【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。更多还原

【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.更多还原