| Objective: Studies have shown that PM2.5 contains a large number of mutagenic and teratogenic substances,which can cause DNA damage and malignant tumors.However,the mechanism of DNA damage and response induced by PM2.5 has not been fully elucidated.Hypoxia is a symptom of many respiratory diseases,so it can affect the body at the same time as PM2.5.The formation of reactive oxygen species(ROS)is closely related to DNA damage,studies have confirmed that A-Acetylcysteine(NAC)has antioxidant and cytokine inhibiting effects,but whether NAC can reduce DNA damage through antioxidant effects is not clear.In this study,we investigate the effects of PM2.5 combined with hypoxia exposure on DNA damage in rat lung tissues and the roles of DNA damage response pathway ATM-Chk2 pathway,as well as the protective effect of NAC.Methods:1.Animal grouping: healthy SPF SD male rats were randomly divided into control group,PM2.5 group,hypoxia group,PM2.5+hypoxia group,PM2.5+NAC group,PM2.5+hypoxia+NAC group,6 rats in each group.The control group inhaled the clean air filtered by the air filter,the hypoxia group was kept in the normal pressure hypoxic chamber(oxygen concentration 10%±0.5%)for 1 month,and the PM2.5 group was exposed to outdoor air for 2 months.The PM2.5+hypoxia group was kept in a hypoxic chamber for 1 month and then exposed to outdoor air for 1 month.The PM2.5+NAC group was exposed to outdoor air for 2 months while daily N-acetylcysteine(150mg/kg/d)gavage,PM2.5+hypoxia+NAC group was kept in a hypoxic chamber for 1 month and then exposed to outdoor air for 1 month while daily N-acetylcysteine(150mg/kg)/d)gavage.2.The histopathological changes of rat lung were observed by HE staining.3.Single-cell gel electrophoresis experiment,also known as Comet Assay,is a sensitive technique for detecting DNA damage at a single cell level.Comet assays were used to detect DNA damage levels in lung tissue of each group to observe the genotoxicity of PM2.5.4.Lung tissues were extracted,the expression levels of ATM and Chk2 m RNA were detected by RT-PCR.The relative expression levels of ATM and Chk2 proteins were observed by immunofluorescence staining,and the expression levels of ATM and Chk2 proteins were detected by Western blot.Results:1.Pathological morphological changes of lung tissue: compared with the control group,alveolar and interstitial inflammatory lesions,alveolar structural destruction were observed in PM2.5 group,hypoxia group and PM2.5+hypoxia group.The alveolar structure and inflammatory infiltration were improved after NAC intervention.2.Results of comet assay: compared with the control group,Olive tail moment of the PM2.5 group and the PM2.5+hypoxia group were significantly increased(P<0.05).It was observed that Olive tail moment increased in the hypoxic group,but the difference was not statistically significant.After NAC intervention,Olive tail moment in PM2.5+NAC group was significantly reduced compared with that in PM2.5 group(P<0.05).Olive tail moment in PM2.5+hypoxia+NAC group was significantly reduced than that in PM2.5+ hypoxia group(P<0.05).3.Determination of ATM and Chk2 m RNA expression in rat lung tissues: The expression levels of ATM and Chk2 m RNA in lung tissues of the PM2.5 group,the hypoxia group and the PM2.5+hypoxia group were significantly higher than those in the control group(P<0.05).After NAC intervention,the expression levels of ATM and Chk2 m RNA in PM2.5+NAC group were significantly lower than those in PM2.5 group(P<0.05).The expression levels of ATM and Chk2 m RNA in PM2.5+hypoxia+NAC group were significantly lower than those in PM2.5+hypoxia group(P<0.05).4.Determination of ATM and Chk2 protein expression in rat lung tissues:4.1 The results of immunofluorescence staining: The relative expression levels of ATM and Chk2 protein in lung tissues of the PM2.5 group,the hypoxia group and the PM2.5+hypoxia group were significantly higher than those in the control group(P<0.05).After NAC intervention,the relative expression levels of ATM and Chk2 protein in PM2.5+NAC group were significantly lower than those in PM2.5 group(P<0.05).The relative expression levels of ATM and Chk2 protein in PM2.5+hypoxia+NAC group were significantly lower than those in PM2.5+hypoxia group(P<0.05).4.2 The results of Western bolt: The expression levels of ATM and Chk2 protein in lung tissues of the PM2.5 group,the hypoxia group and the PM2.5+hypoxia group were significantly higher than those in the control group(P<0.05).After NAC intervention,the expression levels of ATM and Chk2 protein in PM2.5+NAC group were significantly lower than those in PM2.5 group(P<0.05).The expression levels of ATM and Chk2 protein in PM2.5+hypoxia+NAC group were significantly lower than those in PM2.5+hypoxia group(P<0.05).Conclusions:1.PM2.5,hypoxia and PM2.5 combined with hypoxia can cause pulmonary inflammatory lesions and alveolar structural destruction in rats.NAC intervention can improve alveolar structure,inflammatory infiltration and other pathological changes.2.PM2.5,hypoxia and PM2.5 combined with hypoxia can cause DNA damage in rat lung tissue cells and mediate the DNA damage response through the ATM-Chk2 pathway.3.NAC intervention can reduce the level of DNA damage in rat lung tissue,and thereby inhibit the expression of ATM-Chk2 pathway.This further demonstrates the protective effect of NAC on rat lung tissues. |
