absorbance was measured at490nm on a microplate reader. The viabilities of treated cells were expressed as the percentage of control cells, which was assumed to be100%.After treatment with GdCl3for24hours, RAW264.7cell pellets were obtained. The apoptotic rate was analyzed by flow cytometry.8. LPS treatment and cytokine analysisCells (3.0×104) in500μL medium were added to24-well plates. After12hours, LPS at a concentration of100ng/mL with or without GdCl3(100μM) was added to the wells. The supernatants were collected24hours,48hours and72hours after stimulation. The production levels of TNFα, IL-1β and IL-6in the supernatant were detected by ELISA.9. Western blot analysisTotal protein was extracted from mucosal samples of mice and from RAW264.7cell lysates in RIPA buffer. Protein was quantified by using a BCA protein quantification kit. Protein was separated by SDS-PAGE, and was transferred to PVDF membrane (0.22μm pore).The membrane was incubated with primary antibodies (anti-NF-κB p65monoclonal antibody) at4℃overnight. Horseradish peroxidase-conjugated secondary antibodies were probed the next day. Densitometry of protein bands was quantified by use of Quantity One4.6.2.Results1. GdCl3has no elimination effect on mucosal macrophages in colon in miceIt was revealed that intravenous treatment of GdCl3could not deplete macrophages in colonic mucosa (p>0.05). In serum and colonic mucosa tissue, the expression profiles of TNFα, IL-1β and IL-6were not obviously elevated after GdCl3treatment (p>0.05for all three cytokine types).2. GdCl3is protective against colitis induced by TNBS and DSSTo evaluate the potential role of GdCl3in colitis in vivo, we used two murine models of colitis induced by TNBS and DSS. In TNBS colitis, GdCl3was given to mice on day3of TNBS application intravenously. The mortality of TNBS colitis mice was improved by about20%after GdCl3treatment. Treatment with GdCl3resulted in prominent protection from colitis as assessed by body weight, disease activity index (DAI) scores, colon length and histopathological damage of the colon. Control mice presented weight loss after TNBS administration, whilst colitis mice treated with GdCl3showed markedly less body weight loss. GdCl3significantly improved DAI scores and shortening of colon length in TNBS colitis. Histopathological analysis of the colon, examined on day7and14, showed marked crypt architecture damage, inflammatory cell infiltration and ulceration in colitis mice. GdCl3treatment significantly improved these damages of colon as assessed by the histopathological scores. Intrarectal treatment of GdCl3began on day3of TNBS application and continued until day7. Colitis was also significantly ameliorated after GdCl3administration as assessed by changes of body weight, DAI scores, and mucosal damage, which were similar to mice treated with GdCl3intravenously, and there was no significant difference between mice treated with GdCl3through intravenous route and intrarectal route.Mice were exposed to3%DSS in drinking water for7days in DSS colitis. GdCl3was administrated to mice through either intravenous or intrarectal route similar to the treatment in TNBS colitis mice. The results revealed that GdCl3treatment also resulted in striking protection from DSS-induced colitis.3. GdCl3suppresses TNBS-and DSS-induced proinflammatory cytokine secretionsIn TNBS colitis mice treated with GdCl3intravenously or intrarectally, the proportion of macrophages in colonic mucosa was not altered. GdCl3treatment significantly reduced TNFa, IL-1β and IL-6levels in serum and colonic mucosa as compared to those of vehicle control mice. Similar results were observed in DSS colitis mice.4. GdCl3reduces proinflammatory cytokine production by LPS-stimulated RAW264.7cellsThe anti-inflammatory activity of GdCl3was also confirmed in LPS-stimulated RAW264.7cells in vitro. GdCl3did not exert a toxicity in RAW264.7cells. Flow-cytometric analysis also showed a lack of pro-apoptotic effect of GdCl3on the cell. In RAW264.7cells, TNFa, IL-1β and IL-6production was markedly increased in the culture supernatant after LPS stimulation, whereas the levels of these proinflammatory cytokines were significantly reduced by GdCl3.5. GdCl3suppresses NF-κB activation in mucosa in colitis mice and also inLPS-stimulated RAW264.7cellsIn TNBS colitis mice, the NF-κB p65expression in colonic mucosa was significantly attenuated with GdCl3treatment through either intravenous or intrarectal route. Similar results were observed in DSS colitis mice. Also, in vitro studies showed that GdCl3markedly reduced NF-κB p65expression in LPS-stimulated RAW264.7cells. Therefore, GdCl3may down-regulate the secretion of proinflammatory cytokines by macrophages through suppression of NF-κB activation.ConclusionGdCl3might down-regulate the production of proinflammatory cytokines by macrophages through inhibition of the NF-κB signaling pathway. Therefore, intervention with mucosal inflammatory macrophages may be a promising therapeutic target in IBD.