目的 探究黄芩素对急性肺损伤（acute lung injure，ALI）小鼠的治疗作用以及对NOD样受体热蛋白结构域3（NOD-like receptor family pyrin domain containing 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）焦亡通路的调控作用。方法 60只雄性C57BL/6小鼠随机分为对照组、模型组、Caspase-1抑制剂VX-765（30 mg/kg）组和黄芩素低、中、高剂量（10、20、40 mg/kg）组，每组10只。除对照组外，其余各组ip脂多糖（lipopolysaccharide，LPS，15 mg/kg），分别于造模前24 h和造模0.5 h后给予黄芩素或VX-765（30 mg/kg）干预。造模24 h后，检测小鼠肺湿干质量比以评价肺肿胀；苏木素-伊红（HE）染色法检测肺组织病理变化；透射电子显微镜观察肺组织中巨噬细胞损伤情况；ELISA法检测小鼠血清和肺泡灌洗液中白细胞介素-1β（interleukin-1β，IL-1β）、IL-6、IL-18、IL-1α和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）水平；采用Western blotting检测肺组织NLRP3、凋亡相关的斑点样蛋白（apoptosis-associated speck-like protein containing CARD，ASC）、cleaved Caspase-1、pro Caspase-1、GSDMD-N以及GSDMD蛋白表达。人源单核细胞白血病THP-1细胞加入100 nmol/L佛波酯诱导24 h，贴壁后随机分为对照组、模型组、VX-765（500 nmol/L）组和黄芩素低、中、高剂量（3、10、30 μmol/L）组，除对照组外，采用1 μg/mL LPS联合5 mmol/L三磷酸腺苷（adenosine triphosphate，ATP）刺激THP-1细胞建立细胞炎症模型，给予黄芩素或VX-765干预，采用CCK-8法检测细胞活力；ELISA法检测上清液中IL-1β、IL-18和IL-1α水平；LDH试剂盒检测上清液中乳酸脱氢酶（lactate dehydrogenase，LDH）活力；免疫荧光评估NLRP3炎症小体组装情况；采用膜联蛋白-V-PE（Annexin-V-PE）/7-氨基放线菌素（7-aminoactinomycin，7-AAD）试剂盒评价细胞焦亡情况；Western blotting检测细胞NLRP3/Caspase-1/GSDMD通路相关蛋白表达。结果 在ALI小鼠模型中，与模型组比较，VX-765组和黄芩素组小鼠肺肿胀程度明显减轻（P<0.01），肺组织损伤评分显著降低（P<0.01），肺组织中巨噬细胞炎性病变得到缓解，血清及肺泡灌洗液中IL-6、IL-1β、TNF-α、IL-18、IL-1α水平明显降低（P<0.05、0.01），肺组织NLRP3、ASC、cleaved Caspase-1、pro Caspase-1、GSDMD-N以及GSDMD蛋白表达水平均显著降低（P<0.05、0.01）。在细胞炎症模型中，与模型组比较，VX-765组和黄芩素组THP-1细胞IL-1β、IL-18、IL-1α分泌量显著降低（P<0.01），NLRP3/Caspase-1/GSDMD通路相关蛋白表达水平明显降低（P<0.05、0.01），NLRP3炎症小体组装明显被抑制（P<0.05、0.01），LDH释放量及细胞焦亡率显著降低（P<0.01）。结论 黄芩素通过抑制NLRP3/Caspase-1/GSDMD通路介导的细胞焦亡，从而改善LPS诱导的小鼠ALI。

Objective To investigate the therapeutic effect of baicalein on acute lung injure (ALI) mice and its regulatory effect on NOD-like receptor family pyrin domain containing 3 (NLRP3)/cysteine-aspartate protease-1 (Caspase-1)/gasdermin D (GSDMD) pyroptosis pathway. Methods A total of 60 male C57BL/6 mice were randomly divided into control group, model group, Caspase-1 inhibitor (VX-765, 30 mg/kg), baicalein low-, medium- and high-dose (10, 20, 40 mg/kg) groups, with 10 mice in each group. Except for the control group, mice in the other groups were ip lipopolysaccharide (LPS, 15 mg/kg) to establish a mouse model of ALI, baicalein or VX-765 was given 24 h before and 0.5 h after modeling. The lung wet/dry weight ratio of mice was measured 24 h after LPS administration to evaluate lung swelling; The hematoxylin-eosin (HE) staining was used to detect lung tissue pathological changes; The injury of macrophages in lung tissue was observed by transmission electron microscopy (TEM); ELISA method was applied to detect interleukin-1β (IL-1β), IL-6, IL-18, IL-1α and tumor necrosis factor-α (TNF-α) levels in alveolar lavage fluid and serum of mice; Protein expressions of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), cleaved Caspase-1, pro Caspase-1, GSDMD-N, and GSDMD in lung tissue were determined by Western blotting. THP-1 cells were induced with 100 nmol/L phorbol ester for 24 h. After adherence, cells were randomly divided into control group, model group, VX-765 (500 nmol/L) group and baicalein low-, medium- and high-dose (3, 10, 30 μmol/L) groups. Except for the control group, THP-1 cells were stimulated with 1 μg/mL LPS combined with 5 mmol/L adenosine triphosphate (ATP) to replicate inflammatory model, baicalein or VX-765 was given for intervention, CCK-8 method was applied to determine the cell ability; IL-1β, IL-18, and IL-1α levels in supernatant were detected by ELISA; The lactate dehydrogenase (LDH) kit was used to measure LDH activity; The assembly of NLRP3 inflammasome was evaluated by immunofluorescence; Annexin V-PE/7-aminoactinomycin (7-AAD) kit was used to evaluate the cell pyroptosis; The expressions of NLRP3/Caspase-1/GSDMD pathway protein were detected by Western blotting. Results In ALI mice model, compared with model group, lung swelling of mice in VX-765 group and baicalein group weas significantly attenuated (P < 0.01), lung injure score was decreased (P < 0.01), inflammatory changes of macrophages in lung tissue were alleviated, levels of IL-6, IL-1β, TNF-α, IL-18 and IL-1α in serum and bronchoalveolar lavage fluid were significantly decreased (P < 0.05, 0.01), NLRP3, ASC, cleaved Caspase-1, pro Caspase-1, GSDMD-N, and GSDMD protein expressions in lung tissues were decreased (P < 0.05, 0.01). In cell inflammatory model, compared with model group, IL-1β, IL-18 and IL-1α secretions of THP-1 cells in VX-765 group and baicalein group were significantly decreased (P < 0.01), the expressions of NLRP3/Caspase-1/GSDMD pathway related proteins were obviously decreased (P < 0.05, 0.01), NLRP3 inflammasome assembly was inhibited (P < 0.05, 0.01), LDH release and pyroptosis rate were significantly decreased (P < 0.01). Conclusion Baicalein can improve LPS-induced ALI in mice by inhibiting NLRP3/Caspase-1/GSDMD pathway-mediated pyroptosis.

R285.5

国家自然科学基金青年基金项目（82104491）；四川省自然科学基金面上项目（2023NSFSC0674）；中国博士后科学基金面上资助地区专项支持计划项目（2021M693789）；成都中医药大学“杏林学者”学科人才科研提升计划项目（XKTD2022013）