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SiO2 诱导巨噬细胞线粒体分裂在肺成纤维细胞活化中的作用 |
马洁,孟祥敬,厉铭,李超,翟美玲,杨晓涵,贾强
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山东省职业卫生与职业病防治研究院/山东第一医科大学/山东省医学科学院,山东 济南 250062
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摘 要: 目的 探讨巨噬细胞线粒体过度分裂调控的核苷酸结合寡聚化结构域样受体家族含热蛋白结构域3(NLRP3)炎症小体激活在矽肺纤维化中的作用机制。方法 以不同浓度二氧化硅(SiO2)染毒大鼠肺巨噬细胞NR8383 12h,采用CCK8检测SiO2对NR8383 细胞存活率的影响;活细胞荧光探针法检测细胞活性氧(ROS)水平;ELISA法测定细胞上清液中白细胞介素-18(IL-18)、IL-1β和转化生长因子-β1(TGF-β1)的水平;Western blot法检测NLRP3炎症小体相关蛋白(NLRP3、Cleaved caspase-1、ASC)以及线粒体分裂蛋白Drp1的表达水平;Western blot法检测不同染毒时间点(0、12、24、48h)100 μg/ml SiO2 浓度下大鼠成纤维细胞 RFL-6COLⅢ、NLRP3、Drp1、α-SMA蛋白的表达水平;使用 Transwell小室将100 μg/ml SiO2处理过的NR8383细胞与RFL-6细胞进行共培养,检测不同处理时间点共培养上清液中IL-18、IL-1β和TGF-β1的水平及NR8383细胞NLRP3、Cleaved caspase-1、ASC、Drp1和RFL-6细胞COLⅢ、α-SMA的表达水平;使用线粒体分裂抑制剂Mdivi-1(10 μmol/L)预处理共培养细胞,观察线粒体分裂抑制后对NR8383细胞NLRP3炎症小体激活、ROS 水平、TGF-β1释放水平及RFL-6细胞纤维化的影响。结果 随着SiO2染毒浓度增加,NR8383细胞存活率逐渐下降,确定100 μg/ml SiO2为最高染毒浓度;随SiO2染毒剂量的增加,NR8383细胞中ROS水平和细胞培养上清液中IL-18、IL-1β、TGF-β1水平明显增加(P<0.05);与对照组相比,100 μg/ml SiO2染毒后NR8383细胞NLRP3、Cleaved caspase-1、ASC和Drp1蛋白表达显著增加(P<0.05);随着SiO2染毒时间的增加,RFL-6细胞中COLⅢ、NLRP3、Drp1、α-SMA的表达水平无显著变化。在100 μg/ml SiO2染毒NR8383与RFL-6细胞共培养体系中,随着染毒时间的增加,NR8383细胞中ROS产生和上清液中IL-18、IL-1β、TGF-β1水平显著增加(P<0.05);与对照组相比,SiO2染毒24 h后NR8383细胞中NLRP3、Cleaved caspase-1、ASC 及Drp1表达水平显著增加(P<0.05);RFL-6细胞中COLⅢ、α-SMA蛋白表达显著增加(P<0.05)。Mdivi-1可使SiO2处理的NR8383细胞中ROS和上清液中IL-18、IL-1β、TGF-β1水平的升高受到抑制(P<0.05),并下调NR8383细胞中NLRP3、Cleaved caspase-1、ASC、Drp1和RFL-6细胞中COLⅢ、α-SMA的蛋白表达(P<0.05)。结论 SiO2 可以促进大鼠肺巨噬细胞线粒体过度分裂、NLRP3炎症小体激活,进而诱导共培养体系大鼠成纤维细胞纤维化;抑制大鼠肺巨噬细胞线粒体过度分裂可以减轻NLRP3炎症小体活化,并抑制大鼠成纤维细胞纤维化,提示SiO2诱导肺巨噬细胞线粒体过度分裂可能是导致矽肺炎症过程和纤维化的重要原因。 |
关键词: 矽肺 线粒体分裂 核苷酸结合寡聚化结构域样受体家族含热蛋白结构域 3(NLRP3) 纤维化 |
中图分类号: R135. 2
文献标识码: A
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基金项目: 国家重点研发计划 (2022YFC2503202);山东省自然科学基金 (ZR2022QH041, ZR2022MH293) |
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Role of SiO2 induced mitochondrial division in the activation of pulmonary fibroblasts in macrophages |
MA Jie,MENG Xiangjing,LI Ming,LI Chao,ZHAI Meiling,YANG Xiaohan,JIA Qiang
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Shandong Provincial Academy of Occupational Health and Occupational Medicine/Shandong First Medical University/Shandong Provincial Academy of Medical Sciences, Jinan,Shandong 250062, China
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Abstract: Objective To explore the mechanism of nucleotide binding oligmerization domain like receptor family pyrin domain-containing 3(NLRP3)inflammasome activation by mitochondrial division in macrophages in silicosis fibrosis.Methods Rat lung macrophages NR8383 were treated with different concentrations of silica(SiO2)for 12 hours,then,the cell survival rate of NR8383 cells was detected by CCK8 kit,the cellular levels of reactive oxygen species(ROS) were detected by fluorescence probe method, the levels of interleukin-18(IL-18),IL-1β and transforming growth factors-β1(TGF-β1)in cell supernatant were measured by enzyme-linked immunosorbent assay(ELISA), Western blot was used to detect the expressions of NLRP3 inflammasome associated proteins(NLRP3,Cleaved caspase-1,ASC)and mitochondrial division protein Drp1;the expressions levels ofCOLⅢ,NLRP3,Drp1 and α-SMA proteins in rat fibroblast RFL-6 under the exposed concentration of 100 μg/ml SiO2 at different exposure time points(0,12,24,48h) were detected by Western blot; NR8383 cells were co-cultured with RFL-6 cells exposed to 100 μg/ml SiO2 using Transwell chamber,then detect the levels of IL-18, IL-1β and TGF-β1 in co-culture cell supernatant, the expressions of NLRP3,Cleaved caspase-1,ASC,Drp1 in NR8383 cells and the expression levels of COLⅢ,α-SMA in RFL-6 cells at different exposure time points(0,12,24,48h);the co-cultured cells were preteated with mitochondrial division inhibitor Mdivi-1(10 μmol/L),detect the effects of mitochondrial division inhibition on NLRP3 inflammasome activation,ROS and TGF-β1 levels in NR8383 cells and on the fibrosis process of RFL-6 cells. Results The results showed that with the increase of SiO2 exposed concentrations (0,50,100,200 μg/ml),the survival rate of NR8383 cells gradually decreased,too,and 100 μg/ml of SiO2 was determined as the highest exposure concentration, the ROS levels in NR8383 cells and the levels of IL-18、IL-1β、TGF-β1 in cell supernatant all significantly increased(P<0.05);compared with the control group,the protein expressions of NLRP3,Cleaved caspase-1,ASC,and Drp1 in NR8383 cells significantly increased in 100 μg/ml SiO2 group(P<0.05),while there was no significant change in the protein expressions of COLⅢ, NLRP3, Drp1 and α-SMA in RFL-6 cells with increase of SiO2 treatment time(0,12,24,48h).In the co-culture model of RFL-6 cells with NR8383 cells exposed to 100 μg/ml SiO2,the ROS production in NR8383 cells and the levels of IL-18, IL-1β,TGF-β1 in supernatant all were significantly increased(P<0.05)with the time(0,12,24,48h);compared with the control group,after 24 hours of SiO2 exposure,the expressions of NLRP3,Cleaved caspase-1,ASC and Drp1 in NR8383 cells(P<0.05) and the protein expressions of COLⅢ, α-SMA in RFL-6 cells were significantly increased as well(P<0.05).However,the treatment of Mdivi-1 could make the production of ROS in SiO2 exposed NR8383 cells and the levels of IL-18,IL-1β,TGF-β1 in supernatant of co-culture system inhabited (P<0.05),and down-regulate the protein expressions of NLRP3,Cleaved caspase-1,ASC,and Drp1 in NR8383 cells and COLⅢ,α-SMA expressions in RFL-6 cells(P<0.05).Conclusion The results suggested that SiO2 can promote excessive mitochondrial division and NLRP3 inflammasome activation in rat pulmonary macrophages,thereby inducing fibrosis related proteins expression in rat fibroblasts;while inhibiting the excessive mitochondrial division of rat pulmonary macrophages can alleviate the activation of NLRP3 inflammasome by SiO2 and the fibrosis of RFL-6 cells,which suggested that SiO2 induced excessive mitochondrial division in pulmonary macrophages might play an important role in the development of inflammation and fibrosis in the process of silicosis. |
Keywords: silicosis mitochondrial division nucleotide binding oligomerization domain like receptor family pyrin domaincontaining 3(NLRP3) fibrosis |
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