基于NF-κB/NLRP3/caspase-1通路研究白及多糖对溃疡性结肠炎大鼠肠黏膜炎症损伤的保护作用

Study on protective effect of Bletilla striata polysaccharide on intestinal mucosal inflammatory injury in rats with ulcerative colitis based on NF-κB/NLRP3/caspase-1 pathway

目的:探究白及多糖对溃疡性结肠炎(UC)大鼠核因子κB(NF-κB)/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)/半胱天冬酶-1(caspase-1)通路蛋白表达及对肠道屏障损伤的影响。方法:采用三硝基苯磺酸(TNBS)+乙醇法建立UC模型,并将SD大鼠随机分为空白组、模型组、白及多糖低(100 mg/kg)、中(200 mg/kg)、高(400 mg/kg)剂量组、柳氮磺胺吡啶阳性组(0.67 g/kg),除空白组外,其余各组均建立UC模型。白及多糖低、中、高剂量组和阳性组分别灌胃给予相应剂量白及多糖和柳氮磺胺吡啶,空白组和模型组灌胃给予10 ml/kg生理盐水,各组大鼠连续给药2周,1次/d。末次给药12 h后,观察大鼠一般行为并对大鼠进行疾病活动指数(DAI)评分;取大鼠结肠组织,苏木精-伊红染色观察组织病理形态;ELISA检测结肠组织髓过氧化物酶(MPO)、炎症因子IL-1β、肿瘤坏死因子-α(TNF-α)水平及肠道屏障功能受损指标二胺氧化酶(DAO)和肠型脂肪酸结合蛋白(i-FABP)水平;Western blot检测结肠组织通路蛋白NF-κB p65、磷酸化NF-κB p65(p-NF-κB p65)、NLRP3、caspase-1、cleaved caspase-1蛋白表达。结果:与空白组比较,模型组大鼠形体消瘦、大便稀溏、结肠组织可见黏膜溃疡、充血、扩张及炎症细胞浸润等病理损伤,DAI评分、结肠组织MPO、IL-1β及TNF-α含量、p-NF-κB p65/NF-κB p65、NLRP3、caspase-1、cleaved caspase-1蛋白表达均升高(P<0.05),DAO和i-FABP含量均降低(P<0.05)。与模型组比较,白及多糖各剂量组及柳氮磺胺吡啶组大鼠DAI评分、结肠组织病理损伤程度、MPO、IL-1β及TNF-α含量、p-NF-κB p65/NF-κB p65、NLRP3、caspase-1、cleaved caspase-1蛋白表达均降低(P<0.05),DAO和i-FABP含量均升高(P<0.05),且呈剂量依赖性,高剂量白及多糖改善效果与柳氮磺胺吡啶相近(P>0.05)。结论:白及多糖可抑制NF-κB/NLRP3/caspase-1通路激活,降低炎症反应和肠屏障损伤,改善UC症状和肠黏膜损伤。

Objective:To investigate effects of Bletilla striata polysaccharide on expressions of nuclear factor kappa B(NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)/caspase-1(caspase-1)pathway proteins and intestinal barrier damage in rats with ulcerative colitis(UC).Methods:UC model was induced by trinitrobenzene sulfonic acid(TNBS)+ethanol,SD rats were randomly divided into:blank group,model group,Bletilla striata polysaccharide low(100 mg/kg),medium(200 mg/kg),high(400 mg/kg)doses groups and sulfasalazine positive group(0.67 g/kg),and model was established in all groups except blank group.Bletilla striata polysaccharide low,middle,high groups and sulfasalazine group were given low,middle and high doses of Bletilla striata polysaccharide and sulfasalazine respectively by gavage,and rats in blank group and model group were administrated with 10 ml/kg saline by gavage for 2 weeks,once a day,and 12 hours after the last administration,general behavior of rats was observed and disease activity index(DAI)was scored;colonic tissues of rats were taken and histopathology was observed by hematoxylin-eosin staining;levels of myeloperoxidase(MPO),IL-1β,tumor necrosis factor-α(TNF-α),damage indexes of intestinal barrier function:diamine oxidase(DAO)and intestinal fatty acid binding protein(i-FABP)were detected by ELISA;Western blot was used to detect expressions of NF-κB,phosphorylated NF-κB p65(p-NF-κB p65),NLRP3,caspase-1 and cleaved caspase-1 proteins in colonic tissues.Results:Compared with blank group,rats in model group were emaciated,had loose stool,with pathological damages such as mucosal ulcer,hyperemia,dilatation and inflammatory cell infiltration,DAI score,contents of MPO,IL-1βand TNF-α,protein expressions of NF-κB,p-NF-κB p65,NLRP3,caspase-1 and cleaved caspase-1 were all increased(P<0.05),and contents of DAO and i-FABP were decreased(P<0.05).Compared with model group,DAI score,degree of pathological damage of colon tissue,contents of MPO,IL-1βand TNF-α,p-NF-κB p65/NF-κB p65,NLRP3,caspase-1 and cleaved caspase-1 protein expressions were all decreased(P<0.05),and contents of DAO and i-FABP were increased(P<0.05),which were in a dose-dependent manner,improvement effect of high dose of Bletilla striata polysaccharide was similar to sulfasalazine(P>0.05).Conclusion:Bletilla striata polysaccharides can inhibit activation of NF-κB/NLRP3/caspase-1 pathway,reduce inflammation and intestinal barrier damage,and improve UC symptoms and intestinal mucosal damage.