Effect And Mechanism Of DNA Damage And Repair In Silicotic Mice Treated With Polyguanylic Acid

Background Silicosis is a type of pulmonary disease that are caused by inhalation of crystalline silica particles.It is marked by large-scale destruction of the lung structure,inflammation and nodular fibrosis.Although silicosis is currently considered to be preventable and treatable,existing drug treatments have failed to effectively delay the progression of the disease.Therefore,it is urgent to clarify the mechanism of pulmonary fibrosis,especially to develop new effective treatment strategies in the early stage.Previous studies have found that polyguanylic acid which has an anti-fibrotic G-quadruplex(G4)structure could inhibit the expression of nucleolin,a protein related to DNA damage and repair.But it is not clear whether its effect in anti-fibrosis is related to the regulation of nucleolin.Therefore,the study want to further explore the role and mechanism of PolyG in DNA damage repair and in anti-fibrosis,to provide the theoretical basis for elucidating the pathogenesis of silicosis and finding an new drug target to prevent and treat silicosis.Objective The silicosis models of C57/B6 mice were gained by inhaled tracheal instillation.The aims of the study are to explore:(1)whether PolyG could mitigate the fibrosis and DNA damage of silicotic mice;(2)the mechanism of molecular biology.Methods Forty-eight male C57BL/6 mice(4–5 weeks old)were purchased from Vital River Laboratory Animal Technology Co.Ltd.(Beijing,China)weight 17–20g.All animals were fed in a specific-pathogen-free environment which was maintained a temperature of24±1°C and 12/12 h light/dark cycles,with libitum access to water.All animal procedures were approved by the institutional animal care and treatment committee of Public Health at Xinxiang University and were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.Mice were acclimated for 1 week before the experiments.Mice were anesthetized with 0.3% pentobarbital sodium(2ml/100g)and were either administered 50?l crystalline silica particle suspension(50mg/ml)by intratracheal instillation as the model group(n=16)or sterile saline as the control group(n=16).Additional mice treated with the crystalline silica suspension were also given an intravenous injection of PolyG(2.5mg/kg body weight)as the PolyG group(n=16);PolyG was dissolved in sterile saline at a concentration of 1mg/ml.Eight mice,which were chosen from each group randomly,were sacrificed on day 28 and day 56 after treatment separately.The middle lobes of the right lungs were removed and frozen in liquid nitrogen for western blot analysis,and the inferior lobes of the right lung were removed and fixed in 4% formaldehyde for morphological analysis(Masson &HE).The expression of nucleolin,?-SMA,vimentin and DNA damage and repair proteins(?-H2 AX,DDB2,p53R2 and GADD45)were detected by Western blotting.Extraction of RNA in the left lung tissue in mice detect the expression of mRNA level of Collagen?a1,Collagen?a1,ATM,P53,P21,cyclinD1,CDK4 and CDK6 by qPCR.Results1.The pathological images of lung tissue stained by HE showed that the number of infiltrating cells in the alveoli of the silicosis model group was significantly higher than that of the control group.In addition,spindle-shaped fibroblasts,damage to the alveolar structure,and thickening of the interstitial lung could be found,and compared with 28 days,the fibrosis degree of the 56-day group was more obvious.In Masson-stained images,the blue collagen in the experimental group increased significantly compared to the control group,suggesting collagen fibrosis.The pathology of the PolyG intervention group showed a marked remission of the fibrotic phenotype.2.Expression of ?-H2 AX,Vimentin and ?-SMA in mouse lung tissues: expression levels of Vimentin and ?-SMA in silicosis model group were higher than those in control group(P<0.05),and the expression levels of them were all down-regulated after PolyG intervention(P<0.05)at both time points.At the time point of 56 day,the expression of?-H2 AX in the lung tissues of the silicosis model group was increased,compared with the control group(P<0.05),the expression of protein ?-H2 AX in the PolyG intervention group was decreased(P<0.05).3.The results of qPCR showed that at different time points,the mRNA expression levels of Collagen?a1,Collagen?a1,ATM,ATR and p53 in the lung tissue of the silicosis model group were higher than those in the control group(P <0.05),while the expression levels in the PolyG intervention group lower than the model group(P <0.05).4.The expression of nucleolin,DDB2,RRM2 and GADD45? protein in mouse lung tissues of each group: at both time points,expression levels of nucleolin in silicosis model group were higher than those in control group(P<0.05),and the expression levels were all down-regulated after PolyG intervention(P<0.05).At both time points,the expression level of GADD45? in lung tissue of mice after silica exposure was reduced compared with the control group(P <0.05),while the expression level in the PolyG intervention group was increased compared with the model group(P <0.05).5.The expression of cyclinD1,CDK4 and CDK6 mRNA in mouse lung tissues of each group: At the time point of 28 day,the transcription level of cyclinD1 of the silicosis model group was higher than that of the control group(P<0.05).However at the time point of 56 day,the transcription level of cyclinD1 of the silicosis group was lower than that of control group(P<0.05).The transcription level of CDK4 and CDK6 in silicosis group and PolyGgroup were higher than in normal saline group,except at the time point of 56 day,the differences transcription level of CDK4 between silicosis group and the other groups in the same period.were not statistically significant(P>0.05),and,at the time point of 28 day,the difference of CDK6 mRNA level between silicosis group and PolyG group was no statistically significant(P>0.05).Conclusion1.silica particles could induce marker protein of DNA damage: ?-h2 ax and nucleolin expression in mouse lung tissue,and up-regulated mRNA expression of DNA damage repair related genes,indicating that DNA injury repair related signal pathway mediated by nucleolin may play an important role in silicosis fibrosis;2.PolyG could down-regulate ?-H2 AX and nucleolin expression,up-regulate GADD45? expression,regulate the transcription and translation of DNA damage repair related genes in silicotic mice,and mitigate fibrosis.The research has important theoretical significance and practical value for the prevention and treatment of pneumoconiosis.