吕达, 杨慧楠, 刘春城, 王乐, 赵宏宇, 蔡禄. 二氧化硅染尘小鼠肺纤维化相关炎症和氧化应激因子的响应及关键基因表达[J]. 环境与职业医学, 2021, 38(10): 1150-1155. DOI: 10.13213/j.cnki.jeom.2021.21061
引用本文: 吕达, 杨慧楠, 刘春城, 王乐, 赵宏宇, 蔡禄. 二氧化硅染尘小鼠肺纤维化相关炎症和氧化应激因子的响应及关键基因表达[J]. 环境与职业医学, 2021, 38(10): 1150-1155. DOI: 10.13213/j.cnki.jeom.2021.21061
LYU Da, YANG Huinan, LIU Chuncheng, WANG Le, ZHAO Hongyu, CAI Lu. Responses of inflammatory factors and oxidative stress factors and expressions of key genes associated with pulmonary fibrosis in silica-exposed mice[J]. Journal of Environmental and Occupational Medicine, 2021, 38(10): 1150-1155. DOI: 10.13213/j.cnki.jeom.2021.21061
Citation: LYU Da, YANG Huinan, LIU Chuncheng, WANG Le, ZHAO Hongyu, CAI Lu. Responses of inflammatory factors and oxidative stress factors and expressions of key genes associated with pulmonary fibrosis in silica-exposed mice[J]. Journal of Environmental and Occupational Medicine, 2021, 38(10): 1150-1155. DOI: 10.13213/j.cnki.jeom.2021.21061

二氧化硅染尘小鼠肺纤维化相关炎症和氧化应激因子的响应及关键基因表达

Responses of inflammatory factors and oxidative stress factors and expressions of key genes associated with pulmonary fibrosis in silica-exposed mice

  • 摘要: 背景

    二氧化硅(SiO2)长期沉积在肺组织中能够诱发炎症与纤维化反应,进而导致尘肺病,但其发病机制尚未明确。

    目的

    探究SiO2对小鼠肺部纤维化的影响及其相关机制。

    方法

    将35只C57BL/6小鼠随机分成空白对照组、生理盐水对照组、20 g·L-1 SiO2染尘组、60 g·L-1 SiO2染尘组4个组别,其中空白对照组5只,其余组别各10只。采用非气管暴露法在第0天和第14天进行2次染尘,按照小鼠体重灌注不同体积的SiO2悬浊液。在染尘第28天和第56天时取小鼠肺组织和血清。10倍光学显微镜下观察肺组织HE切片;ELISA法检测肺组织中小鼠成纤维生长因子(FGF)、肿瘤坏死因子-α(TNF-α)和转化生长因子-β(TGF-β)3种炎症因子的质量分数(后简称含量),检测血清中超氧化物歧化酶(SOD)、活性氧(ROS)、丙二醛(MDA)3种氧化应激因子的水平;10倍显微镜下观察Masson染色切片胶原纤维分布;分光光度计检测肺组织中羟脯氨酸含量;实时荧光定量PCR技术检测肺组织中Co/1a2α-SMA mRNA表达水平的变化。

    结果

    HE染色结果显示:SiO2染尘组与生理盐水对照组相比有明显的炎症细胞浸润,部分出现纤维结节。炎症因子检测结果显示:20 g·L-1和60 g·L-1染尘组小鼠在染尘第56天时,TGF-β、FGF、TNF-α含量分别是3.42、3.62 ng·g-1,15.20、19.78 ng·g-1和9.02、11.30 ng·g-1,较生理盐水组均有增高,差异有统计学意义(P < 0.01)。氧化应激因子检测结果显示:小鼠20 g·L-1染尘组在染尘第28和56天时,MDA浓度分别是0.23、0.26μmol·L-1,ROS水平均为11U·L-1,SOD质量浓度分别0.43、0.44μg·L-1;小鼠60g·L-1染尘组在染尘第28和56天时,MDA浓度为0.28、0.34 μmol·L-1,ROS水平分别是13、14 U·L-1,SOD质量浓度分别0.38、0.36 μg·L-1;相比于生理盐水对照组,在两个时间点两个浓度染尘组小鼠血清中MDA、ROS水平增加,SOD水平下降,差异均有统计学意义(P < 0.01)。在染尘第28和56天时,20 g·L-1染尘组的小鼠羟脯氨酸含量为0.25、0.28 mg·g-1,60 g·L-1染尘组为0.32、0.43 mg·g-1,较生理盐水组升高,差异均有统计学意义(P < 0.01)。观察Masson染色切片,在第28、56天时,染尘组小鼠肺组织相较于生理盐水对照组胶原面积增大。基因表达水平检测结果发现:小鼠60 g·L-1染尘组在染尘第28、56天时,相较于生理盐水组,Co/1a2基因表达量上升至1.62倍和3.08倍,α-SMA基因表达量分别上升至2.05倍和3.34倍,差异均有统计学意义(P < 0.01)。

    结论

    SiO2染尘小鼠肺部产生纤维化的进程中,可导致肺部出现炎症反应、氧化应激反应以及肺组织中胶原沉积等综合效应。

     

    Abstract: Background

    It is well known that long-term deposition of silicon dioxide (SiO2) in lung tissues can induce inflammation and fibrosis and result in pneumoconiosis, but pathogenesis of pneumoconiosis remains elusive.

    Objective

    This experiment investigates the effect of SiO2 on pulmonary fibrosis in mice.

    Methods

    Thirty-five C57BL/6 mice were randomly divided into four groups: a blank control group (5 mice), a normal saline control group (10 mice), a 20 g·L-1 SiO2 group (10 mice), and a 60 g·L-1 SiO2 group (10 mice). The mice were exposed by intratracheal instillation of SiO2 suspension on day 0 and day 14, and the lung tissue and serum samples were collected on day 28 and day 56 after silica or saline instillation. HE sections of lung tissues were observed under optical microscope (10×magnification). ELISA was used to detect the levels of three inflammatory cytokines including fibroblast growth factor (FGF), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β) in lung tissues, and three oxidative stress factors including superoxide dismutase (SOD), reactive oxygen species (ROS), and malondialdehyde (MDA) in serum. The distributions of collagen fibers in Masson-stained sections were observed under microscope (10×magnification). The level of hydroxyproline (HYP) in lung tissues was detected with a spectrophotometer. The expressions of Co/1a2 and α-SMA mRNA in lung tissues were measured by real-time PCR.

    Results

    The results of HE staining showed that there were obvious infiltration of inflammatory cells and some fibrous nodules in the two SiO2 groups compared with the normal saline control group. The detection results of inflammatory factors indicated that on day 56, the levels of TGF-β in lung tissues of the 20 g·L-1 SiO2 group and the 60 g·L-1 SiO2 group were 3.42 and 3.62 ng·g-1, the levels of FGF were 15.20 and 19.78 ng·g-1, the levels of TNF-α were 9.02 and 11.30 ng·g-1, respectively, all higher than those of the normal saline group (P < 0.01). The detection results of oxidative stress factors showed that the levels of MDA in serum of the 20 g·L-1 SiO2 group on day 28 and day 56 were 0.23 and 0.26 μmol·L-1, the levels of ROS were both 11 U·L-1, and the levels of SOD were 0.43 and 0.44 μg·L-1, respectively; in the 60g·L-1 SiO2 group on day 28 and day 56, the levels of MDA were 0.28 and 0.34μmol·L-1, the levels of ROS were 13 and 14U·L-1, and the levels of SOD were 0.38 and 0.36 μg·L-1, respectively. The serum levels of MDA and ROS were higher and the level of SOD was lower in the two SiO2 groups than in the normal saline control group (P < 0.01). The levels of HYP on day 28 and day 56 were 0.25 and 0.28 mg·g-1 in the 20 g·L-1 SiO2 group, and 0.32 and 0.43 mg·g-1 in the 60 g·L-1 SiO2 group, respectively, all higher than those in the normal saline control group (P < 0.01). Compared with the normal saline control group, the Masson staining sections demonstrated that the collagen areas in lung tissues of the mice exposed to SiO2 on day 28 and day 56 were obviously increased. The relative expression levels of Co/1a2 mRNA in the 60 g·L-1 SiO2 group on day 28 and day 56 were 1.62 and 3.08 times higher than those in the normal saline control group, respectively; the relative expression levels of α-SMA mRNA were 2.05 and 3.34 times higher, respectively (P < 0.01).

    Conclusion

    There exist combined effects of inflammation, oxidative stress, and collagen deposition in lung tissues during silica-induced pulmonary fibrosis in mice.

     

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