青黛及其有效成分靛玉红通过NF-κB信号传导调控溃疡性结肠炎小鼠细胞焦亡的作用机制

Mechanism of action of Qingdai and its active ingredient indigofera in regulating cellular pyroptosis in mice with ulcerative colitis through NF-rB signaling

目的:探讨中药青黛及其有效成分靛玉红对溃疡性结肠炎(UC)小鼠细胞焦亡的影响,并通过NF-κB通路阐释其治疗UC的作用机制。方法:将50只C57BL/6J小鼠随机分为5组:正常组、模型组、清肠栓组、青黛组和美沙拉嗪组,每组10只。除正常组外,其余4组分别予以3%葡聚糖硫酸钠盐(DSS)自由饮用5 d制备UC小鼠模型。成功造模后,各组灌胃治疗7 d。观察各组治疗后疾病活动指数(DAI),HE染色观测结肠组织病理学改变,IHC检测IL-1β、IL-18的表达,Western Blot检测结肠组织p-NF-κB p65、GSDMD、Caspase-1蛋白的表达。RAW264.7细胞采用LPS/ATP共同诱导建立炎症模型,分别以靛玉红及5-ASA干预24 h。应用Western Blot检测各组细胞中p-NF-κB p65、GSDMD、Caspase-1蛋白的表达。结果:与模型组比较,清肠栓组、青黛组和美沙拉嗪组小鼠DAI评分显著降低(P<0.01),病理损伤明显改善,炎症因子IL-1β、IL-18的表达显著减少(P<0.01),结肠组织中p-NF-κB p65、GSDMD、Caspase-1蛋白的表达水平显著下调(P<0.01,P<0.05)。细胞实验结果示经靛玉红及5-ASA干预后,细胞中p-NF-κB p65、GSDMD、Caspase-1蛋白的表达水平较模型组显著下调(P<0.01,P<0.05)。结论:青黛及其有效成分靛玉红通过NF-κB信号传导抑制UC小鼠细胞焦亡。

Objective:To explore the effect of Qingdai and its active ingredient indigofera on pyrocytosis in mice with ulcerative colitis(UC),and to explain their mechanism in the treatment of UC based on NF-κB signaling.Methods:Fifty C57BL/6J mice were randomly divided into 5 groups:normal group,model group,Qingchangshuan group,Qingdai group and mesalazine group,with 10 mice in each group.Except for the normal group,the other four groups were given 3% dextran sulfate sodium(DSS)for 5 d to prepare a mouse model of experimental UC.After successful moulding,the mice were treated by intragastric administration for 7 consecutive days.The disease activity index(DAI)of each group was observed after the treatment;HE staining was used to observe histopathological changes;The immunohistochemical staining was used to detect the expressions of IL-1β and IL-18.Western Blot was used to detect the expression of p-NF-κB p65,GSDMD and Caspase-1 protein in colon tissue of each group.RAW 264.7 cells were co-induced with LPS/ATP for inducing the inflammation and then treated with Indirubin and 5-ASA for 24 h.The protein expression levels of p-NF-κB p65,GSDMD and Caspase-1 were measured by Western Blot.Results:Compared with the model group,the DAI score in Qingchangshuan group,Qingdai group and mesalazine group decreased significantly(P<0.01),and the pathological changes of colon tissue were improved.The expression of inflammatory factors IL-1β and IL-18 was clearly reduced(P<0.01).The expression levels of p-NF-κB p65,GSDMD,Caspase-1 protein in colon tissue were significantly down-regulated(P<0.01,P<0.05).Results of cellular experiments showed that,after Indirubin and 5-ASA's intervention,the expression levels of p-NF-κB p65,GSDMD and Caspase-1 proteins were obviously decreased in the cells(P<0.01,P<0.05).Conclusion:Qingdai and its active components Indirubin can inhibit pyrocytosis in mice with ulcerative colitis by regulating NF-κB signaling.