目的 在小鼠骨髓源性巨噬細(xì)胞（bone marrow derived macrophage，BMDM）中構(gòu)建溶酶體損傷激活的NOD樣受體熱蛋白結(jié)構(gòu)域相關(guān)蛋白3（NOD-like receptor thermal protein domain associated protein 3，NLRP3）炎癥小體模型，探究蝙蝠葛堿對(duì)NLRP3炎癥小體激活模型的抑制作用。方法 使用脂多糖（lipopolysaccharide，LPS）和亮氨酸-亮氨酸甲酯鹽（Leu-Leu-O-methyl ester，LLOME）聯(lián)合刺激BMDM細(xì)胞，設(shè)置對(duì)照組、LPS組、模型（LPS＋LLOME）組和蝙蝠葛堿低、中、高劑量組。采用細(xì)胞計(jì)數(shù)試劑盒-8（cell counting kit-8，CCK-8）檢測(cè)細(xì)胞活力；Western blotting檢測(cè)剪切的半胱氨酸天冬氨酸蛋白酶-1（cleaved cysteinyl aspartate specific protease-1，cleaved Caspase-1）、剪切的白細(xì)胞介素-1β（cleaved interleukin-1β，cleaved IL-1β）、Gasdermin D的N端片段（Gasdermin D N-terminal，GSDMD-N）蛋白表達(dá)；ELISA檢測(cè)細(xì)胞上清液中IL-1β水平；熒光顯微鏡結(jié)合碘化丙錠（propidium iodide，PI）染色檢測(cè)焦亡細(xì)胞比例變化；采用RNA-seq測(cè)序結(jié)合生物信息學(xué)分析蝙蝠葛堿干預(yù)BMDM細(xì)胞的轉(zhuǎn)錄特征；利用galectin-3抗體結(jié)合免疫熒光分析蝙蝠葛堿對(duì)溶酶體膜損傷的影響；采用流式細(xì)胞術(shù)檢測(cè)蝙蝠葛堿對(duì)溶酶體生成的影響。結(jié)果 蝙蝠葛堿顯著抑制NLRP3炎癥小體激活后的cleaved Caspase-1、cleaved IL-1β及GSDMD-N蛋白表達(dá)（P＜0.05、0.001），并顯著抑制IL-1β的分泌（P＜0.001）。蝙蝠葛堿明顯改善細(xì)胞損傷，抑制細(xì)胞焦亡及焦亡小泡的形成，降低PI陽性細(xì)胞比例（P＜0.001）。蝙蝠葛堿能夠上調(diào)溶酶體生成相關(guān)基因表達(dá)，促進(jìn)溶酶體生成，抑制溶酶體膜損傷后形成的galectin-3（P＜0.05、0.001）。結(jié)論 蝙蝠葛堿通過促進(jìn)溶酶體生物發(fā)生，減少溶酶體膜損傷，從而抑制溶酶體損傷激活的NLRP3炎癥小體。

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

國(guó)家自然科學(xué)基金面上項(xiàng)目（82474153）；北京市自然科學(xué)基金資助項(xiàng)目（7242239）；中華中醫(yī)藥學(xué)會(huì)青年人才托舉項(xiàng)目（CACM-2023-QNRC2-A02）；北京市科技新星計(jì)劃課題（20230484342）