雷公藤多苷通过NOXs-ROS-NLRP3炎症小体信号通路抑制结肠炎症

目的:观察雷公藤多苷对右旋葡聚糖硫酸钠(dextran sulphate sodium,DSS)诱导的小鼠溃疡性结肠炎(ulcerative colitis,UC)的防治作用及对黏膜组织NADPH氧化酶(NADPH oxidases,NOXs)-活性氧簇(reactive oxygen species,ROS)-NOD样受体蛋白3(NOD-like receptor protein 3,NLRP3)炎症小体信号通路表达的影响,探讨其治疗溃疡性结肠炎的作用及机制。方法:BALB/c小鼠随机分为5组:模型组;低、中及高剂量雷公藤多苷灌胃组;正常组。采用DSS诱发UC动物模型,雷公藤多苷灌胃21 d后,取结肠组织,运用实时荧光定量PCR法检测结肠黏膜组织NLRP3、ASC及caspase-1的mRNA表达,免疫组化法检测结肠黏膜组织caspase-1的表达,ELISA法检测结肠组织匀浆上清IL-1α、TNF-α及IL-13的含量,鲁米诺化学发光法检测结肠黏膜组织ROS的生成和经DPI(NOXs抑制剂)抑制后的NADPH消耗率来分析NOXs活性;体外分离结肠组织中性粒细胞,检测分离的中性粒细胞中ROS的生成、NOXs活性以及NLRP3、ASC、caspase-1的mRNA表达。结果:雷公藤多苷灌胃各组小鼠结肠黏膜组织病理均存在不同程度异常,但组织病理学评分均低于模型组;雷公藤多苷灌胃各组结肠黏膜组织和分离的中性粒细胞中,除高剂量组结肠黏膜组织caspase-1的mRNA表达与正常组相比差异无统计学显著性外,其余各组ROS生成、NOXs活性及NLRP3、ASC、caspase-1的mRNA表达水平低于模型组(P0.05),高于正常组(P0.05);雷公藤多苷灌胃各组间两两比较发现,除中、高剂量组结肠黏膜组织及分离的中性粒细胞caspase-1的mRNA表达差异无统计学显著性外,其它指标相比均有统计学显著性(P0.05);雷公藤多苷灌胃各组小鼠结肠组织匀浆上清中促炎因子(IL1α和TNF-α)含量低于模型组(P0.05),高于正常组(P0.05),抑炎因子(IL-13)含量各组间比较差异无统计学显著性。结论:雷公藤多苷可能通过抑制NOXs-ROS-NLRP3炎症小体信号通路来降低IL-1α、TNF-α等促炎因子的表达从而对DSS诱导的UC小鼠起保护作用,中性粒细胞可能是参与其保护作用的主要炎性细胞。

Tripterygium glycosides suppress colitis via NOXs-ROS-NLRP3 inflammatory signaling pathways

AIM: To observe the effect of Tripterygium glycosides on NOXs-ROS-NLRP3 inflammatory signaling pathways in the colon tissue in dextran sulphate sodium (DSS)-induced ulcerative colitis (UC) mice, and to investigate the underlying mechanisms. METHODS: BALB/c mice were used and the mouse model of UC was established by DSS induction. The mice were randomly divided into 5 groups (model group, low-, medium- and high-dose Tripterygium glycosides groups, and normal group). The colon tissues were collected 21 d after Tripterygium glycosides gavage. The mRNA expression of NLRP3, ASC and caspase-1 in the colon tissues was detected by real-time PCR. The caspase-1 expression in the colorectal mucosa was observed by immunohistochemical method. ELISA was used to detect the protein le-vels of IL-1α, TNF-α and IL-13. The production of reactive oxygen species (ROS) was measured by chemiluminescence technique, and the consumption rate of NADPH, which was inhibited by DPI, was analyzed to determine the activity of NADPH oxidases (NOXs). The neutrophils were isolated, and the ROS production, NOXs activity, and the mRNA expression of NLRP3, ASC and caspase-1 were also detected. RESULTS: The colon tissues were abnormal with different degrees in Tripterygium glycosides groups, and histopathological scores were lower than that in model group. In Tripterygium glycosides groups, in addition to the mRNA expression levels of caspase-1 in the colon tissues between normal group and high-dose group, ROS production, NOXs activity and the mRNA expression levels of NLRP3, ASC and caspase-1 in the colon tissues and colon-isolated neutrophils were lower than those in model group (P<0.05), and higher than those in normal group (P<0.05). The results of pairwise comparison for the efficacy of Tripterygium glycosides administration showed that the above indexes were statistically significant except the mRNA expression levels of caspase-1 between middle-dose group and high-dose group. Tripterygium glycosides administration significantly decreased the expression levels of proinflammatory cytokines IL-1α and TNF-α in the homogenates of colon tissues in the model mice (P<0.05). No difference of IL-13 expression among the groups was observed.CONCLUSION: Tripterygium glycosides inhibits NOXs-ROS-NLRP3 inflammatory signaling pathways to reduce the expression of IL-1α, TNF-α and other proinflammatory cytokines, and attenuates DSS-induced ulcerative colitis in mice, by which the neutrophils might be involved in the process.