甲醛对嗜酸性粒细胞EOL-1的急性损伤作用机制

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。

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

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.