臭氧暴露诱发小鼠急性肺部损伤及抗氧化剂的拮抗作用

Acute Exposure of Ozone Induced Pulmonary Injury in Mice and the Antagonism of Antioxidant

为了研究臭氧(ozone,O_(3))诱发小鼠肺损伤的作用以及褪黑素(melatonin,MT)的保护机制,本研究以雄性Balb/c小鼠为受试动物,进行连续7 d、每天3 h的急性O_(3)暴露,在保护组每天口服暴露5 mg·kg^(-1)MT。实验小鼠随机分为4组:(1)生理盐水对照组(Control);(2)褪黑素对照组(MT);(3)臭氧暴露组(2.14 mg·m^(-3)O_(3));(4)臭氧暴露+褪黑素保护组(2.14 mg·m^(-3)O_(3)+MT)。最后一次臭氧暴露后,制备小鼠肺泡灌洗液,采用酶联免疫吸附法(ELISA)检测肺组织中IL-1β、IL-33、IL-4和IL-17A的含量;取部分肺组织制作肺匀浆并提取总mRNA,用于检测肺组织中丙二醛(MDA)和还原型谷胱甘肽(GSH)的含量,以及核因子E2相关因子2(Nrf2)、血红素加氧酶1(HO-1)、NADPH:醌氧化还原酶1(NQO-1)mRNA的表达。此外,采用苏木精-伊红染色(H&E stain)观察小鼠肺组织病理学改变。结果表明,与对照组相比,2.14 mg·m^(-3)O_(3)暴露组小鼠肺组织MDA含量上升,IL-1β、IL-33、IL-4和IL-17A表达增加;GSH含量以及Nrf2、HO-1、NQO-1的mRNA相对表达量下降,且差异具有统计学意义(P<0.05或P<0.01)。与O_(3)暴露组相比,MT保护组小鼠肺组织MDA含量下降,IL-1β、IL-33、IL-4和IL-17A表达下降,且GSH含量以及Nrf2、HO-1和NQO-1的mRNA相对表达量相应上升。此外,小鼠肺组织病理切片观察结果表明,O_(3)暴露组小鼠肺组织出现炎细胞浸润和气道重塑现象,使用抗氧化剂MT处理后可减轻相应病理学改变。综上所述,2.14 mg·m^(-3)O_(3)暴露可造成小鼠肺组织氧化损伤,诱发炎症,导致肺组织病理学改变;抗氧化剂的使用可增加Nrf2及其下游抗氧化酶的表达,缓解氧化应激,降低炎症因子水平,这验证了O_(3)暴露引起的肺部损伤是通过氧化性损伤机制介导的。

To investigate the effect of ozone(O_(3))exposure on lung injury in mice and the antagonism mechanism of melatonin(MT),male Balb/c mice were used for ozone exposure(2.14 mg·m^(-3))for 3 h per day,for seven days in this study.And melatonin(5 mg·kg^(-1))was administered as an antioxidant at 3 h after exposure to O_(3).The mice were randomly divided into four groups:(1)control group(Control);(2)melatonin control group(MT);(3)Ozone exposure group(2.14 mg·m^(-3)O_(3));(4)Ozone exposure+melatonin protection group(2.14 mg·m^(-3)O_(3)+MT).After the last-time ozone exposure,bronchoalveolar lavage fluid was obtained and used for detecting the contents of IL-1β,IL-33,IL-4 and IL-17A by ELISA.Part of lung tissue was prepared to make lung homogenate which was used to detect the contents of malondialdehyde(MDA)and glutathione(GSH).Nrf2 pathway involved mRNA were evaluated by RT-qPCR,such as nuclear factor E2-related factor^(2)(Nrf2),heme oxygenase 1(HO-1)and NADPH:quinone acceptor oxidoreductase 1(NQO-1).In addition,histopathological changes were also determined in lung tissues by Hematoxylin-Eosin stain(H&E stain).This study showed that O_(3)exposure increased the content of MDA,the mRNA expressions of IL-1β,IL-33,IL-4 and IL-17A.O_(3)exposure decreased GSH content and the mRNA expressions of Nrf2,NQO-1 and HO-1.Interestingly,melatonin not only markedly decreased the levels of oxidative stress and lung damage,but also enhanced the expression of Nrf2,NQO-1 and HO-1.Taken together,our results demonstrate that melatonin administration could antagonize ozone-induced lung inflammation and damage by stabilizing the Nrf2 pathway.