Nanoplastics aggravate severe asthma by inducing DNA damage of alveolar type Ⅱ epithelial cells

doi:10.3969/j.issn.1674-8115.2024.11.006

Abstract

Abstract:

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.

Key words: severe asthma, nanoplastic, alveolar type Ⅱ epithelial cell, DNA damage, ATR/Chk1/p53 signaling pathway

CLC Number:

R726.6

SHI Zelun, WANG Qing, HE Wen, FU Weijia, WANG Yingwen, HAN Xiao, ZHANG Xiaobo. Nanoplastics aggravate severe asthma by inducing DNA damage of alveolar type Ⅱ epithelial cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(11): 1391-1405.

Figures/Tables 9

Tab 1 Primer sequences for qPCR

Gene	Forward primer (5′→3′)	Reverse primer (5′→3′)
β-actin	CACCATTGGCAATGAGCGGTTC	AGGTCTTTGCGGATGTCCACGT
Ki67	GAGGAGAAACGCCAACCAAGAG	TTTGTCCTCGGTGGCGTTATCC
Bax	AGGATGCGTCCACCAAGAAGCT	TCCGTGTCCACGTCAGCAATCA
Bcl2	CCTGTGGATGACTGAGTACCTG	AGCCAGGAGAAATCAAACAGAGG
Trp53	CCCCTGTCATCTTTTGTCCCT	AGCTGGCAGAATAGCTTATTGAG
Bid	CCACAACATTGCCAGACATCTCG	TCACCTCATCAAGGGCTTTGGC
Cdk6	ACCTCTGGAGTGTCGGTTGCAT	TTCCTCTCCTGGGAGTCCAATG
Atr	GAAAGAGGCTCCTACCAACGAG	CAACTGTCACCTGGAGACTTGC
Ccnd1	GCAGAAGGAGATTGTGCCATCC	AGGAAGCGGTCCAGGTAGTTCA

Fig 1 Results of BALF inflammatory cell counts, lung tissue pathological sections and histopathological scores

Fig 2 Effect of exposure to PS-NPs on BALF inflammatory cell counts, lung tissue pathological sections and histopathological scores in mice with severe asthma

Fig 3 Histopathological changes of mice lung alveolars among various groups after exposure to PS-NPs

Fig 4 Effects of PS-NPs on the proliferation, apoptosis and mRNA expression levels of related gene of AT2 cells

Fig 5 Level of cellular apoptosis in lung tissue of severe asthma mice after PS-NPs exposure

Fig 6 In vitro experiments confirmed the DNA damage induction and the upregulation of the ATR/Chk1/p53 signaling pathway in AT2 cells after PS-NPs exposure

Fig 7 In vivo experiments confirmed that the upregulated expression levels of ATR and p53 in Sftpc＋ AT2 cells of severe asthma mice after PS-NPs exposure

Fig 8 Ceralasertib restores the effects induced by PS-NPs on proliferation and apoptosis of AT2 cells

TrendMD

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