谢利, 李伟, 马继轩, 程曼, 徐以菊, 樊烈阳, 陈卫红. TAM/Gas6通路在石英粉尘致小鼠肺组织细胞凋亡中的作用[J]. 环境与职业医学, 2020, 37(6): 573-578. DOI: 10.13213/j.cnki.jeom.2020.19878
引用本文: 谢利, 李伟, 马继轩, 程曼, 徐以菊, 樊烈阳, 陈卫红. TAM/Gas6通路在石英粉尘致小鼠肺组织细胞凋亡中的作用[J]. 环境与职业医学, 2020, 37(6): 573-578. DOI: 10.13213/j.cnki.jeom.2020.19878
XIE Li, LI Wei, MA Ji-xuan, CHENG Man, XU Yi-ju, FAN Lie-yang, CHEN Wei-hong. Role of TAM/Gas6 pathway in apoptosis in mouse lung tissues induced by silica[J]. Journal of Environmental and Occupational Medicine, 2020, 37(6): 573-578. DOI: 10.13213/j.cnki.jeom.2020.19878
Citation: XIE Li, LI Wei, MA Ji-xuan, CHENG Man, XU Yi-ju, FAN Lie-yang, CHEN Wei-hong. Role of TAM/Gas6 pathway in apoptosis in mouse lung tissues induced by silica[J]. Journal of Environmental and Occupational Medicine, 2020, 37(6): 573-578. DOI: 10.13213/j.cnki.jeom.2020.19878

TAM/Gas6通路在石英粉尘致小鼠肺组织细胞凋亡中的作用

Role of TAM/Gas6 pathway in apoptosis in mouse lung tissues induced by silica

  • 摘要: 背景

    石英粉尘吸入肺部后被肺泡巨噬细胞吞噬,触发内源性或外源性凋亡信号通路,导致肺损伤。近年来TAM/生长抑制特异性蛋白6(Gas6)通路与细胞凋亡的关系成为研究热点。Mer是TAM的主要受体之一,前期研究显示Mer可能参与石英粉尘诱导的肺炎性反应及纤维化。

    目的

    通过动物实验探究TAM/Gas6通路在石英粉尘致细胞凋亡中的作用机制。

    方法

    实验采用3种雄性C57BL/6小鼠:野生型(WT),Gas6-/-Mer-/-基因敲除小鼠,各种小鼠按照体重随机分为对照组和石英染尘组,共6组,每组24只。对照组气管单次滴注50 μL生理盐水,石英染尘组单次滴注50 μL的石英粉尘悬液(50 μg·μL-1),分别在第7天、第28天、第84天各组处死8只小鼠并收集肺泡灌洗液(BALF)和肺组织。采用相应试剂盒检测BALF中的总蛋白(TP)、乳酸脱氢酶(LDH)、碱性磷酸酶(AKP)、酸性磷酸酶(ACP)水平;采用实时荧光定量PCR检测肺组织细胞凋亡因子Bcl2Bax mRNA表达水平;采用Western blotting检测Bcl2、Bax、半胱天冬酶-3(caspase-3)、活化型半胱天冬酶-3(cleaved-caspase-3)蛋白表达水平。

    结果

    与WT小鼠对照组相比,WT小鼠染尘组在上述三个时间点BALF中TP、LDH、AKP、ACP水平均升高,抗凋亡因子Bcl2蛋白水平降低,促凋亡因子Bax蛋白水平升高,Bcl2与Bax的mRNA和蛋白比值均下降(P < 0.05)。与WT小鼠染尘组比较,Gas6Mer基因敲除小鼠在染尘后各时间点BALF中TP(除第84天)、LDH(除第28、84天)、AKP、ACP水平均明显下降,抗凋亡因子Bcl2蛋白表达升高,促凋亡因子Bax蛋白水平下降,Bcl2与Bax的mRNA和蛋白比值均升高,cleaved-caspase-3与caspase-3蛋白比值下降(P < 0.05)。

    结论

    石英粉尘可诱导小鼠肺损伤和肺组织细胞凋亡水平升高。阻断Gas6Mer基因后,可以抑制肺组织细胞凋亡水平,缓解石英粉尘引起的小鼠肺组织损伤。

     

    Abstract: Background

    After being inhaled into the lungs, silica dust is ingested by alveolar macrophages, which triggers endogenous or exogenous apoptotic signaling pathways and leading to lung injury. In recent years, the relationship between TAM/growth arrest-specific protein 6 (Gas6) pathway and apoptosis has become a research hotspot. Previous studies have shown that Mer, one of TAM's main receptors, may be involved in the inflammatory response and fibrosis induced by silica.

    Objective

    This animal experiment aims to investigate the mechanism of TAM/Gas6 pathway on the apoptosis induced by silica.

    Methods

    Three kinds of male C57BL/6 mice were used in the experiment, including wild-type (WT) mice, Gas6-/- mice, and Mer-/- mice. The mice of each kind were randomly divided into a control group and a silica group with body weight matched, and there were a total of six groups with 24 mice in each group. The mice in the control groups were subjected to 50 μL normal saline instillation, while the mice in the silica groups received 50 μL silica suspension instillation (50 μg·μL-1). Eight mice were sacrificed on day 7, 28, and 84, respectively, and bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected. Total protein (TP), lactate dehydrogenase (LDH), alkaline phosphatase (AKP), and acid phosphatase (ACP) levels were measured with corresponding kits; mRNA expression levels of apoptotic factors Bcl2 and Bax were detected by quantitative real-time PCR; protein expression levels of Bcl2, Bax, caspase-3, and cleaved-caspase-3 were detected by Western blotting.

    Results

    Compared with the WT control group, the levels of TP, LDH, AKP, and ACP in BALF were significantly elevated, the protein expression level of anti-apoptotic factor Bcl2 was decreased, the protein expression level of pro-apoptotic factor Bax was increased, and the mRNA and protein expression ratios of Bcl2 and Bax were decreased in the WT silica group at the three designed time points (P < 0.05). Compared with the WT silica group, the levels of TP (except day 84), LDH (except day 28 and 84), AKP, and ACP were largely decreased, the protein expression level of anti-apoptotic factor Bcl2 was increased, the protein expression level of pro-apoptotic factor Bax was decreased, the mRNA and protein expression ratios of Bcl2 to Bax were increased, and the protein expression ratio of cleaved-caspase-3 to caspase-3 was decreased in the Gas6-/- or Mer-/- silica group at the three designed time points (P < 0.05).

    Conclusion

    Silica can induce injury and apoptosis in mouse lung tissues. Blockade of Gas6 or Mer gene can partially inhibit the level of apoptosis and alleviate the lung tissue damage in mice caused by silica.

     

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