目的 基于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）/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）通路研究木犀草素對(duì)脂多糖（lipopolysaccharide，LPS）聯(lián)合三磷腺苷（adenosine triphosphate，ATP）誘導(dǎo)的小鼠單核巨噬細(xì)胞J774A.1焦亡的作用及其機(jī)制。方法 利用分子對(duì)接技術(shù)預(yù)測(cè)木犀草素與焦亡相關(guān)蛋白結(jié)合的可能性。將對(duì)數(shù)生長(zhǎng)期的小鼠單核巨噬細(xì)胞J774A.1細(xì)胞分為對(duì)照組、LPS＋ATP組、木犀草素（8 μmol/L）組和木犀草素低、中、高（2、4、8 μmol/L）＋LPS＋ATP組。采用LPS＋ATP誘導(dǎo)J774A.1細(xì)胞焦亡，采用木犀草素預(yù)防治療12 h。通過碘化丙啶（propidium iodide，PI）染色、乳酸脫氫酶（lactate dehydrogenase，LDH）釋放和細(xì)胞計(jì)數(shù)（cell counting kit-8，CCK-8）法檢測(cè)細(xì)胞活力和細(xì)胞膜損傷，利用ELISA法測(cè)定白細(xì)胞介素-1β（interleukin-1β，IL-1β）和IL-18的水平。采用Western blotting檢測(cè)細(xì)胞中NLRP3、凋亡相關(guān)顆粒樣蛋白（apoptosis-associated speck-like protein containing a CARD，ASC）、Caspase-1、GSDMD的蛋白表達(dá)。同時(shí)，將ICR雄性小鼠隨機(jī)分為對(duì)照組、LPS（50 mg/kg）組、木犀草素（80 mg/kg）組和木犀草素低、中、高劑量（40、60、80 mg/kg）＋LPS（50 mg/kg）組，進(jìn)行木犀草素干預(yù)LPS誘導(dǎo)小鼠脾臟細(xì)胞焦亡的體內(nèi)實(shí)驗(yàn)，檢測(cè)血清中IL-1β、IL-18的水平和脾臟中NLRP3、ASC、Caspase-1、GSDMD的表達(dá)。結(jié)果 分子對(duì)接結(jié)果表明，木犀草素與細(xì)胞焦亡相關(guān)蛋白Caspase-1、NLRP3、ASC、GSDMD的結(jié)合良好，結(jié)合能均小于−5 kcal/mol。體外實(shí)驗(yàn)表明，經(jīng)過木犀草素預(yù)處理（2～8 μmol/L）12 h能顯著提高LPS＋ATP誘導(dǎo)的J774A.1細(xì)胞活力（P＜0.05），顯著減少PI染色陽性率和LDH釋放（P＜0.05），顯著降低IL-1β和IL-18水平（P＜0.01），并顯著下調(diào)NLRP3、Caspase-1 p10及GSDMD p30的蛋白表達(dá)（P＜0.01）。體內(nèi)實(shí)驗(yàn)表明，木犀草素可以顯著降低小鼠血清中IL-1β和IL-18水平（P＜0.01），并顯著下調(diào)NLRP3、Caspase-1 p20及GSDMD p30的蛋白表達(dá)（P＜0.01）。結(jié)論 木犀草素能夠顯著改善LPS＋ATP誘導(dǎo)的J774A.1細(xì)胞焦亡模型的形態(tài)學(xué)損傷，抑制促炎性細(xì)胞因子IL-1β和IL-18的釋放，并通過NLRP3/Caspase-1/GSDMD信號(hào)通路抑制細(xì)胞焦亡的發(fā)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

國家自然科學(xué)基金面上項(xiàng)目（32273047）