Gasdermin D蛋白对内毒素导致肺泡巨噬细胞焦亡的影响

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

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。

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-1βlevel 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-1βand 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-1βinduced by LPS.