褪黑素通过调控Nrf2通路对甲醛暴露致大鼠急性肺损伤的保护作用

Protective effect of melatonin on formaldehyde exposure-induced acute lung injury in rats through modulation of the Nrf2 pathway

目的探讨褪黑素(MT)通过调控核因子E2相关因子2(Nrf2)信号通路对甲醛(FA)吸入致大鼠急性肺损伤(ALI)的保护作用及其机制。方法50只雌性Wistar大鼠随机分为Control组、FA组、FA+MT 5 mg/kg组、FA+MT 10 mg/kg组和FA+MT 20 mg/kg组,每组10只。除Control组外,其他各组连续21 d每天吸入3 mg/m 3 FA以构建染毒模型,然后用不同MT剂量治疗14 d,MT治疗期间继续染毒。苏木精-伊红(HE)染色观察肺组织病理学变化,称重测肺水含量和肺系数,吸光光度法测肺组织谷胱甘肽(GSH)、超氧化物歧化酶(SOD)和8-羟基脱氧鸟苷(8-OHdG)水平,酶联免疫吸附实验(ELISA)测肺泡灌洗液肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-6和IL-1β的浓度,Western blot检测肺组织Nrf2、血红素氧合酶-1(HO-1)、核因子-κB(NF-κB)和磷酸化核因子-κB(p-NF-κB)的蛋白表达水平,定量聚合酶链反应(qPCR)检测肺组织Nrf2、HO-1及Kelch样ECH相关蛋白1(Keap1)的mRNA表达水平。结果与Control组相比,FA组大鼠出现明显肺损伤;肺组织GSH、SOD水平下降,8-OHdG水平升高(P<0.05);肺泡灌洗液TNF-α、IL-6和IL-1β水平升高(P<0.05);肺组织Nrf2、HO-1蛋白表达水平降低(P<0.05),p-NF-κB蛋白表达水平升高(P<0.05);肺组织Nrf2、HO-1的mRNA相对表达量降低,Keap1的mRNA相对表达量升高(P<0.05)。与FA组相比,MT组大鼠肺损伤有好转;肺组织GSH、SOD水平升高(P<0.05),8-OHdG水平下降(P<0.05);肺泡灌洗液TNF-α、IL-6和IL-1β水平下降(P<0.05);肺组织Nrf2、HO-1蛋白表达水平升高(P<0.05),p-NF-κB蛋白表达水平降低(P<0.05);肺组织Nrf2、HO-1的mRNA相对表达量升高(P<0.05),Keap1的mRNA相对表达量降低(P<0.05),且均呈剂量依赖性。结论MT可以通过调控Nrf2/Keap1/HO-1信号通路减轻氧化应激和炎症反应,缓解FA暴露诱导的急性肺损伤。

Objective To investigate the protective effect of melatonin(MT)on formaldehyde(FA)inhalation-induced acute lung injury(ALI)in rats and its mechanism through the regulation of nuclear factor E2-related factor 2(Nrf2)signaling pathway.Methods Fifty female Wistar rats were randomly divided into Control group,FA group,FA+MT 5 mg/kg group,FA+MT 10 mg/kg group and FA+MT 20 mg/kg group,with 10 rats in each group.Except for the Control group,all other groups inhaled 3 mg/m 3 FA daily for 21 d consecutively to construct the tainted model,and then treated with different MT doses for 14 d.The tainting was continued during the MT treatment.Hematoxylin-eosin(HE)staining was used to observe the histopathological changes in lung tissue,lung water content and lung coefficient were weighed and measured,glutathione(GSH),superoxide dismutase(SOD)and 8-hydroxydeoxyguanosine(8-OHdG)levels were measured by absorbance photometric method,and enzyme linked immunosorbent assay(ELISA)was used to measure the levels of tumor necrosis factor-alpha(TNF-α),interleukin(IL)-6,and IL-1βconcentrations,Western blot to detect the protein expression levels of Nrf2,heme oxygenase-1(HO-1),nuclear factor-κB(NF-κB),and phosphorylated nuclear factor-κB(p-NF-κB)in lung tissues,and quantitative polymerase chain reaction(qPCR)to detect the Nrf2,HO-1,and Kelch-like ECH-associated protein 1(Keap1)mRNA expression levels.Results Compared with the control group,lung injury was obvious in rats in the FA group;lung tissue GSH and SOD levels were reduced,and 8-OHdG levels were elevated(P<0.05);alveolar lavage fluid TNF-α,IL-6,and IL-1βlevels were elevated(P<0.05);Nrf2 and HO-1 protein expression levels were reduced in the lung tissue(P<0.05),and p-NF-κB protein expression levels were was elevated(P<0.05);the relative mRNA expression of Nrf2 and HO-1 in lung tissue was decreased,and the relative mRNA expression of Keap1 was elevated(P<0.05).Compared with the FA group,the lung injury of rats in the MT group was improved;the levels of GSH and SOD in the lung tissue were increased(P<0.05),and the level of 8-OHdG was decreased(P<0.05);the levels of TNF-α,IL-6,and IL-1βin the alveolar lavage fluid were decreased(P<0.05);and the expression levels of the Nrf2 and HO-1 proteins in the lung tissue were increased(P<0.05).p-NF-κB protein expression level was decreased(P<0.05);the relative mRNA expression levels of Nrf2 and HO-1 in lung tissues were increased(P<0.05),and the relative mRNA expression level of Keap1 was decreased(P<0.05)in lung tissues,and all of them were in a dose-dependent manner.Conclusion MT can alleviate oxidative stress and inflammatory responses and mitigate FA exposure-induced acute lung injury by regulating the Nrf2/Keap1/HO-1 signaling pathway.