抑制肺泡上皮细胞焦亡对支气管肺发育不良新生大鼠肺泡化阻滞的改善作用

doi:10.3969/j.issn.1674-8115.2023.02.005

摘要/Abstract

摘要：

目的·研究gasdermin D（GSDMD）抑制剂necrosulfonamide（NSA）通过抑制肺上皮细胞焦亡对脂多糖（lipopolysaccharide，LPS）诱导的新生大鼠支气管肺发育不良（bronchopulmonary dysplasia，BPD）肺泡化阻滞的影响。方法·将孕SD大鼠随机分为对照组、BPD组、BPD+NSA组和NSA组，羊膜腔注射LPS建立新生鼠BPD模型。取各组出生后第1、3、7日新生鼠肺组织，通过苏木精-伊红（H-E）染色观察肺泡化情况；利用免疫荧光法检测各组新生鼠肺部GSDMD-N端蛋白表达情况；荧光定量PCR法检测新生鼠肺组织中炎症因子白介素-1β（interleukin-1β，IL-1β）mRNA水平。体外培养小鼠肺泡上皮细胞系MLE-12，给予LPS以及腺苷三磷酸（adenosine triphosphate，ATP）刺激和NSA干预，CCK-8法检测MLE-12细胞活力，Hoechst 33342和碘化丙啶（propidium iodide，PI）染色法检测细胞焦亡水平，免疫荧光法检测MLE-12细胞表面活性剂蛋白C（surfactant protein C，SFTPC）和GSDMD-N端蛋白的表达情况。结果·体内实验结果显示：羊膜腔内注射LPS可导致肺发育受阻，模拟BPD的病理改变；羊膜腔内注射LPS建立的BPD模型中肺泡上皮细胞GSDMD-N端表达升高；NSA干预明显改善了BPD新生鼠的肺发育阻滞并抑制了IL-1β的mRNA表达（均P<0.05）。体外实验结果显示：LPS/ATP刺激下肺泡上皮细胞MLE-12活力下降，发生焦亡；NSA干预提高了肺泡上皮细胞活力并且抑制了焦亡（均P<0.05）；NSA上调了LPS/ATP刺激下肺泡上皮细胞SFTPC的表达，抑制了LPS/ATP刺激下肺泡上皮细胞GSDMD-N端表达的上调（均P<0.05）。结论·抑制肺泡上皮细胞焦亡可改善LPS诱导的BPD新生鼠肺组织病理学改变。

关键词: 支气管肺发育不良, 细胞焦亡, gasdermin D, 肺泡上皮细胞

Abstract:

Objective ·To study the effect of gasdermin D (GSDMD) inhibitor necrosulfonamide (NSA) on alveolarization arrest in lipopolysaccharide (LPS)-induced bronchopulmonary dysplasia (BPD) newborn rats via inhibiting alveolar epithelial cell pyroptosis. Methods ·Pregnant SD rats were randomly assigned to four groups as follows: control, BPD, BPD with NSA and NSA group, and then were prepared to receive intra-amniotic injection of LPS. Lung tissues of newborn rats on the first, third and seventh day after birth were stained by hematoxylin-eosin (H-E) to observe lung development. The expressions of GSDMD-N-terminal in lungs of newborn rats in each group were detected by immunofluorescence. The mRNA levels of interleukin-1β (IL-1β) of newborn rats' lungs was detected by real-time PCR. In vitro, the mouse alveolar epithelial cell line MLE-12 was cultured and treated with LPS/adenosine triphosphate (ATP) and NSA. The cell viability of MLE-12 cells was detected by CCK-8 method, the pyroptosis was detected by Hoechst 33342 and propidium iodide (PI) staining, and the expressions of surfactant protein C (SFTPC) and GSDMD-N protein in MLE-12 cells were detected by immunofluorescence. Results ·In vivo, intra-amniotic injection of LPS hindered lung development, resulting in the pathological hallmarks of BPD. The GSDMD-N expression of alveolar epithelial cells increased in the BPD rat model established by intra-amniotic injection of LPS, while NSA treatment significantly improved the lung development of BPD rats and inhibited the IL-1β mRNA expression (both P<0.05). In vitro, the study confirmed that LPS/ATP treatment decreased the viability of alveolar epithelial cells MLE-12 and induced pyroptosis, while NSA treatment increased alveolar epithelial cell viability and inhibited pyroptosis (both P<0.05). In addition, NSA treatment upregulated the SFTPC expression and inhibited the GSDMD-N expression in LPS/ATP-stimulated alveolar epithelial cells (both P<0.05). Conclusion ·Inhibiting the alveolar epithelial cell pyroptosis can improve the alveolar development in BPD newborn rats.

Key words: bronchopulmonary dysplasia, pyroptosis, gasdermin D (GSDMD), alveolar epithelial cell

中图分类号:

R722

郑小雁, 王星云, 张拥军. 抑制肺泡上皮细胞焦亡对支气管肺发育不良新生大鼠肺泡化阻滞的改善作用[J]. 上海交通大学学报（医学版）, 2023, 43(2): 171-179.

ZHENG Xiaoyan, WANG Xingyun, ZHANG Yongjun. Improvement of alveolarization arrest in newborn rats with bronchopulmonary dysplasia via inhibiting alveolar epithelial cell pyroptosis[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(2): 171-179.

图/表 6

图1 羊膜腔内注射LPS导致的新生鼠肺发育阻滞观察Note： A. The representative lung sections stained with H-E staining. B. The quantification of the terminal air spaces of lung tissues from newborn rats. C. The quantification of the secondary septa of lung tissues from newborn rats. D. The quantification of the MLI of lung tissues from newborn rats. ①P=0.000, ②P=0.010, ③P=0.038, ④P=0.004, compared with the control group.

Fig 1 Alveolar developmental arrest caused by intra-amniotic LPS injection

图2 BPD新生鼠肺泡上皮细胞焦亡现象观察Note：A. Representative immunofluorescence images of GSDMD-N terminal (red) in the lungs of newborn rats. B. Quantitation of GSDMD-N terminal fluorescence intensity. ①P=0.007, ②P=0.015, ③P=0.033, compared with the control group.

Fig 2 Alveolar epithelial cell pyroptosis in BPD newborn rats

图3 NSA对新生鼠肺部病理状态和 IL-1β mRNA表达的影响Note：A. The representative lung sections stained with H-E staining. B. The quantification of the terminal air spaces of lung tissues from newborn rats. C. The quantification of the secondary septa of lung tissues from newborn rats. D. The quantification of the MLI of lung tissues from newborn rats. E. Comparison of IL-1β mRNA among the groups. ①P=0.000, compared with the control group; ②P=0.031, ③P=0.049, ④P=0.002, ⑤P=0.040, ⑥P=0.020, ⑦P=0.024, ⑧P =0.026, ⑨P =0.014, compared with the BPD group.

Fig 3 Effects of NSA on lung pathology and expression of IL-1β mRNA in newborn rats

图4 NSA对LPS/ATP诱导的肺泡上皮细胞MLE-12焦亡的抑制作用Note：A. Cell viability of MLE-12 detected by CCK-8 assay. B. Percentages of PI+ cells. C. Pyroptotic cells detected by Hoechst 33342 and PI staining. ①P=0.000, compared with the control group; ②P=0.003, ③P=0.008, compared with the LPS/ATP group.

Fig 4 Inhibition effect of NSA on LPS/ATP-induced pyroptosis in MLE-12 cells

图5 NSA对肺泡上皮细胞MLE-12中SFTPC表达的影响Note：A. Representative immunofluorescence images of SFTPC (green) in MLE-12. B. Quantitation of SFTPC fluorescence intensity. ①P=0.002, compared with the control group; ②P=0.035, compared with the LPS/ATP group.

Fig 5 Effect of NSA treatment on SFTPC expression in MLE-12 cells

图6 NSA对肺泡上皮细胞MLE-12中GSDMD-N端表达的影响Note：A. Representative immunofluorescence images of GSDMD-N terminal (green) in MLE-12. B. Quantitation of GSDMD-N terminal fluorescence intensity. ①P=0.000, compared with the control group; ②P=0.010, compared with the LPS/ATP group.

Fig 6 Effect of NSA treatment on GSDMD-N expression in MLE-12 cells

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