双硫仑抑制细胞焦亡改善脓毒症小鼠急性肺损伤的作用机制

Mechanism of disulfiram in alleviating sepsis‐induced acute lung injury by suppressing pyroptosis in mice

摘要

目的：探讨双硫仑（DSF）是否通过抑制细胞焦亡减轻脓毒症所致急性肺损伤（ALI）的作用机制。方法:采用6周龄雄性C57BL/6小鼠，腹腔注射脂多糖（LPS）15 mg·kg^-1构建脓毒症急性肺损伤模型。随机分为对照组、LPS组及LPS+DSF干预组（25、50、100 mg·kg^-1），检测小鼠72 h存活率；于LPS注射24 h后取肺组织行HE染色并进行Smith病理评分，测定肺湿干质量比；采用qRT-PCR与ELISA法分别检测血清及肺组织中炎症因子IL-6、TNF-α、IL-1β的蛋白与mRNA表达水平。体外培养THP-1细胞分别经0、0.5、1、2、5、10 μg·mL^-1 DSF处理24 h后，采用CCK-8法检测细胞活力。将THP-1细胞分为对照组、LPS组及LPS+DSF组，LPS+DSF组先以1 μmol·L^-1 DSF预处理4 h，再与1 μg·mL^-1 LPS共刺激6 h，采用qRT-PCR、ELISA及Western Blot检测炎症因子及焦亡相关蛋白cleaved caspase-1、GSDMD-N、NLRP3、IL-1β的表达；Western Blot检测小鼠肺组织中上述焦亡相关蛋白的表达。结果：与对照组相比，LPS组小鼠死亡率升高，肺湿干质量比增加，血清及肺组织中IL-6、TNF-α、IL-1β水平均显著上升（P<0.05）；DSF干预可改善上述病理改变并降低炎症因子水平（P<0.05）。细胞实验显示，在0.5~10 μmol·L^-1浓度范围内，DSF对THP-1细胞无明显毒性，仅10 μmol·L^-1时细胞活力轻微下降（P<0.05）。DSF可有效抑制LPS诱导的炎症因子释放及NLRP3/Caspase-1/GSDMD焦亡通路相关蛋白的表达（P<0.05）。结论：双硫仑可能通过抑制NLRP3炎症小体激活及后续的细胞焦亡通路，缓解脓毒症引起的急性肺损伤。

Abstract

Objective: To investigate the mechanism by which disulfiram (DSF) alleviates sepsis-induced acute lung injury (ALI) through the inhibition of pyroptosis. Methods: A sepsis-induced ALI model was established in 6-week-old male C57BL/6 mice by intraperitoneal injection of lipopolysaccharide (LPS) 15 mg·kg^-1. The mice were randomly divided into a control group, an LPS group, and LPS+DSF intervention groups (DSF at 25, 50, 100 mg·kg^-1). The 72-hour survival rate was recorded. At 24 hours after LPS injection, lung tissues were collected for hematoxylin and eosin (HE) staining and Smith pathological scoring; the lung wet/dry weight (W/D) ratio was measured. The mRNA and protein expression levels of inflammatory cytokines IL-6, TNF-α, and IL-1β in serum and lung tissues were detected by qRT-PCR and ELISA, respectively. THP-1 cells were culturedin vitro and treated with 0, 0.5, 1, 2, 5, or 10 μg·mL^-1 DSF for 24 hours, followed by cell viability assessment using the CCK-8 assay. For mechanistic studies, THP-1 cells were divided into control, LPS, and LPS+DSF groups. The LPS+DSF group was pretreated with 1 μmol·L^-1 DSF for 4 hours and then co-stimulated with 1 μg·mL^-1 LPS for 6 hours. The expression of inflammatory cytokines and pyroptosis-related proteins, including cleaved caspase-1, Gasdermin D (GSDMD)-N, NLRP3, and IL-1β, was detected by qRT-PCR, ELISA, and Western Blot. Western Blot was also used to examine the expression of pyroptosis-related proteins in mouse lung tissues. Results: Compared with the control group, the LPS group exhibited increased mortality, elevated lung W/D ratio, and significantly higher levels of IL-6, TNF-α, and IL-1β in serum and lung tissues (P<0.05). DSF intervention alleviated these pathological changes and reduced the levels of inflammatory cytokines (P<0.05).In vitro, DSF showed no obvious cytotoxicity to THP-1 cells within the concentration range of 0.5-10 μmol·L^-1, except for a slight decrease in cell viability at 10 μmol·L^-1 (P<0.05). DSF effectively suppressed LPS-induced release of inflammatory cytokines and the expression of pyroptosis-related proteins in the NLRP3/caspase-1/GSDMD pathway (P<0.05). Conclusion: Disulfiram may alleviate sepsis-induced acute lung injury by inhibiting NLRP3 inflammasome activation and the subsequent pyroptosis pathway.

关键词

脓毒症 / 急性肺损伤 / 双硫仑 / 细胞焦亡 / NLRP3炎症小体 / Gasdermin D(GSDMD)

Key words

Sepsis / Acute Lung Injury / Disulfiram / Pyroptosis / NLRP3 Inflammasome / Gasdermin D（GSDMD）

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基金资助

新发突发与重大传染病防控国家科技重大专项(2025ZD01902600)

国家自然科学基金面上项目(82272226)

国家自然科学基金面上项目(82572461)

国家自然科学基金青年科学基金项目（C类）(82502662)

国家自然科学基金青年科学基金项目（C类）(82202409)