冬凌草甲素通过NLRP3途径抑制脂多糖诱导的巨噬细胞炎症反应  

Oridonin inhibits LPS-induced macrophage inflammatory response by NLRP3 pathway

作　　者：宋沁岚 刘云奇[1] 段晓莹 殷胜利[1] SONG Qin-lan;LIU Yun-qi;DUAN Xiao-ying;YIN Sheng-li(Department of Cardiac Surgery,the First Affiliated Hospital of Sun Yat-sen University,Guangzhou,Guangdong 510080,China)

机构地区：[1]中山大学附属第一医院心外科,广东广州510080

出　　处：《热带医学杂志》2021年第4期433-437,463,F0003,共7页Journal of Tropical Medicine

基　　金：广州市科技计划项目(201803010077)。

摘　　要：目的研究冬凌草甲素(Oridonin)对脂多糖(LPS)诱导的巨噬细胞炎症反应中核苷酸结合寡聚化结构域样受体3(NLRP3)的作用。方法将Raw264.7巨噬细胞分成空白组、对照组、实验组(终浓度分别为4、10、15、20μmol/L Oridonin组),采用CCK8法检测细胞存活率,筛选Oridonin药物浓度。将小鼠Raw264.7巨噬细胞分为正常对照组、LPS组和LPS+Oridonin组,采用RT-qPCR法检测NLRP3、肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)、半胱氨酸蛋白水解酶-1(Caspase-1)和白介素-1β(IL-1β)mRNA的表达;Western blot法检测NLRP3、Caspase-1和IL-1β蛋白表达;免疫荧光染色检测NLRP3和Caspase-1蛋白共定位。结果Oridonin浓度为20μmol/L细胞存活率急剧减少,浓度在4~15μmol/L之间细胞存活率较高,实验选用10μmol/L Oridonin处理小鼠巨噬细胞。与正常对照组比较,LPS组NLRP3、TNF-α、IL-6、Caspase-1和IL-1βmRNA基因表达显著上调(P<0.05);与LPS组比较,LPS+Oridonin组NLRP3、TNF-α、IL-6、Caspase-1和IL-1βmRNA基因表达明显减少(P<0.05)。与正常对照组比较,LPS组NLRP3、Caspase-1和IL-1β蛋白表达显著增多(P<0.05),与LPS组比较,LPS+Oridonin组巨噬细胞内NLRP3、Caspase-1和IL-1β蛋白表达明显减少(P<0.05)。与正常对照组比较,LPS组NLRP3和Caspase-1蛋白共定位明显增多,LPS+Oridonin组蛋白共定位明显减少。结论Oridonin通过抑制NLRP3途径减轻LPS诱导的巨噬细胞炎症反应。Objective This study investigated the effect of Oridonin on nucleotide-binding oligomerization domain-like receptor 3(NLRP3)in lipopolysaccharides(LPS)-induced macrophage inflammatory response.Methods Mouse RAW264.7 macrophage cells were divided into three groups:normal control group,LPS-induced group and LPS+Oridonin group.The optional concentration of Oridonin was determined by CCK8 method.The mRNA expressions of NLRP3,tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),Caspase-1 and interleukin-1β(IL-1β)were detected by RT-qPCR.The protein expressions of NLRP3,caspase-1 and IL-1βwere detected by western blot.Colocalization of NLRP3 and Caspase-1 was detected by immunofluorescence staining.Results The viability of macrophage was significantly reduced when Oridonin concentration was 20μmol/L.The cell survival rate was high when Oridonin concentration was 4-15μmol/L.For further experiments,macrophages were treated with 10μmol/L Oridonin.Compared with the normal control group,the mRNA expressions of NLRP3,TNF-α,IL-6,Caspase-1 and IL-1βwere significantly up-regulated in the LPS group,and the expressions of these genes were significantly inhibited in the LPS+Oridonin group.Compared with the normal control group,the protein expressions of NLRP3,Caspase-1 and IL-1βwere significantly increased in the LPS group,and the expression of these proteins were significantly inhibited in the LPS+Oridonin group.Compared with the normal control group,the colocalization of NLRP3 and caspase-1 was significantly increased in the LPS group,while the colocalization in LPS+Oridonin group was significantly decreased.Conclusion Oridonin inhibits LPS-induced macrophage inflammatory response and might be via NLRP3 pathway.

关 键 词：巨噬细胞 NLPR3 CASPASE-1 IL-1Β 

分 类 号：Q538[生物学—生物化学]