目的 基于CD39-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)-GSDMD焦亡路径探究表小檗碱改善脓毒症肺损伤的作用机制。方法 通过AMP-Glo TM实验考察黄连异喹啉生物碱成分小檗碱、黄连碱、巴马汀、表小檗碱及药根碱的CD39酶促活性。小鼠ip脂多糖(lipopolysaccharide，LPS，12 mg/kg)建立脓毒症肺损伤模型，分别于造模前24 h和造模0.5 h后给予表小檗碱或MCC950，造模后24 h，采用苏木素-伊红(HE)染色法观察小鼠肺组织病理变化；计算肺组织湿质量/干质量，评价肺肿胀；ELISA法检测肺泡灌洗液中白细胞介素-1β(interleukin-1β，IL-1β)、单核细胞趋化蛋白-1(monocyte chemotactic protein-1，MCP-1)和IL-17A水平。采用1 μg/mL LPS联合5 mmol/L三磷酸腺苷(adenosine triphosphate，ATP)/5μmol/L nigericin/150 μg/mL MSU或转染0.5 μg/mL poly(dA:dT)刺激人源单核细胞白血病THP-1细胞复制炎症模型，给予表小檗碱或VX-765干预，采用Western blotting法检测细胞CD39-NLRP3-GSDMD路径蛋白表达；ELISA法检测上清液中IL-1β水平；采用免疫荧光评估NLRP3炎症小体组装情况；采用扫描电镜评价细胞焦亡情况；采用BzATP介导配体门控离子通道受体7(ligand-gated ion channel receptor 7，P2X7)通道开放，考察表小檗碱对P2X7功能的作用。结果 表小檗碱的CD39酶促活性最佳，半数致死浓度(median effective concentration，EC₅₀)值为14.23 μmol/L。与对照组比较，模型组小鼠肺组织结构异常并显示炎性浸润，且肺肿胀明显(P＜0.01)，肺泡灌洗液中炎性因子水平显著上调(P＜0.01)；表小檗碱干预后，以上变化得到了有效逆转(P＜0.05、0.01)，并呈剂量相关性。以上药效与表小檗碱上调CD39蛋白表达水平、抑制P2X7过表达、干扰NLRP3炎症小体组装、降低GSDMD-N蛋白表达水平、逆转GSDMD介导的巨噬细胞焦亡并降低IL-1β水平有关(P＜0.05、0.01)。结论 表小檗碱通过抑制脓毒症肺损伤小鼠肺泡灌洗液中IL-1β、MCP-1和IL-17A水平逆转肺损伤和肺肿胀，进一步改善脓毒症肺损伤，可能与调控CD39-NLRP3-GSDMD焦亡路径相关。

Objective To explore the mechanism of epiberberine on improving sepsis-induced lung injury based on CD39-NOD-like receptor thermal protein domain associated protein 3 (NLRP3)-GSDMD and downstream pyroptosis. Methods CD39 enzymatic activity of isoquinoline alkaloids from Coptis chinensis such as berberine, coptisine, palmatine, epiberberine and jatrorrhizine, were investigated by AMP-Glo^TM experiment. Mice were ip lipopolysaccharide (LPS, 12 mg/kg) to establish sepsis-induced lung injury model, epiberberine or MCC950 were given 24 h before and 0.5 h after modeling, and pathological changes of lung tissue in mice were observed by hematoxylin-eosin (HE) staining at 24 h after modeling; Wet weight/dry weight ratio of lung tissue was calculated to evaluate lung swelling; Levels of interleukin-1β (IL-1β), monocyte chemotactic protein-1 (MCP-1) and IL-17A in alveolar lavage fluid were detected by ELISA. The 1 μg/mL LPS combined with 5 mmol/L adenosine triphosphate (ATP)/5 μmol/L nigericin/150 μg/mL MSU or transfection of 0.5 μg/mL poly(dA:dT) were used to stimulate THP-1 cells to replicate inflammatory model, epiberberine or VX-765 was given to intervene. The expressions of CD39-NLRP3-GSDMD pathway protein were detected by Western blotting; IL-1β level in supernatant was detected by ELISA; Immunofluorescence was used to evaluate the assembly of NLRP3 inflammatory bodies; Scanning electron microscope was used to evaluate the cell pyroptosis. BzATP-mediated channel opening of ligand-gated ion channel receptor 7 (P2X7) was used to investigate the effect of epiberberine on P2X7 function. Results Enzymatic activity of CD39 in epiberberine was the best, with median effective concentration (EC₅₀) of 14.23 μmol/L. Compared with control group, mice in model group showed abnormal lung tissue structure and inflammatory infiltration, and lung swelling was obvious (P < 0.01), and levels of inflammatory factors in alveolar lavage fluid were significantly increased (P < 0.01); After epiberberine intervention, above changes were effectively reversed (P < 0.05, 0.01), and with a dose-dependent relationship. Above effects were related to up-regulation of CD39 protein expression, inhibition of P2X7 over-expression, interference of NLRP3 inflammatory body assembly, reduction of GSDMD-N protein expression, reversal of GSDMD-mediated macrophage scorch and reduction of IL-1β level by epiberberine (P < 0.05, 0.01). Conclusion Epiberberine can reverse lung injury and lung swelling by inhibiting IL-1β, MCP-1 and IL-17A levels in alveolar lavage fluid of mice with septic lung injury, which may be related to regulating CD39-NLRP3-GSDMD and downstream pyroptosis.

国家自然科学基金青年基金项目(82104491)；中国博士后科学基金面上资助地区专项支持计划(2021M693789)；四川省自然科学基金面上项目(2023NSFCSC2036)；成都中医药大学杏林学者提升计划项目(BSH2020024)