摘要
目的探究姜黄素抑制游离二氧化硅(SiO2)诱导的小鼠肺泡巨噬细胞(AM)内核苷酸结合寡聚化结构域样受体家族含热蛋白结构域蛋白3(NLRP3)炎性小体活化的分子机制。方法取自无特定病原体C57BL/6小鼠支气管肺泡灌洗液中分离的AM分为6组;其中,对照组AM不予任何刺激;SiO2刺激组AM予终质量浓度为50 mg/L的游离SiO2混悬液刺激;核转录因子-κB(NF-κB)抑制组AM予终浓度为200 nmol/L的5-(4-氟苯基)-2-脲基噻吩-3-甲酰胺预处理1 h,姜黄素低、中、高剂量组AM分别予终浓度为20、40、50μmol/L的姜黄素预处理1 h,其后均以终质量浓度为50 mg/L的游离SiO2混悬液刺激。孵育6 h后收集样品,以实时荧光定量聚合酶链式反应法检测细胞内NLRP3炎性小体相关基因NLRP3、半胱氨酸蛋白酶-1(Caspase-1)和白细胞介素(IL)-1β的mRNA表达;以酶联免疫吸附实验检测AM中成熟IL-1β(mIL-1β)和IL-18分泌水平;以免疫印迹法检测各组细胞中Caspase-1剪切体(cleaved Caspase-1)、IL-1β前体(pro-IL-1β)、mIL-1β蛋白的表达和分泌水平。结果与对照组比较,SiO2刺激组AM中NLRP3、Caspase-1、IL-1β mRNA相对表达水平和mIL-1β、IL-18分泌水平,cleaved Caspase-1、pro-IL-1β、mIL-1β蛋白的相对表达水平与cleaved Caspase-1、mIL-1β蛋白的分泌水平均升高(P<0.05)。除cleaved Caspase-1蛋白的相对表达水平和分泌水平外,NF-κB抑制组AM中其余8个指标均低于SiO2刺激组(P<0.05)。除姜黄素低剂量组cleaved Caspase-1和mIL-1β蛋白相对表达水平外,3个姜黄素剂量组AM中上述10个指标均低于SiO2刺激组(P<0.05);且上述10个指标均随姜黄素干预剂量的增加而下降(P<0.05)。与NF-κB抑制组比较,姜黄素中剂量组AM中NLRP3、IL-1β mRNA相对表达水平和pro-IL-1β蛋白相对表达水平均升高(P<0.05),mIL-1β和IL-18分泌水平,以及cleaved Caspase-1、mIL-1β蛋白的相对表达水平与分泌水平均下降(P<0.05)。结论姜黄素可抑制SiO2诱导
Objective To explore the molecular mechanism of curcumin in inhibiting the nucleotide-binding oligomerization domain like receptor family pyrin domain-containing(NLRP3)inflammatory bodies induced by silica(SiO2)in mouse alveolar macrophages(AM).Methods AMs were isolated from the bronchoalveolar lavage fluid of specific pathogen free C57BL/6 mice and divided into 6 groups.Among them,the AM of the control group received no stimulation;the AM in the SiO2 stimulation group was stimulated with SiO2 suspension at the final mass concentration of 50 mg/L;the AM in nuclear factor(NF-κB)inhibition group was pretreated with 5-(4-fluorophenyl)-2-urea-thiophene-3-formamide with a final concentration of 200 nmoL/L for 1 hour,the AM in the low-,medium-and high-dose curcumin groups were pretreated with curcumin with the final concentrations of 20,40 and 50μmol/L for 1 hour,respectively,and then stimulated with SiO2 suspension with a final concentration of 50 mg/L.Samples were collected after 6 hours of incubation.The mRNA expression of NLRP3 inflammasome related genes such as NLRP3,Caspase-1 and interleukin(IL)-1βwas detected by real-time fluorescence quantitative polymerase chain reaction.The secretion level of maturation IL-1β(mIL-1β)and IL-18 in AM was detected by enzyme-linked immunosorbent assay.The protein expression and secretion level of cleaved Caspase-1,precursor-IL-1β(pro-IL-1β)and mIL-1βwere analyzed by Western blotting.Results The mRNA relative expression of NLRP3,Caspase-1 and IL-1β,and the secretion levels of mIL-1βand IL-18,and the protein relative expression of Caspase-1,pro-IL-1βand mIL-1β,as well as the secretion levels of cleaved Caspase-1 and mIL-1βincreased in the SiO2 stimulated group compared with the control group(P<0.05).Except for the relative expression and the secretion level of cleaved Caspase-1,the other 8 indexes in the NF-κB inhibition group were lower than that in the SiO2 stimulation group(P<0.05).Except for the relative expression of cleaved Caspase-1 and mIL-1βproteins in the l
作者
宋楠楠
杜忠君
贾强
陈尚雅
朱文文
杨旭
侯珊珊
邵华
SONG Nannan;DU Zhongjun;JIA Qiang;CHEN Shangya;ZHU Wenwen;YANG Xu;HOU Shanshan;SHAO Hua(Shandong University of Traditional Chinese Medicine,Jinan,Shandong 250355,China;不详)
出处
《中国职业医学》
CAS
北大核心
2020年第2期121-128,共8页
China Occupational Medicine
基金
国家自然科学基金(81872575)
山东省自然科学基金(ZR2018MH036,ZR2017MH020)
山东省重点研发计划(2019GSF107055)。
