臭氧暴露导致人鼻黏膜上皮细胞中氧化应激相关基因表达的改变

Expression of oxidative stress-related genes in human nasal epithelium after ozone exposure

目的通过分析臭氧暴露后人鼻黏膜上皮细胞中氧化应激相关基因表达的变化,初步探索臭氧暴露引发慢性鼻窦炎的机制。方法建立人鼻黏膜上皮细胞臭氧暴露体外培养模型,用高通量PCR芯片技术对臭氧暴露前后鼻黏膜氧化应激相关基因进行检测,并用qRT-PCR进行验证。采用qRT-PCR、蛋白质印迹法及流式细胞术检测臭氧暴露前后培养细胞中环氧化酶2(Cox2)和活性氧(ROS)的表达。结果用PCR芯片对鼻黏膜上皮细胞中84个氧化应激相关基因进行筛查,发现高强度臭氧暴露后人鼻黏膜上皮细胞中醛酮还原酶家族1成员C2(AKR1C2)、谷氨酸半胱氨酸连接酶(GCLM)、谷胱甘肽过氧化物酶2(GPX2)、谷胱甘肽还原酶(GSR)、血红素加氧酶1(HMOX1)、NADPH氧化酶(NOX5)、前列腺素内过氧化物酶合酶2(PTGS2)、超氧化物歧化酶2(SOD2)及丝氨酸肽酶抑制剂(Kazal型)1(SPINK1)9个基因表达上调,而趋化因子配体5(CCL5)、细胞球蛋白(CYGB)、可溶性环氧化酶水解酶2(EPHX2)、谷胱甘肽S-转移酶zeta 1(GSTZ1)、角蛋白1(KRT1)、组氨酸磷酸酶(LHPP)、肌球蛋白(MB)、MPV17线粒体内膜蛋白(MPV17)、硒蛋白P(SEPP1)及转运蛋白颗粒复合物6A(TRAPPC6A)10个基因表达下调。同时,臭氧暴露导致ROS大量生成(暴露组及对照组荧光强度:184.3±6.8 vs 13.0±1.4,P<0.05),Cox2 mRNA及蛋白表达水平升高(暴露组与对照组mRNA相对表达量:6.4±1.2 vs 1.0±0.0,P<0.05;暴露组与对照组蛋白相对表达量:11.7±2.6 vs 13.8±1.5,P<0.05)。结论臭氧暴露后GSTZ1、NOX5、SOD2等相关基因参与鼻黏膜氧化应激调控,臭氧暴露引发鼻黏膜炎症反应可能与这些基因调控Cox2表达的相关信号通路有关。

Objective To explore the mechanism of ozone exposure-induced chronic rhinosinusitis by analyzing the alterations in the expression of oxidative stress-related genes in human nasal epithelium.Methods An in vitro cultured ozone exposure-induced human nasal epithelium model was established.The oxidative-stress-related genes of human nasal epithelium were detected by high-throughput polymerase chain reaction(PCR)chip before and after ozone exposure,and were verified by quantitative real-time polymerase chain reaction(qRT-PCR).The expressions of cyclooxygenase 2(Cox2)and reactive oxide species(ROS)were detected by qRT-PCR,Western blotting and flow cytometry.Results A total of 84 oxidativestress-related genes were screened by PCR chip in the human nasal epithelium.It was found that the expression of aldo-keto reductase family 1 member C2(AKR1 C2),glutamate-cysteine ligase modifier subunit(GCLM),glutathione peroxidase 2(GPX2),glutathione-disulfide reductase(GSR),heme oxygenase 1(HMOX1),NADPH oxidase 5(NOX5),prostaglandin-endoperoxide synthase 2(PTGS2),superoxide dismutase 2(SOD2),and serine peptidase inhibitor Kazal type 1(SPINK1)was up-regulated after intensive ozone exposure,while the expression of C-C motif chemokine ligand 5(CCL5),cytoglobin(CYGB),epoxide hydrolase 2(EPHX2),glutathione S-transferase zeta 1(GSTZ1),keratin 1(KRT1),phospholysine phosphohistidine inorganic pyrophosphate phosphatase(LHPP),myoglobin(MB),mitochondrial inner membrane protein MpV17(MPV17),selenoprotein P(SEPP1),and trafficking protein particle complex 6 A(TRAPPC6 A)was down-regulated.At the same time,ozone exposure resulted in a large amount of ROS(fluorescence intensity of the exposure group and control group:184.3±6.8 vs 13.0±1.4,P<0.05)and increased expressions of Cox2 mRNA and protein(relative expression of mRNAs:6.4±1.2 vs 1.0±0.0,P<0.05;relative protein expression:11.7±2.6 vs 13.8±1.5,P<0.05).Conclusion After ozone exposure,GSTZ1,NOX5,SOD2 and other related genes are involved in the regulation of oxidative stress in nasal epithelium.The inflammatory response of nasal epithelium induced by ozone exposure may be related to the signaling pathways of Cox2 expression that regulated by these genes.