目的 基于核酸結(jié)合寡聚結(jié)構(gòu)域（NOD）樣受體熱蛋白結(jié)構(gòu)域3（NLRP3）炎癥小體的激活，探究漢黃芩素對(duì)脂多糖（LPS）誘導(dǎo)的小鼠急性肺損傷的治療作用。方法 C57BL/6J小鼠隨機(jī)分為對(duì)照組、模型組和漢黃芩素低、高劑量（40、80 mg·kg^-1）組，漢黃芩素ip給藥3 d，對(duì)照組和模型組ip等量0.9%氯化鈉溶液。第3次給藥1 h后給予模型組和漢黃芩素低、高劑量組小鼠ip 25 mg·kg^-1的LPS，對(duì)照組ip等量0.9%氯化鈉溶液。統(tǒng)計(jì)小鼠死亡情況，繪制生存曲線； LPS刺激12 h后監(jiān)測(cè)小鼠呼吸頻率；蘇木精-伊紅（HE）染色檢測(cè)肺組織病理變化；實(shí)時(shí)熒光定量PCR（qRT-PCR）檢測(cè)肺組織炎癥相關(guān)白細(xì)胞介素-1β（Il1b）、腫瘤壞死因子α（Tnfα） mRNA水平； Western blotting檢測(cè)小鼠肺組織NLRP3、凋亡相關(guān)的斑點(diǎn)樣蛋白（ASC）、天冬氨酸特異性的半胱氨酸蛋白水解酶-1（cleaved Caspase-1）、Gasdermin D-NT（GSDMD-NT）的蛋白表達(dá)。采用200 ng·mL^-1 LPS分別聯(lián)合4 mmol·L^-1三磷酸腺苷（ATP）、500 mg·mL^-1明礬（Alum）或1 μmol·L^-1短桿菌肽（ Gra）刺激PMs細(xì)胞構(gòu)建經(jīng)典的NLRP3炎癥小體活化模型，Western blotting檢測(cè)漢黃芩素（5、10、20 μmol·L^-1）對(duì)NLRP3炎癥小體相關(guān)蛋白NLRP3、cleaved Caspase-1、IL-1β和細(xì)胞焦亡相關(guān)蛋白GSDMD-NT表達(dá)的影響。結(jié)果 在小鼠急性肺損傷模型中，與模型組比較，漢黃芩素80 mg·kg^-1組小鼠的死亡率顯著降低（P＜0.05）；漢黃芩素顯著改善小鼠呼吸頻率降低（P＜0.05），顯著改善小鼠肺組織損傷（P＜0.001），顯著降低肺組織Il1b和Tnfα mRNA表達(dá)水平（P＜0.001），顯著降低肺組織NLRP3、GSDMD-NT蛋白的表達(dá)量（P＜0.01、0.001）。在細(xì)胞模型中，與模型組比較，漢黃芩素顯著降低IL-1β、cleavedCaspase-1、GSDMD-NT蛋白的表達(dá)水平（P＜0.05、0.01、0.001）。結(jié)論 漢黃芩素通過(guò)抑制NLRP3炎癥小體活化改善LPS誘導(dǎo)的小鼠急性肺損傷。

Objective To explore the therapeutic effects of wogonin on lipopolysaccharide (LPS) - induced acute lung injury in mice based on the activation of the nucleotide oligomerization domain (NOD) like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. Methods C57BL/6J mice were randomly divided into the control group, model group, and low-dose (40 mg·kg^-1) and high-dose (80 mg·kg^-1) wogonin groups. Wogonin was ip administered for 3 d, while the control and model groups were ip injected with the same volume of 0.9% sodium chloride solution. One hour after the third administration, the model group and the low-dose and high-dose wogonin groups were ip injected with 25 mg·kg^-1 LPS, and the control group was ip injected with the same volume of 0.9% sodium chloride solution. The mortality of mice was recorded, and the survival curve was plotted. The respiratory rate of mice was monitored 12 hours after LPS stimulation. Hematoxylin-eosin (HE) staining was used to detect the pathological changes of lung tissue. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the mRNA levels of inflammation-related interleukin-1β (Il1b) and tumor necrosis factor α (Tnfα) in lung tissue. Western blotting was used to detect the protein expression of NLRP3, apoptosis-associated speck-like protein (ASC), cleaved caspase-1, and gasdermin D-NT (GSDMD-NT) in mouse lung tissue. PMs cells were stimulated with 200 ng·mL^-1 LPS in combination with 4 mmol·L^-1 ATP, 500 mg·mL^-1 Alum, or 1 μmol·L^-1 nigericin to establish a classic NLRP3 inflammasome activation model. Western blotting was used to detect the effects of wogonin (5, 10, and 20 μmol·L^-1) on the expression of NLRP3 inflammasome-related proteins NLRP3, cleaved caspase-1, IL-1β, and pyroptosisrelated protein GSDMD-NT. Results In the acute lung injury model of mice, compared with the model group, the mortality rate of the 80 mg·kg^-1 wogonin group was significantly reduced (P < 0.05); wogonin significantly improved the decreased respiratory rate of mice (P < 0.05), significantly improved lung tissue injury (P < 0.001), significantly reduced the mRNA expression levels of Il1b and Tnfα in lung tissue (P < 0.001), and significantly reduced the protein expression levels of NLRP3 and GSDMD-NT in lung tissue (P < 0.01, 0.001). In the cell model, compared with the model group, wogonin significantly reduced the expression levels of IL-1β, cleaved caspase-1, and GSDMD-NT proteins (P < 0.05, 0.01, 0.001). Conclusion Wogonin ameliorates LPS induced acute lung injury in mice through the inhibition of NLRP3 inflammasome activation.

R285.5

國(guó)家自然科學(xué)基金面上項(xiàng)目（82474153）；2023年度北京中醫(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）