Gasdermin D蛋白对内毒素导致肺泡巨噬细胞焦亡的影响

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

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。

关键词: 急性肺损伤/急性呼吸窘迫综合征, 焦亡, GSDMD, 内毒素

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

图1 各组MH-S细胞GSDMD mRNA表达；注：*与相同处理条件下WT组相比，P<0.05；#与相同处理条件下NC组相比，P<0.05

图2 对照组及焦亡刺激物处理后各组MH-S细胞GSDMD-NT蛋白表达

图3 对照组及焦亡刺激物处理后各组MH-S细胞GSDMD-NT相对表达量；注：*与WT组比较，P<0.05；#与NC组相比，P<0.05

表1 焦亡刺激物处理后MH-S细胞死亡率及炎症因子释放水平

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

表1 焦亡刺激物处理后MH-S细胞死亡率及炎症因子释放水平

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

图4 不同焦亡刺激物处理后MH-S细胞的PI染色阳性百分率；注：*与相同处理条件下WT组相比，P<0.05；#与相同处理条件下NC组相比，P<0.05；

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

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