DDAH1通过抑制氧化应激及炎症反应缓解PM2.5诱导的肺损伤机制研究

DDAH1 alleviates PM2.5-induced lung injury by inhibiting oxidative stress and inflammatory response

目的探讨二甲基精氨酸二甲胺水解酶1(DDAH1)通过抑制氧化应激及炎症反应缓解PM2.5诱导的小鼠肺损伤的保护作用。方法32只雌性C57BL/6野生型小鼠,按照随机数字表法将其分为4组,分别为对照组、模型组、DDAH1高剂量组、DDAH1低剂量组,每组各8只。除对照组滴注0.9%氯化钠溶液外,其余各组室温下用无菌0.9%氯化钠溶液将PM2.5稀释至终浓度220μg/100μL,小鼠气管滴注PM2.5溶液60μL,每天1次,连续暴露7 d。其中,DDAH1高剂量组气管滴注PM2.5溶液前30 min,给予300 ng重组人DDAH1蛋白进行滴鼻处理。DDAH1低剂量组气管滴注PM2.5溶液前30 min,给予100 ng重组人DDAH1蛋白进行滴鼻处理。检测小鼠支气管灌洗液中炎症因子[白细胞介素(IL)-6、IL-8、IL-1β、肿瘤坏死因子-α(TNF-α)]及肺组织中丙二醛、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)含量;观察各实验组小鼠肺组织的病理学改变,肺组织中的细胞黏附分子-1(ICAM-1)、血管细胞黏附因子1(VCAM-1)、不对称二甲基精氨酸(ADMA)、转化生长因子-β(TGF-β)、诱导型一氧化氮合酶(iNOS)蛋白表达。结果与对照组相比,模型组肺泡灌洗液中IL-6、IL-8、TNF-α及IL-1β水平均明显升高,差异均有统计学意义(P<0.05);与模型组相比,DDAH1高剂量组、DDAH1低剂量组肺泡灌洗液中IL-6、IL-8、TNF-α及IL-1β水平均明显降低,差异均有统计学意义(P<0.05)。与对照组相比,模型组肺组织中丙二醛水平明显升高,SOD及CAT水平均明显降低,差异均有统计学意义(P<0.05);与模型组相比,DDAH1高剂量组、DDAH1低剂量组肺组织中丙二醛水平明显降低,SOD及CAT水平均明显升高,差异均有统计学意义(P<0.05)。与对照组相比,模型组肺组织中ICAM-1、VCAM-1、TGF-β、iNOS、ADMA蛋白表达水平均明显升高,差异均有统计学意义(P<0.05);与模型组相比,DDAH1高剂量组、DDAH1低剂量组肺组织中ICAM-1、VCAM-1、TGF-β、iNOS、ADMA蛋白表达水平均明显降低,差异均有统计学意义(P<0.05)。结论PM2.5诱导的肺损伤可增加肺泡灌洗液中炎症因子表达,加重氧化应激反应,并影响血管肌化和血管内皮屏障功能障碍相关蛋白的表达。而外源性DDAH1对PM2.5所致小鼠肺损伤具有一定的保护作用,其机制可能与抑制氧化应激和炎症反应有关。

Objective To investigate the protective effect of dimethylarginine dimethylamine hydrolase 1(DDAH1)on PM2.5-induced lung injury in mice by inhibiting oxidative stress and inflammatory response.Methods Thirty-two female C57BL/6 wild type mice were divided into control group,model group,high-dose DDAH1 group and low-dose DDAH1 group according to the random number table,with 8 mice in each group.Except for the control group,PM2.5 was diluted to a final concentration of 220μg/100μL with sterile normal saline at room temperature,and 60μL PM2.5 solution was instilled into the trachea once a day for 7 consecutive days.In the DDAH1 high dose group,300 ng recombinant human DDAH1 protein was intranasal instilled 30 minutes before intratracheally instilled PM2.5 solution.The low-dose DDAH1 group was given 100 ng recombinant human DDAH1 protein intranasal 30 minutes before intratracheally instilled PM2.5 solution.The contents of inflammatory factors[interleukin(IL)-6,IL-8,IL-1β,tumor necrosis factor-α(TNF-α)]in bronchial lavage fluid and malondialdehyde,superoxide dismutase(SOD),catalase(CAT)in lung tissue were detected.The pathological changes of lung tissue in each experimental group were observed,and the protein expressions of cell adhesion molecule-1(ICAM-1),vascular cell adhesion molecule-1(VCAM-1),asymmetric dimethylarginine(ADMA),transforming growth factor-β(TGF-β)and inducible nitric oxide synthase(iNOS)in lung tissue were observed.Results Compared with the control group,the levels of IL-6,IL-8,TNF-αand IL-1βin the alveolar lavage fluid of the model group were significantly increased,the differences were statistically significant(P<0.05);compared with the model group,the levels of IL-6,IL-8,TNF-αand IL-1βin the alveolar lavage fluid of the DDAH1 high-dose group and DDAH1 low-dose group were significantly decreased,the differences were statistically significant(P<0.05).Compared with the control group,the level of malondialdehyde in the lung tissue was significantly increased,and the levels of SOD and CAT were significantly decreased,the differences were statistically significant(P<0.05);compared with the model group,the level of malondialdehyde in the lung tissue was significantly decreased,the levels of SOD and CAT were significantly increased,the differences were statistically significant(P<0.05).Compared with the control group,the expression levels of ICAM-1,VCAM-1,TGF-β,iNOS and ADMA proteins in the lung tissue of the model group were significantly increased,the differences were statistically significant(P<0.05);compared with the model group,the expression levels of ICAM-1,VCAM-1,TGF-β,iNOS,and ADMA proteins in the lung tissue of the high-dose and low-dose DDAH1 groups were significantly decreased,the differences were statistically significant(P<0.05).Conclusion PM2.5-induced lung injury can increase the expression of inflammatory factors in alveolar lavage fluid,aggravate oxidative stress response,and affect the expression of vascular muscularization and vascular endothelial barrier dysfunction related proteins.Exogenous DDAH1 has a certain protective effect on PM2.5-induced lung injury in mice,and its mechanism may be related to inhibiting oxidative stress and inflammatory response.