【摘要】 目的·探讨纳米塑料（nanoplastics,NPs）对重症哮喘发生发展的影响及潜在分子机制。方法·建立屋尘螨（house dust mite,HDM）和脂多糖（lipopolysaccharide,LPS）联合诱导的重症哮喘小鼠模型，并给予聚苯乙烯纳米塑料（polystyrene nanoplastics,PS-NPs）气道滴注，收集小鼠肺泡灌洗液及制作肺组织切片。通过流式细胞术、苏木精-伊红（hematoxylin-eosin,H-E）染色、过碘酸希夫（periodic acid-Schiff,PAS）染色、免疫组织化学染色、脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（terminal dexynucleotidyl transferase-mediated dUTP nick-end labeling,TUNEL）染色，观察PS-NPs对重症哮喘小鼠气道炎症、黏液分泌、肺泡结构，以及肺泡Ⅱ型上皮细胞（alveolar typeⅡepithelial cells,AT2cells）增殖和凋亡的影响。采用CCK-8法及AnnexinⅤ/PI双染色法测定PS-NPs对小鼠AT2细胞系MLE-12细胞增殖、凋亡的影响。使用γ-H2A.X免疫荧光染色检测PS-NPs对AT2细胞的DNA损伤作用。通过实时荧光定量聚合酶链反应（realtime fluorescent quantitative polymerase chain reaction,qPCR）、蛋白质印迹法（Western blotting）、酪胺信号放大（Tyramide signal amplification,TSA）多重荧光染色及免疫荧光共定位检测PS-NPs对AT2细胞ATR/Chk1/p53信号通路相关基因和蛋白表达的影响。使用ATR特异抑制剂Ceralasertib (AZD6738)与PS-NPs共同处理MLE-12细胞，以测定其对细胞增殖、凋亡的恢复作用。结果·流式细胞术显示，重症哮喘小鼠暴露于PS-NPs后，其肺泡灌洗液中炎症细胞总数及各炎症细胞数量均有增加，以中性粒细胞增多为主；肺组织H-E染色及PAS染色显示，气道炎症细胞浸润及气道黏液分泌显著增加，肺泡结构被破坏。在体外试验中，CCK-8结果显示PS-NPs显著抑制MLE-12细胞的增殖能力并呈现浓度依赖性；AnnexinⅤ/PI双染色结果显示，PS-NPs暴露后的细胞凋亡率[（56.20±3.84）%]相比于未暴露组[（23.22±2.52）%]显著上升；免疫荧光染色显示PS-NPs能被MLE-12细胞所吞噬并定位在细胞核周围。TUNEL染色结果表明，在体内PS-NPs同样促进AT2细胞凋亡的发生。免疫荧光染色结果显示，与对照组相比，实验组DNA损伤标志物γ-H2A.X表达增加。qPCR、Western blotting、TSA多重荧光染色显示，PS-NPs诱导MLE-12细胞ATR/Chk1/p53信号通路相关基因和蛋白表达水平升高。免疫荧光共定位实验也证实在小鼠体内，PS-NPs诱导AT2细胞表达ATR、p53蛋白。而ATR特异抑制剂Ceralasertib能部分恢复由PS-NPs引起的MLE-12细胞增殖的抑制和凋亡的增强。结论·NPs暴露能够造成AT2细胞DNA损伤，激活ATR/Chk1/p53信号通路，进而加重重症哮喘小鼠气道炎症反应及肺泡结构损伤。更多还原

【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) costimulation. 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.更多还原