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山东大学学报 (医学版) ›› 2022, Vol. 60 ›› Issue (11): 54-62.doi: 10.6040/j.issn.1671-7554.0.2022.0797

• 基础医学 • 上一篇    下一篇

甲醛对嗜酸性粒细胞EOL-1的急性损伤作用机制

李娜1,郭增丽2,迟令懿3,杨立卓2,马志勇1,付志婕2   

  1. 1.山东大学齐鲁医院心内科 教育部和国家卫健委心血管重构与功能研究重点实验室, 山东 济南 250012;2.山东第一医科大学第一附属医院耳鼻咽喉科, 山东 济南 250014;3.山东大学齐鲁医院神经外科, 山东 济南 250012
  • 出版日期:2022-11-10 发布日期:2022-11-04
  • 通讯作者: 付志婕. E-mail:fuzhijie104@126.com
  • 基金资助:
    国家自然科学基金(81700891);山东省自然科学基金(ZR2020MH038);山东第一医科大学学术提升计划(2019QL015)

Mechanism of acute injury of eosinophil EOL-1 induced by formaldehyde

LI Na1, GUO Zengli2, CHI Lingyi3, YANG Lizhuo2, MA Zhiyong1, FU Zhijie2   

  1. 1. Department of Cardiology, Qilu Hospital of Shandong University, The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, Jinan 250012, Shandong, China;
    2. Department of Otorhinolaryngology, The First Affiliated Hospital of Shandong First Medical University, Jinan 250014, Shandong, China;
    3. Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan 250012, Shandong, China
  • Online:2022-11-10 Published:2022-11-04

摘要: 目的 探讨甲醛对人嗜酸性粒细胞(EOL-1)的急性损伤作用及相关机制。 方法 体外培养EOL-1细胞,将甲醛室温处理2 h的EOL-1细胞设置为0 mmol组、5 mmol组、10 mmol组、25 mmol组及50 mmol组;同时将3种活性氧(ROS)通路抑制剂(NADPH氧化酶抑制剂DPI、细胞透性超氧化物清除剂Tiron、谷胱甘肽稳定前体NAC)和 25 mmol甲醛共同处理后的细胞设置为对照组、甲醛组、甲醛+DPI组、甲醛+Trion组和甲醛+NAC组;依据NAC浓度的不同,设置为对照组、甲醛组、甲醛+0.01 mmol NAC组、甲醛+0.1 mmol NAC组和甲醛+1 mmol NAC组。采用碘化丙啶(PI)和Hoechst荧光染色法检测各组细胞凋亡和死亡的表达,采用罗丹明123(Rho-123)荧光标记法检测细胞线粒体功能损伤,采用Western blotting法检测损伤信号通路蛋白Bax、Bcl-2的表达。 结果 不同浓度甲醛组(0、5、10、25、50 mmol)细胞死亡率分别为(3.313±2.395)%、(8.205±5.719)%、(20.335±5.167)%、(19.387±6.056)%、(28.043±8.851)%,与0 mmol组比较,5 mmol组细胞凋亡和死亡无增加(P=1.00),但是10 mmol组(P=0.030)、25 mmol组(P=0.033)和50 mmol组(P=0.001)甲醛处理细胞死亡显著增加,其半数效应浓度EC50=25 mmol;甲醛组、甲醛+DPI组、甲醛+Trion组、甲醛+NAC组细胞死亡率分别为(61.430±9.885)%、(57.907±13.619)%、(55.700±18.487)%、(21.837±6.674)%,与甲醛组比较,甲醛+DPI组(P=1.00)、甲醛+Trion组(P=1.00)对甲醛诱导的细胞损伤无影响,但是甲醛+NAC组逆转甲醛诱导的细胞死亡(P=0.01);与甲醛组(52.853±11.338)%对比,随着NAC浓度的不同,细胞死亡率不同[甲醛+0.01 mmol NAC组(10.620±4.483)%,甲醛+0.1 mmol NAC组(6.257±6.265)%,甲醛+1 mmol NAC组(4.002±2.50)%],NAC对甲醛诱导的细胞死亡呈浓度依赖性的逆转作用。Rho-123荧光标记结果显示,与0 mmol组相比, 10 mmol组、25 mmol组、50 mmol组可以降低线粒体功能(P<0.001),而甲醛+NAC组逆转甲醛诱导的线粒体功能损伤(809.339±163.210 vs 675.552±126.993,P=0.021)。Western blotting结果显示,与对照组比较,甲醛25 mmol组能下调Bcl-2蛋白表达(0.401±0.122,P<0.001),上调Bax蛋白表达(2.937±1.388,P=0.006),甲醛+NAC组可明显减轻甲醛诱导的Bax蛋白的表达上调(1.196±0.597,P=0.018)和Bcl-2蛋白的表达下调(0.717±0.246,P=0.018)。 结论 甲醛≥10 mmol通过抑制线粒体功能和调控Bcl-2/Bax信号通路诱导嗜酸性粒细胞EOL-1的损伤,而抗氧化剂NAC可减轻甲醛对EOL-1细胞的损伤及其信号通路。

关键词: 甲醛, EOL-1细胞, 细胞死亡, 线粒体功能, 信号通路

Abstract: Objective To explore the acute injury effect of formaldehyde on human eosinophils(EOL-1)and the mechanism. Methods EOL-1 cells were cultured in vitro. Eol-1 cells treated with formaldehyde for 2 hours at room temperature were set as the 0 mmol, 5 mmol, 10 mmol, 25 mmol and 50 mmol groups. Cells co-treated with DPI, Tiron and NAC and 25 mmol formaldehyde were divided into control group, formaldehyde group, formaldehyde + DPI group, formaldehyde + Trion group, and formaldehyde + NAC group. According to the different concentrations of NAC, the cells were set as the control group, formaldehyde group, formaldehyde + 0.01 mmol NAC group, formaldehyde + 0.1 mmol NAC group, and formaldehyde +1 mmol NAC group. Cell apoptosis and death were detected with propidium iodide(PI)and Hoechst fluorescent staining. The mitochondrial function damage was detected with rhodamine 123(Rho-123)fluorescent labeling method. The expressions of Bax and Bcl-2 were detected with Western blotting. Results The cell death rates of different concentrations of formaldehyde(0, 5, 10, 25, 50 mmol)were(3.313±2.395)%,(8.205±5.719)%,(20.335±5.167)%,(19.387±6.056)%, and(28.043±8.851)%, respectively. Compared with the 0 mmol group, the 5 mmol group had unchanged cell apoptosis and death(P=1.00), but the 10 mmol(P=0.030), 25 mmol(P=0.033)and 50 mmol groups(P=0.001)had significantly increased cell death, with EC50=25 mmol. The cell mortality rates of formaldehyde group, formaldehyde + DPI group, formaldehyde + Trion group and formaldehyde + NAC group were(61.430±9.885)%,(57.907±13.619)%,(55.700±18.487)% and(21.837±6.674)%, respectively. Formaldehyde + DPI group(P=1.00)and formaldehyde + Trion group(P=1.00)had no effect on formaldehyde-induced cell damage, but formaldehyde + NAC group reversed the formaldehyde-induced cell death(P=0.01). Compared with formaldehyde group(52.853±11.338)%, with different concentrations of NAC, the cell mortality was different [formaldehyde + 0.01 mmol NAC(10.620±4.483)%, formaldehyde + 0.1 mmol NAC(6.257±6.265)%, formaldehyde + 1 mmol NAC(4.002±2.50)%], and the reverse effect of formaldehyde on cell death was concentration-dependent. The results of Rho-123 fluorescence labeling showed that compared with the 0 mmol group, the 10 mmol, 25 mmol, and 50 mmol groups had reduced mitochondrial function(P<0.001), while formaldehyde + NAC group reversed the mitochondrial function damage induced by formaldehyde(809.339±163.210 vs 675.552±126.993, P=0.021). Western blotting results showed that, compared with the control group, 25 mmol formaldehyde significantly down-regulated the protein expression of Bcl-2(0.401±0.122, P<0.001)but up-regulated the protein expression of Bax(2.937±1.388, P=0.006); formaldehyde + NAC group significantly reduced the protein expressions of Bax(1.196±0.597, P=0.018)and Bcl-2(0.717±0.246, P=0.018). Conclusion Formaldehyde ≥10 mmol can induce eosinophil EOL-1 damage by inhibiting mitochondrial function and regulating Bcl-2/Bax signal pathway, while antioxidant NAC can reduce the damage and signal pathway of eosinophil EOL-1 caused by formaldehyde.

Key words: Formaldehyde, EOL-1 cells, Cell death, Mitochondrial function, Signal pathway

中图分类号: 

  • R392.8
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