目的 基于NOD樣受體蛋白3（NLRP3）炎癥小體與細(xì)胞焦亡，探究金合歡素對急性肺損傷（ALI）的治療作用。方法 將雄性C57BL/6J小鼠適應(yīng)性喂養(yǎng)1周后，隨機(jī)分為對照組、模型組、金合歡素（40 mg·kg^-1）組，除對照組外，ip致死劑量的脂多糖（LPS，20 mg·kg^-1）構(gòu)建ALI小鼠模型，進(jìn)行小鼠生存率實(shí)驗(yàn)； ip致炎劑量的LPS（10 mg·kg^-1）構(gòu)建ALI小鼠模型，蘇木素-伊紅（HE）染色法檢測肺組織病理變化；實(shí)時熒光定量PCR（qRT-PCR）法檢測小鼠肺組織炎癥相關(guān)基因腫瘤壞死因子（Tnf）、白細(xì)胞介素（Il） 6、Il1b的表達(dá)； Western blotting檢測小鼠肺組織中NLRP3通路相關(guān)蛋白NLRP3、消皮素D（GSDMD）的表達(dá)； ELISA法檢測小鼠腹腔灌洗液IL-1β水平。體外培養(yǎng)原代腹腔巨噬細(xì)胞，通過將LPS分別與腺嘌呤核苷三磷酸（ATP）、尼日利亞菌素（Nig）以及短桿菌肽（Gram）聯(lián)合使用，刺激NLRP3炎癥小體激活，給予金合歡素（5、10、20 μmol·L-1）聯(lián)合培養(yǎng)，Western blotting法檢測半胱氨酸蛋白酶-1（Caspase-1）、NLRP3、GSDMD蛋白表達(dá)水平，碘化丙啶（PI）/Hoechst染色觀察細(xì)胞焦亡。結(jié)果 LPS 20 mg·kg^-1造模后45 h，模型組小鼠全部死亡；金合歡素組小鼠最終生存率為60%。與LPS 10 mg·kg^-1模型組相比，金合歡素組的小鼠肺組織結(jié)構(gòu)損傷得到顯著緩解，炎癥細(xì)胞浸潤明顯減少，肺泡壁增厚情況減輕，且肺組織損傷評分顯著降低（P＜0.001）；肺組織Tnf、Il6、Il1b mRNA表達(dá)顯著下降（P＜0.001），腹腔灌洗液IL-1β水平顯著降低（P＜0.001）；肺組織NLRP3、GSDMD-NT蛋白表達(dá)水平明顯降低（P＜0.01、0.001）。體外實(shí)驗(yàn)結(jié)果表明，與模型組比較，金合歡素組NLRP3、GSDMD-NT、Caspase-1（p20）蛋白表達(dá)水平顯著降低（P＜0.01、0.001）；細(xì)胞焦亡率顯著降低（P＜0.001）。結(jié)論 金合歡素可以通過抑制NLRP3炎癥小體與細(xì)胞焦亡，改善ALI小鼠的肺部損傷以及炎癥反應(yīng)。

Objective To investigate the therapeutic effects of acacetin on acute lung injury (ALI) base on the NOD-like receptor protein 3 (NLRP3) inflammasome and pyroptosis. Methods Male C57BL/6J mice were adaptively fed for one week and then randomly divided into control group, model group, and acacetin (40 mg·kg^-1) group. Except for control group, the mice in the other groups were ip with a lethal dose of lipopolysaccharide (LPS, 20 mg·kg^-1) to establish ALI mouse model, and the survival rate of the mice was examined. The mice in the other groups were ip with an inflammatory dose of LPS (10 mg·kg^-1) to establish the ALI mouse model. The pathological changes of lung tissue were detected by hematoxylin-eosin (HE) staining. The expression of inflammationrelated genes tumor necrosis factor (Tnf), interleukin (Il) 6, and Il1b in lung tissue was detected by real-time fluorescence quantitative PCR (qRT-PCR). The expression of NLRP3 pathway-related proteins NLRP3 and gasdermin D (GSDMD) in lung tissue was detected by Western blotting. The level of IL^-1β in the peritoneal lavage fluid was detected by ELISA. Primary peritoneal macrophages were cultured in vitro. LPS was combined with adenosine triphosphate (ATP), nigericin (Nig), and gramicidin (Gram) to stimulate the activation of NLRP3 inflammasome. Acacetin (5, 10, 20 μmol·L^-1) was added for co-culture. The expression levels of caspase-1, NLRP3, and GSDMD proteins were detected by Western blotting, and pyroptosis was observed by propidium iodide (PI)/Hoechst staining. Results 45 h after LPS 20 mg·kg^-1 modeling, all mice in the model group died; The final survival rate of the acacetin group was 60%. Compared with LPS 10 mg·kg^-1 model group, the structural damage of lung tissue in the acacetin group was significantly alleviated, the infiltration of inflammatory cells was significantly reduced, the thickening of alveolar walls was alleviated, and the lung tissue injury score was significantly decreased (P < 0.001); The mRNA expression of Tnf, Il6, and Il1b in lung tissue was significantly decreased (P < 0.001), and the level of IL^-1β in peritoneal lavage fluid was significantly decreased (P < 0.001); The expression levels of NLRP3 and GSDMD-NT proteins in lung tissue were significantly decreased (P < 0.01, 0.001). The in vitro experimental results showed that compared with the model group, the expression levels of NLRP3, GSDMD-NT, and Caspase-1 (p20) proteins in the acacetin group were significantly decreased (P < 0.01, 0.001), and the pyroptosis rate was significantly decreased (P < 0.001). Conclusion Acacetin has the potential to ameliorate lung injury and modulate the inflammatory response in ALI mice by suppressing the NLRP3 inflammasome and pyroptosis.

R285.5

國家自然科學(xué)基金面上項(xiàng)目（82474153）;北京中醫(yī)藥大學(xué)-優(yōu)莎納聯(lián)合研究中心（BURC）基金重點(diǎn)項(xiàng)目（ BUCM-2022-JS-KF-003）;北京市科技新星計(jì)劃課題（20230484342）;北京市自然科學(xué)基金資助項(xiàng)目（7242239）;中華中醫(yī)藥學(xué)會青年人才托舉工程項(xiàng)目(CACM-2023-QNRC2-A02)