目的 在小鼠骨髓源性巨噬细胞（bone marrow derived macrophage，BMDM）中构建溶酶体损伤激活的NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3，NLRP3）炎症小体模型，探究蝙蝠葛碱对NLRP3炎症小体激活模型的抑制作用。方法 使用脂多糖（lipopolysaccharide，LPS）和亮氨酸-亮氨酸甲酯盐（Leu-Leu-O-methyl ester，LLOME）联合刺激BMDM细胞，设置对照组、LPS组、模型（LPS＋LLOME）组和蝙蝠葛碱低、中、高剂量组。采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）检测细胞活力；Western blotting检测剪切的半胱氨酸天冬氨酸蛋白酶-1（cleaved cysteinyl aspartate specific protease-1，cleaved Caspase-1）、剪切的白细胞介素-1β（cleaved interleukin-1β，cleaved IL-1β）、Gasdermin D的N端片段（Gasdermin D N-terminal，GSDMD-N）蛋白表达；ELISA检测细胞上清液中IL-1β水平；荧光显微镜结合碘化丙锭（propidium iodide，PI）染色检测焦亡细胞比例变化；采用RNA-seq测序结合生物信息学分析蝙蝠葛碱干预BMDM细胞的转录特征；利用galectin-3抗体结合免疫荧光分析蝙蝠葛碱对溶酶体膜损伤的影响；采用流式细胞术检测蝙蝠葛碱对溶酶体生成的影响。结果 蝙蝠葛碱显著抑制NLRP3炎症小体激活后的cleaved Caspase-1、cleaved IL-1β及GSDMD-N蛋白表达（P＜0.05、0.001），并显著抑制IL-1β的分泌（P＜0.001）。蝙蝠葛碱明显改善细胞损伤，抑制细胞焦亡及焦亡小泡的形成，降低PI阳性细胞比例（P＜0.001）。蝙蝠葛碱能够上调溶酶体生成相关基因表达，促进溶酶体生成，抑制溶酶体膜损伤后形成的galectin-3（P＜0.05、0.001）。结论 蝙蝠葛碱通过促进溶酶体生物发生，减少溶酶体膜损伤，从而抑制溶酶体损伤激活的NLRP3炎症小体。

Objective To investigate the inhibitory effects of dauricine on NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome model activated by lysosome damage in bone marrow-derived macrophages (BMDM) of mice. Methods Lipopolysaccharide (LPS) and Leu-Leu-O-methyl ester (LLOME) were used in combination to stimulate BMDM cells, control group, LPS group, model (LPS + LLOME) group, and dauricine low-, medium-, and high-dose groups were set up. Cell viability was detected using cell counting kit-8 (CCK-8); Western blotting was used to detect the protein expressiond of cleaved cysteine aspartate specific protease-1 (cleaved Caspase-1), cleaved interleukin-1β (cleaved IL-1β) and Gasdermin D N-terminal (GSDMD-N); ELISA was used to detect IL-1β level in cell supernatant; Fluorescence microscopy combined with propidium iodide (PI) staining were used to detect changes in the proportion of pyroptosis cells; RNA sequencing combined with bioinformatics analysis were used to investigate the transcriptional characteristics of dauricine intervention in BMDM cells; Galectin-3 antibody combined with immunofluorescence analysis were used to investigate the effect of dauricine on lysosomal membrane damage; Flow cytometry was used to detect the effect of dauricine on lysosome production. Results Dauricine significantly inhibited the expressions of cleaved Caspase-1, cleaved IL-1β and GSDMD-N proteins after NLRP3 inflammasome activation (P < 0.05, 0.001), and significantly inhibited the secretion of IL-1β (P < 0.001). Dauricine significantly improved cell damage, inhibited cell pyroptosis and the formation of pyroptosis vesicles, and reduced the proportion of PI positive cells (P < 0.001). Dauricine could upregulate the expressions of genes related to lysosome formation, promote lysosome formation, and inhibit the formation of galectin-3 after lysosome membrane damage (P < 0.05, 0.001). Conclusion Dauricine promotes lysosomal biogenesis, reduces lysosomal membrane damage, and thus inhibits NLRP3 inflammasome activated by lysosomal damage.

R285.5

国家自然科学基金面上项目（82474153）；北京市自然科学基金资助项目（7242239）；中华中医药学会青年人才托举项目（CACM-2023-QNRC2-A02）；北京市科技新星计划课题（20230484342）