何邵波, 蒋传命. 镉诱导小鼠原代神经细胞程序性坏死的机制[J]. 环境与职业医学, 2021, 38(5): 536-541. DOI: 10.13213/j.cnki.jeom.2021.20453
引用本文: 何邵波, 蒋传命. 镉诱导小鼠原代神经细胞程序性坏死的机制[J]. 环境与职业医学, 2021, 38(5): 536-541. DOI: 10.13213/j.cnki.jeom.2021.20453
HE Shaobo, JIANG Chuanming. Mechanism of cadmium-induced necroptosis of mouse primary nerve cells[J]. Journal of Environmental and Occupational Medicine, 2021, 38(5): 536-541. DOI: 10.13213/j.cnki.jeom.2021.20453
Citation: HE Shaobo, JIANG Chuanming. Mechanism of cadmium-induced necroptosis of mouse primary nerve cells[J]. Journal of Environmental and Occupational Medicine, 2021, 38(5): 536-541. DOI: 10.13213/j.cnki.jeom.2021.20453

镉诱导小鼠原代神经细胞程序性坏死的机制

Mechanism of cadmium-induced necroptosis of mouse primary nerve cells

  • 摘要: 背景

    镉是一种常见的重金属污染物,以往的研究主要聚焦其影响细胞自噬和凋亡,但其是否导致细胞程序性坏死尚不明确。

    目的

    研究镉诱导小鼠原代神经细胞程序性坏死的机制。

    方法

    以小鼠原代神经细胞为实验对象,10 μmol·L-1氯化镉分别处理细胞2、4、6、12、24、48 h,用台盼蓝染色法计算细胞存活率;采用流式细胞仪检测经10 μmol·L-1氯化镉处理后细胞周期的变化(24、48 h)及细胞程序性坏死水平(48 h);实时荧光定量PCR法检测经10 μmol·L-1氯化镉处理48 h后细胞程序性坏死相关基因如受体相互作用丝氨酸苏氨酸激酶3(RIPK3)、混合系列蛋白激酶样结构域蛋白(MLKL)的表达;蛋白印迹法检测5、10、20 μmol·L-1镉离子处理后自噬相关蛋白p62的蛋白表达水平;检测p62和自噬微管相关蛋白1轻链3-B(LC3-B)的荧光强度。

    结果

    小鼠原代神经细胞经10 μmol·L-1氯化镉处理4、6、12、24、48 h后细胞活力下降(P < 0.05);相比较对照组(49.62%),细胞经10 μmol·L-1氯化镉处理24 h和48 h后分别有60.88%和82.94%的细胞停留在G0-G1期;10 μmol·L-1氯化镉处理48 h后,细胞程序性坏死水平(47.50%)高于对照组(0.01%),且细胞程序性坏死相关基因RIPK3MLKL转录水平是对照组的6.9倍、3.7倍。经5、10、20 μmol·L-1氯化镉处理后小鼠原代神经元细胞自噬相关蛋白p62表达分别上调(587±17)%、(609±14)%、(893±16)%。经10 μmol·L-1氯化镉处理后细胞内自噬相关蛋白p62和LC3-B在细胞内共定位,且荧光强度高于对照组。

    结论

    镉离子能降低小鼠原代神经元细胞存活率,使细胞周期停滞在G0-G1期且提高细胞程序性坏死水平,可能与p62和LC3-B表达水平升高阻断细胞自噬流有关。

     

    Abstract: Background

    Cadmium is a common heavy metal pollutant. Previous studies mainly focus on cadmium-induced autophagy and apoptosis, but whether it leads to cell necroptosis remains unclear.

    Objective

    This study investigates the mechanism of cadmium-induced necroptosis of mouse primary nerve cells.

    Methods

    Mouse primary neurons were treated with 10 μmol·L-1 cadmium chloride for 2, 4, 6, 12, 24, and 48 h, respectively. The cell survival rate was calculated by Trypan blue staining. The changes of cell cycle (24 and 48 h) and the rate of cell necroptosis (48 h) after treatment with 10 μmol·L-1 cadmium chloride were detected by flow cytometry. The expressions of genes related to cell necroptosis such as receptor interacting serine threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) after being treated with 10 μmol·L-1 cadmium chloride for 48 h were measured by real-time fluorescence quantitative PCR. The protein expression of autophagy-related factor p62 and the fluorescence intensities of p62 and microtubule-associated protein 1 light 3-B (LC3-B) after 10 μmol·L-1 cadmium chloride treatment were analyzed by Western blotting and fluorescence observation.

    Results

    The cell viability of mouse primary nerve cells were decreased after 4, 6, 12, 24, and 48 h treatment with 10 μmol·L-1 cadmium chloride (P < 0.05). Compared with the control group (49.62%), 60.88% and 82.94% of cells were arrested in the G0-G1 phase after treatment with 10 μmol·L-1 cadmium chloride for 24 h and 48 h, respectively. The flow cytometry results showed that when being treated with 10 μmol·L-1 cadmium chloride for 48 h, the rate of cell necroptosis was higher (47.5%) than that of the control group (0.01%), and the transcription of cell necroptosis related genes such as RIPK3 and MLKL were 6.9 and 3.7 times of the control group. The results of Western blotting showed that the expression of autophagyrelated protein p62 in mouse primary neurons were upregulated by (587±17)%, (609±14)%, and (893±16)% when being treated with 5, 10, and 20 μmol·L-1 cadmium chloride respectively. Further fluorescence observation showed that p62 and LC3-B were colocalizated and the fluorescence intensity levels of them were increased after cadmium treatment.

    Conclusion

    Cadmium treatment could reduce the survival rate of mouse primary neurons, arrest the cell cycle in G0-G1 phase, and increase the necroptosis rate. The mechanism by which cadmium induce necroptosis may be related to the increased expressions of p62 and LC3-B and then the blocked autophagy flow.

     

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