烟酰胺腺嘌呤二核苷酸磷酸氧化酶-活性氧氧化应激信号通路在过敏反应中的作用及抗氧化剂干预实验研究

Role of Noxs-ROS oxidative stress signaling pathway in allergic reaction and intervention of antioxidants

目的:探讨烟酰胺腺嘌呤二核苷酸磷酸氧化酶(Noxs)-活性氧(ROS)氧化应激通路在过敏反应中的作用,并考察抗氧化剂对过敏反应的影响。方法:体外建立肥大细胞脱颗粒模型,将细胞分为空白组、阳性对照组[培养基中含有Compound48/80 (C48/80) 100μmol/L]和抗氧化剂组[培养基中含有C48/80 100μmol/L+二苯基氯化碘盐(DPI) 5μmol/L]。利用酶联免疫吸附试剂盒检测各组细胞胞内β氨基己糖苷酶(β-Hex)、组胺、超氧化物歧化酶(SOD)、脂质过氧化物(LPO)及炎症介质白细胞介素-4(IL-4)和IL-6水平。利用荧光染色及流式细胞仪检测胞内活性氧(ROS)水平。采用Western blot检测三组细胞中烟酰胺腺嘌呤二核苷酸磷酸氧化酶1、2、3、4(Nox1、Nox2、Nox3、Nox4)蛋白表达情况。结果:C48/80诱导大鼠原代嗜碱性白血病细胞(RBL-2H3)释放β-Hex、组胺及炎症介质IL-4、IL-6,RBL-2H3细胞脱颗粒过程中胞内的SOD含量呈下降趋势,而LPO及ROS浓度升高。利用DPI处理脱颗粒的RBL-2H3细胞后,可以抑制胞内ROS水平,同时升高胞内SOD含量,降低LPO浓度,并且抑制了β-Hex、组胺、IL-4及IL-6的释放。当肥大细胞脱颗粒时,Nox1-4的蛋白表达均呈上升趋势,抗氧化剂DPI可以抑制这一反应。结论:阻断Noxs-ROS信号通路可以抑制过敏反应,抗氧化剂的应用为抗过敏提供了新的治疗思路。

Objective:To explore the role of nicotinamide adenine dinucleotide phosphate hydrogen oxidases(Noxs)-reactive oxygen species(ROS)oxidative stress signaling pathway in allergic reaction and the effect of antioxidants on allergic reaction.Methods:The degranulation model of mast cells was established in vitro.The cells were divided into blank group,positive control group(the culture medium contained C48/80100μmol/L),and antioxidant group(containing C48/80100μmol/L+DPI 5μmol/L).Enzyme-linked immunosorbent assay kits were used to detect the levels of intracellularβ-Hex,histamine,SOD,L PO and inflammatory mediators IL-4 and IL-6 in each group.The intracellular ROS level was detected by fluorescence staining and flow cytometry.Western blot was used to detect the protein expressions of Nox1,Nox2,Nox3 and Nox4 in the three groups of cells.Results:C48/80 induced the release ofβ-Hex,histamine,IL-4 and IL-6 in RBL-2H3 cells,as well as the concentrations of LPO and ROS,but the content of intracellular SOD was decreased.DPI could inhibit the level of intracellular ROS,β-Hex,histamine,IL-4 and IL-6 in RBL-2H3 cells treated with C48/80 and the content of SOD was increased.The protein expressions of Nox1-4 were increased when mast cells degranulate,and the antioxidant DPI can inhibit this response.Conclusion:Blocking the Noxs-ROS signaling pathway can inhibit allergic response,and the application of antioxidants provides a new idea for anti-allergic treatment.