木香烃内酯通过抑制NLRP3缓解咪喹莫特诱导小鼠银屑病模型中炎性因子的表达

Costunolide Attenuates the Expression of Imiquimod-induced Psoriasis-like Mouse Model by Inhibiting NLRP3

目的探讨木香烃内酯(Cos)通过抑制NOD样受体热蛋白结构域相关蛋白3(NLRP3)炎性小体在咪喹莫特诱导的小鼠银屑病样模型中的作用及可能的机制。方法选取50只雄性小鼠,随机分为5组,每组10只。正常对照组:涂抹等量凡士林,同时灌胃0.2 mL的0.9%氯化钠溶液;模型组:涂抹60 mg咪喹莫特,同时灌胃0.2 mL的0.9%氯化钠溶液;实验组:涂抹60 mg咪喹莫特,同时灌胃0.2 mL的浓度分别为5、10和20μmol/L的Cos。用咪喹莫特诱导小鼠银屑病模型,建模后第6天开始给药,连续7 d,第8天取材。进一步实验,将NLRP3的小分子干扰核糖核酸(si-NLRP3)转染至建模后的小鼠皮肤细胞,再用Cos或MCC950(10μmol/L)处理细胞,分别作为si-NLRP3组、Cos组和MCC950组。实时荧光定量聚合酶链反应(qRT-PCR)法检测NLRP3、胱天蛋白酶-1(caspase-1)、凋亡相关斑点样蛋白(ASC)的mRNA表达;蛋白质印迹法(Western blotting)检测NLRP3的蛋白表达;酶联免疫吸附测定(ELISA)法检测肿瘤坏死因子-α(TNF-α)、白细胞介素-17(IL-17)及IL-1β的含量。结果与正常对照组比较,模型组NLRP3 mRNA、caspase-1 mRNA、ASC mRNA和NLRP3蛋白表达显著升高(均P<0.05);与模型组比较,5、10和20μmol/L的Cos可显著降低NLRP3、caspase-1、ASC mRNA和NLRP3蛋白表达(均P<0.05)。与正常对照组比较,模型组TNF-α、IL-17、IL-1β含量显著升高(P<0.05);与模型组比较,5、10和20μmol/L的Cos可显著降低TNF-α、IL-17、IL-1β含量(P<0.05)。与模型组比较,si-NLRP3组NLRP3 mRNA和蛋白表达显著降低,TNF-α、IL-17、IL-1β含量显著降低(P<0.05);而Cos或MCC950未显著影响NLRP3 mRNA、蛋白表达和炎性因子TNF-α、IL-17、IL-1β含量(均P>0.05)。结论Cos通过抑制NLRP3炎性小体减轻咪喹莫特诱导的小鼠银屑皮肤病。

Objective In order to investigate the role and possible mechanisms of costunolide(Cos)in inhibiting NODlike receptor thermoprotein domain-related protein(NLRP)3 inflammasome in imiquimod-induced psoriasis-likemousemodel.Methods Fifty male mice were randomly divided into five groups,with 10 mice in each group.Normal control group:smear the same amount of petroleum jelly and simultaneously gavage 0.2 mL of 0.9%sodium chloride solution;model group:smear 60 mg imiquimod and simultaneously gavage 0.2 mL of 0.9%sodium chloride solution;experimental group:smear 60 mg imiquimod was given simultaneously with 0.2 mL of Cos at 5,10 and 20μmol/L.Imiquimod was used to induce a mouse psoriasis model,and the administration was started on the 6th day after modeling,for seven consecutive days,and the material was taken on the eighth day.In further experiments,si-NLRP3 was transfected into the mice skin cells,and the cells were treated with Cos or MCC950(10μmol/L),as si-NLRP3 group,Cos group and MCC950 group,respectively.Quantitative realtime polymerase chain reaction(qRT-PCR)was used to detect the mRNA expression levels of NLRP3,cysteine protease caspase-1,and apoptosis-related dot-like protein ASC(ASC).Western blotting was used to detect the protein expression level of NLRP3.Enzyme-linked immunosorbent assay(ELISA)method was used to detect the content of tumor necrosis factor-α(TNF-α),interleukin-17(IL-17),IL-1β.Results Compared with the normal control group,the mRNA and NLRP3 protein expression levels of NLRP3,caspase-1,ASC in the model group were significantly increased(P<0.05).Compared with the model group,5,10 and 20μmol/L Cos were significantly reduced NLRP3,caspase-1,ASC mRNA and NLRP3 protein expression levels(P<0.05).Compared with the normal control group,the contents of TNF-α,IL-17 and IL-1βin the model group were significantly increased(P<0.05).Compared with the model group,5,10 and 20μmol/L Cos significantly reduced TNF-α,IL-17,IL-1β content(P<0.05).Compared with the model group,the expression levels of NLRP3 mRNA and protein in the si-NLRP3 group were significantly reduced,and the levels of TNF-α,IL-17,and IL-1βwere significantly reduced(P<0.05).While the Cos or MCC950 did not significantly affected NLRP3 mRNA and protein expression levels and the levels of inflammatory factors TNF-α,IL-17,IL-1β(all P>0.05).Conclusion Cos could reduce imiquimod-induced psoriasis skin disease in mice by inhibiting NLRP3 inflammasome.