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

doi:10.3877/cma.j.issn.1674-6902.2020.04.002

Abstract

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.

Key words: Acute lung injury/Acute respiratory distress syndrome, Pyroptosis, Gasdermin D, Lipopolysaccharide

Weizheng Shuai, Jun Li, Xuxin Chen, Dawei Li, Zhicheng Zhang. Role of Gasdermin D in pyroptosis of mouse alveolar macrophages induced by lipopolysaccharide[J]. Chinese Journal of Lung Diseases(Electronic Edition), 2020, 13(04): 441-445.

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URL: https://zhfbjbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-6902.2020.04.002

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Figures/Tables 5

References 25

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变　量	con			LPS+CTB			LPS+nigericin
变　量	WT	NC-	KD	WT	NC	KD	WT	NC	KD
死亡率(%)	1.86±1.81	0.14±5.18	3.13±4.05	30.88±4.96	32.97±2.80	19.83±3.20^a	58.33±4.24	66.4±6.94	42.79±6.64^a
IL-1β(pg/ml)	1.78±0.19	1.78±0.51	0.77±0.58	2 374.00±72.11	2 655.67±88.14	1 184.56±36.98^b	4 257.33±117.80	3 883.44±106.06	1 870.11±170.07^b

变　量	con			LPS+CTB			LPS+nigericin
变　量	WT	NC-	KD	WT	NC	KD	WT	NC	KD
死亡率(%)	1.86±1.81	0.14±5.18	3.13±4.05	30.88±4.96	32.97±2.80	19.83±3.20^a	58.33±4.24	66.4±6.94	42.79±6.64^a
IL-1β(pg/ml)	1.78±0.19	1.78±0.51	0.77±0.58	2 374.00±72.11	2 655.67±88.14	1 184.56±36.98^b	4 257.33±117.80	3 883.44±106.06	1 870.11±170.07^b

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