目的 基于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）/半胱氨酸天冬氨酸蛋白酶-1（cystein-asparate protease-1，Caspase-1）/消皮素D（gasdermin D，GSDMD）通路研究木犀草素对脂多糖（lipopolysaccharide，LPS）联合三磷腺苷（adenosine triphosphate，ATP）诱导的小鼠单核巨噬细胞J774A.1焦亡的作用及其机制。方法 利用分子对接技术预测木犀草素与焦亡相关蛋白结合的可能性。将对数生长期的小鼠单核巨噬细胞J774A.1细胞分为对照组、LPS＋ATP组、木犀草素（8 μmol/L）组和木犀草素低、中、高（2、4、8 μmol/L）＋LPS＋ATP组。采用LPS＋ATP诱导J774A.1细胞焦亡，采用木犀草素预防治疗12 h。通过碘化丙啶（propidium iodide，PI）染色、乳酸脱氢酶（lactate dehydrogenase，LDH）释放和细胞计数（cell counting kit-8，CCK-8）法检测细胞活力和细胞膜损伤，利用ELISA法测定白细胞介素-1β（interleukin-1β，IL-1β）和IL-18的水平。采用Western blotting检测细胞中NLRP3、凋亡相关颗粒样蛋白（apoptosis-associated speck-like protein containing a CARD，ASC）、Caspase-1、GSDMD的蛋白表达。同时，将ICR雄性小鼠随机分为对照组、LPS（50 mg/kg）组、木犀草素（80 mg/kg）组和木犀草素低、中、高剂量（40、60、80 mg/kg）＋LPS（50 mg/kg）组，进行木犀草素干预LPS诱导小鼠脾脏细胞焦亡的体内实验，检测血清中IL-1β、IL-18的水平和脾脏中NLRP3、ASC、Caspase-1、GSDMD的表达。结果 分子对接结果表明，木犀草素与细胞焦亡相关蛋白Caspase-1、NLRP3、ASC、GSDMD的结合良好，结合能均小于−5 kcal/mol。体外实验表明，经过木犀草素预处理（2～8 μmol/L）12 h能显著提高LPS＋ATP诱导的J774A.1细胞活力（P＜0.05），显著减少PI染色阳性率和LDH释放（P＜0.05），显著降低IL-1β和IL-18水平（P＜0.01），并显著下调NLRP3、Caspase-1 p10及GSDMD p30的蛋白表达（P＜0.01）。体内实验表明，木犀草素可以显著降低小鼠血清中IL-1β和IL-18水平（P＜0.01），并显著下调NLRP3、Caspase-1 p20及GSDMD p30的蛋白表达（P＜0.01）。结论 木犀草素能够显著改善LPS＋ATP诱导的J774A.1细胞焦亡模型的形态学损伤，抑制促炎性细胞因子IL-1β和IL-18的释放，并通过NLRP3/Caspase-1/GSDMD信号通路抑制细胞焦亡的发生。

Objective Based on NOD-like receptor thermal protein domain associated protein 3 (NLRP3)/cysteinyl aspartate specific proteinase-1 (Caspase-1)/Gasdermin D (GSDMD) pathway to study the intervention effect of luteolin on lipopolysaccharide (LPS) combined with adenosine triphosphate (ATP)-induced pyroptosis in J774A.1 cells and its mechanism. Methods Molecular docking technology was used to predict the possibility of luteolin binding to pyroptosis-related proteins. Mouse mononuclear macrophage J774A.1 cells in logarithmic growth phase were divided into control group, LPS+ATP group, luteolin (8 μmol/L) group, and luteolin low, medium, and high (2, 4, and 8 μmol/L) + LPS + ATP groups. LPS + ATP was used to induce pyroptosis in J774A.1 cells, and luteolin was used for preventive treatment for 12 h. Cell viability and cell membrane damage were detected by propidium iodide (PI) staining, lactate dehydrogenase (LDH) release, and cell counting kit-8 (CCK-8) method. The levels of interleukin-1β (IL-1β) and IL-18 were determined by ELISA. Western blotting was used to detect the protein expressions of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), Caspase-1, and GSDMD in cells. At the same time, ICR male mice were randomly divided into a control group, an LPS (50 mg/kg) group, a luteolin alone (80 mg/kg) group, and a luteolin low-, medium-, and high-dose (40, 60, and 80 mg/kg) + LPS (50 mg/kg) group. An in vivo experiment was performed to investigate the intervention of luteolin in LPS-induced pyroptosis of mouse spleen cells. The levels of IL-1β and IL-18 in serum and the expressions of NLRP3, ASC, Caspase-1, and GSDMD in spleen were detected. Results The results of molecular docking showed that luteolin had good binding activity with pyroptosis-related proteins Caspase-1, NLRP3, ASC and GSDMD, and the binding energy was less than −5 kcal/mol. In vitro experiments showed that 12 hours of luteolin pretreatment (2—8 μmol/L) significantly increased the cell viability induced by LPS + ATP (P < 0.05), significantly reduced the positive rate of PI staining and LDH release (P < 0.05), significantly reduced the release of IL-1β and IL-18 (P < 0.01), and significantly down-regulated the expression of NLRP3, Caspase-1 p10 and GSDMD p30 (P < 0.01). In vivo experiments showed that luteolin could significantly reduce the release of IL-1β and IL-18 in serum (P < 0.01) and significantly down-regulate the expression of NLRP3, Caspase-1 p20 and GSDMD p30 (P < 0.01). Conclusion Luteolin can significantly improve the morphological damage of LPS + ATP-induced J774A.1 cell pyroptosis, inhibit the release of the pro-inflammatory cytokine IL-1β and IL-18, and inhibit the occurrence of pyroptosis through the NLRP3/Caspase-1/GSDMD signaling pathway.

R285.5

国家自然科学基金面上项目（32273047）