Nanoplastics aggravate severe asthma by inducing DNA damage of alveolar type Ⅱ epithelial cells
Objective·To explore the effects and possible molecular mechanisms of nanoplastics(NPs)on severe asthma.Methods·A mouse model of severe asthma was established by using house dust mite(HDM)and lipopolysaccharide(LPS)co-stimulation.Polystyrene nanoplastics(PS-NPs)were instilled into the severe asthma mice's airways.Subsequently,bronchoalveolar lavage fluid(BALF)was collected and lung tissue sections were prepared.Flow cytometry,hematoxylin-eosin(H-E)staining,periodic acid-Schiff(PAS)staining,immunohistochemistry,and terminal dexynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)staining,were used to observe the effects of PS-NPs on airway inflammation,mucus secretion,alveolar structure,and the proliferation and apoptosis of alveolar type Ⅱ epithelial cells(AT2 cells)in severe asthma mice.The CCK-8 assay and Annexin Ⅴ/PI double staining were performed to evaluate the effects of PS-NPs on the proliferation and apoptosis of the mouse AT2 cell line MLE-12.DNA damage in AT2 cells caused by PS-NPs was detected by using anti-γ-H2A.X immunofluorescence staining.The expression of genes in the ATR/Chk1/p53 signaling pathway was detected by real-time fluorescent quantitative polymerase chain reaction(qPCR),Western blotting,Tyramide signal amplification(TSA)multiplex immunofluorescence staining,and immunofluorescence co-localization,respectively.The ATR-specific inhibitor Ceralasertib(AZD6738)was administrated to MLE-12 cells in combination with PS-NPs to evaluate the recovery effect on cell proliferation and apoptosis.Results·Flow cytometry revealed that exposure to PS-NPs increased the total number of inflammatory cells and the number of each type of inflammatory cells in the BALF of mice with severe asthma,with a predominance of neutrophils.H-E and PAS staining showed significant increase in airway inflammatory cell infiltration and mucus secretion,as well as disruption of alveolar structure.In vitro,the CCK-8 assay demonstrated significant,dose-dependent inhibition of MLE-12 cell proliferation by PS-NPs.The Annexin V/PI double staining assay indicated a higher apoptosis rate of(56.20±3.84)%in PS-NP-exposed cells compared to(23.22±2.52)%in the control group.Immunofluorescence staining demonstrated that PS-NPs were phagocytosed by MLE-12 cells and localized around the nucleus.TUNEL staining confirmed enhanced apoptosis in AT2 cells in vivo.The immunofluorescence assay revealed that compared to the control group,the expression of the DNA damage marker γ-H2A.X increased in the experimental group.qPCR,Western blotting,and TSA multiplex staining results showed that PS-NP-induced elevated expression of mRNA and proteins was related to the ATR/Chk1/p53 pathway in MLE-12 cells.Moreover,immunofluorescence co-localization also confirmed the induction of ATR and p53 proteins in AT2 cells in vivo.The ATR-specific inhibitor Ceralasertib partially restored the PS-NP-induced inhibition of cell proliferation and enhancement of apoptosis in MLE-12 cells.Conclusion·NPs exposure leads to DNA damage in AT2 cells,activating the ATR/Chk1/p53 signaling pathway and exacerbating airway inflammation and alveolar damage in mice with severe asthma.
severe asthmananoplasticalveolar type Ⅱ epithelial cellDNA damageATR/Chk1/p53 signaling pathway
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