二氧化硅扰乱P2X7离子通道介导NLRP3炎性小体依赖的巨噬细胞焦亡在矽肺发病中的作用  

Role of silica-induced P2X7 disorder in NLRP3-dependent macrophage pyroptosis and pathogenesis of silicosis

作　　者：田家齐 马兰 韩正朴 殷浩宇 彭延杰 张敬[2,3] 李炜修 张美华 张林 TIAN Jia-qi;MA Lan;HAN Zheng-pu;YIN Hao-yu;PENG Yan-jie;ZHANG Jing;LI Wei-xiu;ZHANG Mei-hua;ZHANG Lin(School of Public Health,Weifang Medical University,Weifang,Shandong 261053,China;不详)

机构地区：[1]潍坊医学院公共卫生学院,山东潍坊261053 [2]山东大学附属山东省妇幼保健院妇女儿童疾病临床医学研究中心 [3]华北理工大学公共卫生学院 [4]山东大学附属山东省妇幼保健院国家卫健委生育调控技术重点实验室

出　　处：《现代预防医学》2022年第9期1664-1670,1719,共8页Modern Preventive Medicine

基　　金：国家自然科学基金(82003405);山东省自然科学基金(ZR2020QH290);山东省医药卫生科技发展计划项目(2018WS059)。

摘　　要：目的利用稳定表达凋亡相关斑点样蛋白(ASC)的小鼠巨噬细胞系(RAW-ASC)构建巨噬细胞焦亡体外模型,深入探讨不同粒径二氧化硅(SiO_(2))粉尘诱导核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎性小体依赖的巨噬细胞焦亡机制及其在矽肺发病中的潜在作用。方法以脂多糖(LPS,1μg/ml)预处理RAW-ASC巨噬细胞6 h,纳米(100μg/ml)或微米(750μg/ml)SiO_(2)粉尘暴露4 h,构建巨噬细胞焦亡体外模型,同时匹配空白对照组、LPS预处理组和焦亡阳性对照组(1μg/ml LPS预处理6 h后,3 mmol/L ATP处理4 h),检测各组细胞活性、增殖能力、破裂状况和焦亡相关蛋白表达情况。针对离子通道和焦亡通路相关靶点,分别加入NLRP3抑制剂和嘌呤能P2X7受体(P2X7)拮抗剂,评估SiO_(2)诱发焦亡改变。结果与对照组相比,纳米和微米SiO_(2)粉尘均可减弱巨噬细胞活力并抑制细胞增殖,还可促进细胞裂解,使细胞外乳酸脱氢酶(LDH)(纳米组:t=78.906,P<0.001;微米组:t=8.123,P<0.001)、细胞内活性氧(ROS)(纳米组:t=5.662,P<0.001;微米组:t=11.761,P<0.001)和钙离子(Ca^(2+))(纳米组:t=13.224,P<0.001;微米组:t=40.733,P<0.001)水平升高,加速ATP耗竭(纳米组:t=4.898,P<0.001;微米组:t=6.029,P<0.001),上调焦亡相关蛋白NLRP3、ASC、半胱天冬氨酸蛋白酶-1(Caspase-1)、Gasdermin D(GSDMD)和白介素-1β(IL-1β)表达(纳米SiO_(2)组:t=5.476,P=0.008;t=5.846,P=0.017;t=8.078,P=0.006;t=8.420,P=0.004;t=5.361,P=0.003;微米SiO_(2)组:t=4.411,P=0.013;t=6.154,P=0.015;t=7.188,P=0.002;t=14.265,P<0.001;t=8.574,P<0.001)。加入NLRP3抑制剂或P2X7拮抗剂后,NLRP3蛋白表达水平下调(纳米组:t=3.894,P=0.007;t=13.954,P<0.001;微米组:t=2.470,P=0.034;t=13.263,P<0.001)。结论纳米和微米级SiO_(2)粉尘暴露均可抑制巨噬细胞活性和增殖能力,扰乱P2X7离子通道对ATP的传递能力,诱发NLRP3依赖巨噬细胞焦亡,相关过程可能是矽肺发病的重要环节。Objective To explore the mechanism of(Nod-like receptor protein 3)NLRP3 inflammasome-dependent macrophage pyroptosis induced by micro and nano silica particles(SiO_(2)),and to further reveal its potential role in the pathogenesis of silicosis by an established macrophage pyroptosis model,which used the RAW 264.7 murine macrophage cell line(RAW-ASC)stably expressing Apoptosis-related spot-like protein(ASC).Methods RAW-ASC macrophages were pretreated with LPS(lipopolysaccharide)for 6 h and then exposed to nano-or micro-SiO_(2) for 4 h,and RAW-ASC macrophage blank control group,LPS pretreatment group and pyroptosis positive control group were set up.The cell activity,proliferation,rupture and the expression of pyroptosis related protein were detected.In order to detect ion channel and pyroptosis pathway related targets,NLRP3 inhibitor and Purinergic P2X7 receptor(P2X7)antagonist were added to the cells,respectively.Results Compared with the control group,both nano and micro SiO_(2) dust could weaken the activity of macrophages,inhibit cell proliferation,promote cell lysis and increase extracellular Lactate dehydrogenase(LDH)(nano-group:t=78.906,P<0.001;micro-group:t=8.123,P<0.001),intracellular Reactive Oxygen Species(ROS)(nano-group:t=5.662,P<0.001;micro-group:t=11.761,P<0.001)and Ca^(2+)levels(nano-group:t=13.224,P<0.001;micro-group:t=40.733,P<0.001),and accelerate ATP depletion(nano-group:t=4.898,P<0.001;micro-group:t=6.029,P<0.001).The expression of NLRP3,ASC,Cysteinyl aspartate-specific proteases 1(Caspase-1),Gasdermin D(GSDMD),and Interleukin-1β(IL-1β)were up-regulated(nano-group:t=5.476,P=0.008;t=5.846,P=0.017;t=8.078,P=0.006;t=8.420,P=0.004;t=5.361,P=0.003;micro-group:t=4.411,P=0.013;t=6.154,P=0.015;t=7.188,P=0.002;t=14.265,P<0.001;t=8.574,P<0.001).After the addition of NLRP3 inhibitor and P2X7 antagonist,the expression levels of NLRP3 protein decreased(nano-group:t=3.894,P=0.007;t=13.954,P<0.001;micro-group:t=2.470,P=0.034;t=13.263,P<0.001).Conclusion Nano SiO_(2) and micro SiO_(2) exposure both inhibit t

关 键 词：矽肺 细胞焦亡 P2X7 NLRP3炎性小体 二氧化硅 

分 类 号：R135.2[医药卫生—劳动卫生]