Effects Of Exposure To Carbon Black Nanoparticles In Pregnant Mice On Bleomycin-induced Lung Fibrosis In Their Offspring

Objective: To explore the effect of carbon black nanoparticles-Printex 90(Pr-CBNPs)exposure on bleomycin(BLM)-induced lung fibrosis in offspring mice and its molecular mechanism.Methods: 36 C57/BL6 J mice were caged at a male to female ratio of2: 1,and the vaginal plugs were observed daily.Pregnant mice are kept separately and recorded as Gestational day 1,GD1.16 pregnant mice were selected and randomly divided into a control group of pregnant rats(Mock,Pr-M)and a carbon black group of pregnant rats(Pr-CBNPs),8mice in each group,which were sterilized by intranasal administration on GD 9-18 days Ultra-pure water and Pr-CBNPs(515 ?g / animal)were used to construct animal models of Pr-CBNPs exposure during pregnancy.After parting day 49(PD49),16 male progeny were selected from each of Pr-M group and Pr-CBNPs group,and were randomly divided into Pr-M group,Pr-CBNPs group,Pr-M + BLM group,Pr-CBNPs + BLM group,8 animals in each group,25 ?L of sterile saline was administeredintra-tracheally to Pr-M group and Pr-CBNPs group,2.5 U / kg in Pr-M +BLM group and Pr-CBNPs + BLM group BLM dissolved in 25 ?L of sterile saline.On the 14 th day after BLM administration,the mice were sacrificed to obtain lung tissue.Lung tissue paraffin sections were subjected to H&E staining,Masson trichrome staining,and immunohistochemical staining(?-SMA,COL1A1);lung tissue Western-blotting(WB)was used to detect lung fibrosis related indicators(Fn1,COL1A1);further through WB detection Autophagy-related indicators(LC3-B,p-AMPK,p-ULK1,p-mTOR,LKB1),using kits to detect lung ATP content,explore related molecular mechanisms.Results: H&E staining results suggest that BLM can cause edema and thickening of the alveolar septum in mice,and the Pr-CBNPs + BLM group is more severely injured than the Pr-M + BLM group.Masson trichrome staining results suggest that BLM can cause a large amount of collagen deposition in the lung tissue of mice,and the Pr-CBNPs + BLM group has more collagen deposition than the Pr-M + BLM group.Immunohistochemical results suggest that BLM can cause the accumulation of ?-SMA and COL1A1 in mouse lung tissue,and the Pr-CBNPs + BLM group accumulated more than the Pr-M + BLM group.WB detection of lung fibrosis-related indicators suggested that BLM can cause increased expression of Fn1,COL1A1 in mouse lung tissue,and the Pr-CBNPs + BLM group increased more than the Pr-M + BLM group,the difference was statistically significant(P < 0.05).WB detection of autophagy related indicators suggested that BLM treatment can increase the degree of autophagy,but Pr-CBNPs + BLM compared with Pr-M +BLM group decreased autophagy activation,the difference was statistically significant(P <0.05).WB test results suggest that BLM can significantly activate the expression of key autophagy regulatory protein p-AMPK,but the activation degree of pr-CBNPs + BLM group is significantly lower than that of Pr-M + BLM group,the difference is statistically significant(P < 0.05);while the expression of p-mTOR was not different among the groups.Further WB detection results suggest that BLM can significantly activate the expression of AMPK upstream activation factor LKB1 and downstream p-ULK1,but the activation degree of pr-CBNPs + BLM group is significantly reduced compared to Pr-M + BLM group,the difference is statistically significant(P <0.05).The results of lung tissue ATP content measurement showed that BLM caused a decrease in lung tissue ATP content,the difference was statistically significant(P <0.05).Conclusion: Comprehensive H&E staining,Masson three-color staining,immunohistochemical staining,and lung tissue WB results showed that exposure of carbon black nanoparticles during pregnancy can aggravate the degree of lung fibrosis induced by bleomycin in progeny mice,and the possible mechanism for this phenomenon is Pr-CBNPsdisrupted LKB1-AMPK-ULK1 axis-mediated autophagy activation.