殷浩宇, 田家齐, 马兰, 张敬, 李炜修, 彭延杰, 张美华, 翟庆峰, 张林. 二氧化硅上调活性氧诱导NLRP3依赖的巨噬细胞焦亡机制[J]. 环境与职业医学, 2022, 39(4): 446-452. DOI: 10.11836/JEOM21363
引用本文: 殷浩宇, 田家齐, 马兰, 张敬, 李炜修, 彭延杰, 张美华, 翟庆峰, 张林. 二氧化硅上调活性氧诱导NLRP3依赖的巨噬细胞焦亡机制[J]. 环境与职业医学, 2022, 39(4): 446-452. DOI: 10.11836/JEOM21363
YIN Haoyu, TIAN Jiaqi, MA Lan, ZHANG Jing, LI Weixiu, PENG Yanjie, ZHANG Meihua, ZHAI Qingfeng, ZHANG Lin. Mechanism of silica-induced ROS over synthsis in NLRP3-dependent macrophage pyroptosis[J]. Journal of Environmental and Occupational Medicine, 2022, 39(4): 446-452. DOI: 10.11836/JEOM21363
Citation: YIN Haoyu, TIAN Jiaqi, MA Lan, ZHANG Jing, LI Weixiu, PENG Yanjie, ZHANG Meihua, ZHAI Qingfeng, ZHANG Lin. Mechanism of silica-induced ROS over synthsis in NLRP3-dependent macrophage pyroptosis[J]. Journal of Environmental and Occupational Medicine, 2022, 39(4): 446-452. DOI: 10.11836/JEOM21363

二氧化硅上调活性氧诱导NLRP3依赖的巨噬细胞焦亡机制

Mechanism of silica-induced ROS over synthsis in NLRP3-dependent macrophage pyroptosis

  • 摘要: 背景 巨噬细胞是天然免疫系统的重要组成成分,其在抵抗呼吸道途径外来异物入侵和维持肺组织内环境稳态中发挥重要作用。

    目的 探讨不同粒径的二氧化硅(SiO2)粉尘诱导巨噬细胞焦亡发生机制。

    方法 将巨噬细胞RAW-ASC分为空白组、脂多糖(LPS)组(1 μg·mL−1 LPS)、纳米SiO2组(1 μg·mL−1 LPS + 100 μg·mL−1 纳米SiO2)、微米SiO2组(1 μg·mL−1 LPS +750 μg·mL−1 微米SiO2)和阳性对照组1 μg·mL−1 LPS+3 mmol·L−1 三磷酸腺苷(ATP)。除空白组外,其他组细胞均以LPS预处理6 h,再以SiO2或ATP处理4 h。锚定焦亡通路分子靶点NOD样受体蛋白3(NLRP3)、活性氧(ROS)施加特异性干预剂NLRP3抑制剂MCC950、ROS清除剂N-乙酰-L-半胱氨酸(NAC)。CCK-8法检测细胞活性;5-乙炔基-2' -脱氧尿苷(EdU)染色检测细胞增殖能力;试剂盒法检测细胞上清乳酸脱氢酶(LDH)水平;钙黄绿素/碘化丙啶(Calcein AM/PI)荧光双染评估细胞破裂状况;二氯二氢荧光素-乙酰乙酸酯(DCFH-DA)荧光探针法检测细胞内ROS水平;Western blotting检测焦亡相关标志NLRP3、凋亡相关斑点样蛋白(ASC)、半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)、gasdermin D(GSDMD)、白介素-1β(IL-1β)的蛋白表达水平。

    结果 与空白组相比,100 μg·mL−1纳米SiO2及750 μg·mL−1微米SiO2处理,分别使细胞活性降低至40%和68%(P<0.05),细胞增殖率降低至30%和33%(P<0.01),并且诱导细胞破裂与ROS释放,上调NLRP3、ASC、Caspase-1、GSDMD、IL-1β蛋白表达水平(P<0.05),诱导巨噬细胞发生焦亡。添加特异性干预剂MCC950(10 μmol·L−1)和NAC(10 mmol·L−1)后,巨噬细胞内NLRP3、ASC、Caspase-1、IL-1β表达水平均较未干预组下降(P<0.05)。此外,NAC还可有效降低细胞内ROS水平(P<0.05)。

    结论 纳米及微米级SiO2粉尘均具细胞毒性,表现为上调巨噬细胞ROS水平,激活NLRP3炎性小体并介导细胞焦亡,促进炎性细胞因子释放等,相关研究结果有助于揭示SiO2粉尘诱导的巨噬细胞焦亡分子机制。

     

    Abstract: Background Macrophages are essential components of the natural immune system. They play a significant role in resisting foreign bodies in the respiratory tract and maintaining the homeostasis of the internal environment of lung tissue.

    Objective To investigate the mechanism of macrophage pyroptosis induced by silica dust with different particle sizes.

    Methods The modified murine macrophage cell line, RAW-ASC cells, was cultured and divided into a blank control group, a lipopolysaccharide (LPS) group (1 μg·mL−1 LPS), a nano-SiO2 group (1 μg·mL−1 LPS+100 μg·mL−1 nano-SiO2), a micro-SiO2 group (1 μg·mL−1 LPS+750 μg·mL−1 micro-SiO2), and a positive control group 1 μg·mL−1 LPS+3 mmol·L−1 adenosine triphosphate (ATP). Apart from the blank control group, cells in other groups were pretreated with LPS for 6 h, and then exposed to SiO2 or ATP for 4 h. According to the molecular target NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and reactive oxygen species (ROS), we applied MCC950 (NLRP3 inhibitor) and N-acetyl cysteine (NAC, ROS scavenger) to macrophages. CCK-8 assay was used to detect cell viability; 5-ethynyl-2'-deoxyuridine (EdU) staining was used to detect cell proliferation; lactate dehydrogenase (LDH) assay kit was used to detect LDH in supernatant; calcein AM/PI fluorescent double-staining was applied to evaluate cell rupture; 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescent probe was used to measure the content of ROS; Western blotting was used to measure the expressions of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), Caspase-1, gasdermin D (GSDMD), and interleukin-1β (IL-1β).

    Results Compared with the blank group, 100 μg·mL-1nano-SiO2and 750 μg·mL-1micro-SiO2 dust exposure reduced the cell viability to 40% and 68% (P<0.05), and the cell proliferation rate to 30% and 33% (P<0.01), respectively; they also induced cell lysis and ROS release, upregulated NLRP3, ASC, Caspase-1, GSDMD, and IL-1β at protein level (P<0.05), and induced macrophage pyroptosis. After intervening with MCC950 (10 μmol·L-1) and NAC (10 mmol·L-1), the expressions of NLRP3, ASC, Caspase-1, and IL-1β decreased (P<0.05), and, specifically, NAC effectively reduced ROS levels (P<0.05).

    Conclusion Both nano- and micro-SiO2 dust have cytotoxicity, can upregulate ROS level, activate NLRP3 inflammasome, and promote the release of cytokines, leading to pyroptosis. These results are helpful to reveal the molecular mechanism of macrophage pyroptosis induced by SiO2 dust.

     

/

返回文章
返回