表小檗碱调控CD39-NLRP3-GSDMD焦亡路径改善脓毒症肺损伤的机制研究

Mechanism of epiberberine on ameliorating sepsis-induced lung injury via modulation of CD39-NLRP3-GSDMD and downstream pyroptosis

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1μg/mL LPS联合5 mmol/L三磷酸腺苷(adenosine triphosphate,ATP)/5μmol/L nigericin/150μg/mL MSU或转染0.5μg/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14.23μmol/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-GloTM 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 CD39in 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 CD39protein 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.