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《中华肺部疾病杂志》[ISSN:1006-6977/CN:61-1281/TN]

卷:

期数:

2020年04期

页码:

441-445

栏目:

论著

出版日期:

2020-08-20

文章信息/Info

Title:

Role of Gasdermin D in pyroptosis of mouse alveolar macrophages induced by lipopolysaccharide

作者:

帅维正¹ 李 军² 陈旭昕³ 李大伟¹ 张志成¹

100048 北京,北京解放军总医院第六医学中心重症医学科¹、卫勤部²

Author(s):

Shuai Weizheng¹;  Li Jun¹; 2;  Chen Xuxin³;  Li Dawei¹;  Zhang Zhicheng¹.

¹Department of Critical Care Medicine, Sixth Medical Center of PLA General Hospital, Beijing 100048, China; ²Department of Medical Administration, Sixth Medical Center of PLA General Hospital, Beijing 100048, China; ³Department of Respiratory and Critical Care Medicine, Sixth Medical Center of PLA General Hospital, Beijing 100048, China
Corresponding author

关键词:

急性肺损伤/急性呼吸窘迫综合征;  焦亡;  GSDMD;  内毒素

Keywords:

Acute lung injury/Acute respiratory distress syndrome;  Pyroptosis;  Gasdermin D;  Lipopolysaccharide

分类号:

R563

DOI:

10.3877/cma.j.issn.1674-6902.2020.04.002

摘要:

目的 探讨Gasdermin D(GSDMD)蛋白在内毒素导致肺泡巨噬细胞焦亡中的作用。方法 使用小干扰RNA技术建立GSDMD蛋白低表达的MH-S细胞株。将细胞分为野生型MH-S细胞组(WT)、转染空病毒载体的空白对照细胞组(NC)和GSDMD敲低MH-S细胞组(KD),分别接受PBS、LPS+尼日利亚菌素和LPS+霍乱毒素B亚单位进行处理。通过测定其乳酸脱氢酶(LDH)和IL-1β,荧光显微镜下观察细胞PI染色情况,RT-qPCR和蛋白免疫印迹检测焦亡相关蛋白表达等指标,来判定MH-S细胞有无焦亡发生和炎症因子水平的差异。结果 在接受LPS+nigericin和LPS+CTB的处理后,三组细胞均出现了焦亡现象。其中KD组接受LPS+nigericin刺激后,细胞培养上清LDH水平和IL-1β浓度明显低于WT和NC组(P<0.05); PI染色阳性百分比明显少于WT和NC组(P<0.05); 而GSDMD mRNA相对表达量和GSDMD-NT蛋白相对表达量也是三组中最低的(P<0.05)。而在经LPS+CTB处理后,WT和NC组细胞死亡发生率明显高于KD组(P<0.01); 细胞上清中IL-1β水平和PI染色阳性百分比增加幅度也是WT和NC组更大(P<0.05)。此外,KD组中GSDMD mRNA相对表达量和GSDMD-NT蛋白相对表达量相较于另两组也是更低的(P<0.05)。结论 下调MH-S细胞GSDMD蛋白表达可以起到抑制LPS导致的MH-S细胞焦亡和IL-1β分泌释放的作用。

Abstract:

Objective To investigate the role of Gasdermin D(GSDMD)in the pyroptosis of mouse alveolar macrophages induced by lipopolysaccharide(LPS). Methods The GSDMD knockdown MH-S cell line was established using small interfering RNA(siRNA)technique. There were three groups of cells including wild-type MH-S cell group(WT), blank control cell group(NC)and GSDMD knockdown MH-S cell group(KD), and they were treated with phosphate buffered solution(PBS), LPS+nigericin and LPS+cholera toxin B subunit, respectively. Then 5 different assays were performed to identify the pyroptosis of MH-S cells which were stimulated by these reagnets. The details of these assays were described as follows: the mortality of the MH-S cells was determined by lactate dehydrogenase(LDH)release assay, PI staining was performed under a fluorescence microscope, IL-1β level in the cell culture supernatant was detected by enzyme-linked immunosorbent assay(ELISA), the mRNA transcriptional level of GSDMD was evaluated by real-time quantitative RT-PCR, and the expression of GSDMD-NT protein level was evaluated by Western blotting method. Results After LPS induction, pyroptosis appeared in the cells of all the three groups. The analyses of LPS plus nigericin-treated or cholera toxin B subunit-treated MH-S cell lines showed that pyroptosis was suppressed in the KD group. In the knockdown cell line, after the treatment of LPS+nigericin, the level of LDH and the concentration of IL-1βwere significant lower than those of the WT group and the NC group(P<0.05). The less positive percentage of PI staining was present in the KD group than the other two groups(P<0.05). Moreover, the expression levels of GSDMD mRNA and GSDMD-NT protein in the KD group were lower than those of the WT group and the NC group(P<0.05). After LPS+CTB stimulation, the cell mortalities were more in the WT group and the NC group than the KD group(P<0.01). Likewise, the level of IL-1β and the positive percentage of PI staining in the KD group were obviously lower compared with those of the NC group and the WT group(P<0.05). The expression levels of GSDMD mRNA and GSDMD-NT protein were lower in the KD group than the WT group and the NC group, respectively(P<0.05). Conclusion Down-regulation of GSDMD protein expression could inhibit the pyroptosis of MH-S cells and the release of IL-1β induced by LPS.

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备注/Memo

备注/Memo:

基金项目: 国家自然科学基金青年科学基金项目(81300050)
通信作者: 张志成, Email: zhangzhichengicu@ hotmail.com

常用功能

更新日期/Last Update: 2020-08-20