| ObjectiveInflammatory bowel disease(IBD)is a chronic and nonspecific inflammatory disease of which the etiology is still not clear,including Crohn`s disease(CD)and ulcerative colitis(UC).Difficult to treat,easy to recurrent and malignant potential are the typical features of IBD which bring the patients long-term and relapsing pain.In recent years,the incidence of IBD is rising worldwide.Despite current medical treatments which focus primarily on immunosuppression,a lot of patients still require surgical treatment.Recent studies have found that silibinin,a plant extract,has antiinflammatory and anti-cancer effect but the effects silibinin exerts on IBD still remain unclear.NLRP3 inflammasome is a multiprotein complex composed of NLRP3,apoptosis associated speck-like protein containing CARD(ASC)and caspase-1,with a molecular weight >700 k D.NLRP3 inflammasome can be activated by multiple stimuli including microbial RNA,ATP,particulate matter and bacterial pore-forming toxins.Activated NLRP3 inflammasome plays an important role in orchestrating innate immune responses to infection through activation of caspase-1 and maturation of inflammatory cytokines pro-interleukin-1β(pro-IL-1β)and pro-IL-18.In the present study,we aim to investigate the effect of silibinin in regulating NLRP3 inflammasome and attenuating DSS-induced colitis and provide experimental basis for the treatment and prevention of IBD in the future.MethodsMouse model of DSS-induced colitis was used in the present study.A total of 48 SPF C57BL/6 mouse(8 weeks old,18-20g)were randomly divided into three groups: 8 in the control group(normal drinking water),20 in DSS group and 20 in silibinin treated group.For the first 7 days,both DSS and silibinin treated group were given ordinary drinking water.Then DSS group was administered 2% DSS in drinking water(ad libitum)for the next 7 days with 0.2ml/mouse/day vehicle control(0.5% carboxymethyl cellulose in distilled water)by gavage(i.g.)and silibinin treated group was administered 2% DSS in drinking water(ad libitum)with silibinin(750mg/kg/day i.g.)Disease activity index(DAI)was used to assess the protective effect of silibinin on DSS-induced colitis.Therefore,weight and condition of loose fecal and fecal blood was recorded every day for the last 7 days and feces of the last day were collected.The mice were sacrificed on the 15 th day after fasting for one night.Colon tissues were collected.HE staining was used to evaluate pathological analysis.Severity degree of inflammation was assessed by colon length and injury score of histology.The expression of proinflammatory cytokine IL-1β and IL-6 in the colon tissue were detected by Realtime-PCR.Mucin from goblet cells and antibacterial peptide from paneth cells detected by Realtime-PCR were used to assess intestinal permeability.The expression of surface molecule F4/80 on macrophages was detected by immunofluorescence.Both Realtime-PCR and western blot were used to detect the expression of NLRP3、ASC and caspase-1 to verify the effect of silibinin on the activity of NLRP3 inflammasome.Results1.During the DSS intervention,most of the mice were well tolerated.Five in DSS group and 4 in silibinin treated group died by the end of the experiment.Weight of both experimental groups were rapidly decreased compared with control group but DSS group was decreased more significant especially on the 5th day(17.65±0.16 g vs 18.36±0.26 g,P<0.05).2.The colon length of silinibin treated group was significantly longer than DSS group [(4.78±0.14)vs(5.34±0.14)cm,P<0.01] and the DAI score was higher,especially on the 5th [(8.74±0.45)vs(6.73±0.72),P<0.05],6th [(9.39±0.32)vs(7.90±0.59),P<0.05] and 7th day [(10.44±0.34)vs(8.53±0.56),P<0.01].HE staining and injury score of histology [(11.40±0.31)vs(7.31±0.50),P<0.001] showed that the inflammation in colon tissue of silibilin treated group was much slighter than DSS group.3.Compared with DSS group,silibinin reduced the expression of MUC2,claudin-1 and occludin in colon tissue(P<0.05).And the concentration of FITC-D in serum is lower in silibinin treated group.This showed that silibinin might improve the intestinal permeability and the functions of epithelial barrier.4.Realtime-PCR showed that IL-1β and IL-6 in colon tissue of silibinin treated group were significantly decreased(P<0.05)and immunofluorescence also displayed less F4/80 in silibinin treated group.All these may prove that silibinin might make a contribution to the remission of colonic inflammation.5.The results of Realtime-PCR and Western blot showed that the expression level of m RNA and proteins of NLRP3、ASC、caspase-1 in mice colon were significantly decreased in silibinin treated group compared with DSS group(P<0.05).ConclusionsSilibinin could improve the function of intestinal mucosal barrier and decrease the release of proinflammatory cytokines by inhibiting the activity of NLRP3 inflammasome to protect colon from DSS-induced colitis. |
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