Wang Yanwei, Liu Huifan, Feng Yali, Wu Shujuan, He Jingxuan, Cao Lei
The first hospital of Hebei Medical University.
Department of Anesthesiology, Research Centre of Anesthesiology and Critical Care Medicine, Zhongnan Hospital of Wuhan University.
Tohoku J Exp Med. 2025 Apr 26;265(4):239-248. doi: 10.1620/tjem.2024.J105. Epub 2024 Oct 3.
Sepsis-induced acute lung injury (ALI) has a high mortality rate, and cytokine storm is its feature. PANoptosis is a new type of cell death including apoptosis, pyroptosis and necroptosis. The aim of this study is to detect the PANoptosis level of lung macrophages, and to elucidate the new mechanism of sulforaphane (SFN) in sepsis-induced ALI. In septic animal model, the fluorescent staining of Caspase-8, GSDMD and p-MLKL and ASC/Caspase-8/RIPK3 PANoptosome in lung macrophages was performed. Lipopolysaccharide (LPS) was used to induce macrophages to construct cell model of sepsis. The proportion of dead cells was detected by PI staining, and the expression of Bax, GSDMD-N, NLRP3 and p-MLKL was detected by western blotting. Search for the target genes of SFN and sepsis by network pharmacology. Molecular docking analysis confirmed the binding between SFN and TLR4. The protein levels of TLR4, P65 and p-P65 were detected by western blotting. The transcriptional levels of inflammatory factors were detected by qPCR. The expression of Caspase-8, GSDMD, p-MLKL and PANoptosome in septic lung macrophages was significantly increased, suggesting PANoptosis was up-regulated. LPS induced macrophages death and increased protein levels of Bax, GSDMD-N, NLRP3 and p-MLKL, which were reversed by pretreatment with SFN. Network pharmacology and molecular docking demonstrated that SFN could bind to TLR4 and inhibit NFκB pathway. The mRNA levels of pro-inflammatory factors IL6, CXCL16, iNOS and IL18 were down-regulated by SFN. SFN might alleviate LPS-induced macrophage PANoptosis through TLR4/NFκB pathway, thereby inhibiting macrophage inflammation and becoming a potential therapeutic drug for sepsis-induced ALI.
脓毒症诱导的急性肺损伤（ALI）死亡率高，细胞因子风暴是其特征。PAN凋亡是一种新型细胞死亡，包括凋亡、焦亡和坏死性凋亡。本研究旨在检测肺巨噬细胞的PAN凋亡水平，并阐明萝卜硫素（SFN）在脓毒症诱导的ALI中的新机制。在脓毒症动物模型中，对肺巨噬细胞中的半胱天冬酶-8（Caspase-8）、Gasdermin D（GSDMD）和磷酸化混合谱系激酶结构域样蛋白（p-MLKL）以及凋亡相关斑点样蛋白（ASC）/半胱天冬酶-8/受体相互作用蛋白激酶3（RIPK3）PAN凋亡小体进行荧光染色。使用脂多糖（LPS）诱导巨噬细胞构建脓毒症细胞模型。通过碘化丙啶（PI）染色检测死细胞比例，通过蛋白质免疫印迹法检测Bax、GSDMD-N、NLRP3和p-MLKL的表达。通过网络药理学寻找SFN和脓毒症的靶基因。分子对接分析证实了SFN与Toll样受体4（TLR4）之间的结合。通过蛋白质免疫印迹法检测TLR4、P65和磷酸化P65（p-P65）的蛋白水平。通过实时定量聚合酶链反应（qPCR）检测炎症因子的转录水平。脓毒症肺巨噬细胞中Caspase-8、GSDMD、p-MLKL和PAN凋亡小体的表达显著增加，提示PAN凋亡上调。LPS诱导巨噬细胞死亡并增加Bax、GSDMD-N、NLRP3和p-MLKL的蛋白水平，而SFN预处理可使其逆转。网络药理学和分子对接表明，SFN可与TLR4结合并抑制核因子κB（NFκB）通路。SFN下调促炎因子白细胞介素6（IL6）、CXC趋化因子配体16（CXCL16）、诱导型一氧化氮合酶（iNOS）和白细胞介素18（IL18）的mRNA水平。SFN可能通过TLR4/NFκB通路减轻LPS诱导的巨噬细胞PAN凋亡，从而抑制巨噬细胞炎症，成为脓毒症诱导的ALI的潜在治疗药物。
