目的 基于腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）/动力相关蛋白1（dynamin-related protein 1，Drp1）通路阐明丹红注射液（Danhong Injection，DHI）改善H9c2心肌细胞缺氧/缺糖模型线粒体动力学失衡并抑制NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor family pyrin domain containing 3，NLRP3）介导焦亡的作用机制。方法 采用超高效液相色谱-质谱联用（ultra-high performance liquid chromatography-mass spectrometry，UHPLC-MS）鉴定DHI核心活性成分，利用AutoDock Vina软件对AMPK、Drp1、视神经萎缩症蛋白1（optic atrophy 1，OPA1）、半胱氨酸天冬氨酸蛋白酶-1（cystein-asparate protease-1，Caspase-1）等靶蛋白与DHI核心活性成分进行分子对接。构建H9c2细胞缺氧/缺糖模型，设置对照组、模型组、DHI组、DHI＋Compound C（AMPK抑制剂）组、AICAR（AMPK激动剂）组、MCC950（NLRP3抑制剂）组和Mdivi-1（Drp1抑制剂）组，采用CCK-8法筛选DHI最佳干预浓度；用JC-1探针检测线粒体膜电位（mitochondrial membrane potential，MMP）；运用MitoSOX^TM测定线粒体活性氧（mitochondrial reactive oxygen species，mtROS）水平；透射电镜观察线粒体超微结构；免疫荧光检测p-AMPK与p-Drp1蛋白表达；Western blotting检测AMPK/Drp1通路相关蛋白、线粒体动力学相关蛋白[线粒体分裂蛋白1（mitochondrial fission 1 protein，FIS1）、线粒体融合蛋白2（mitofusin 2，MFN2）、OPA1]以及焦亡相关蛋白[NLRP3、凋亡相关斑点样蛋白（apoptosis-associated speck-like protein containing a CARD，ASC）、Caspase-1、消皮素D-N端片段（N-terminal fragment of gasdermin D，GSDMD-NT）]表达；ELISA测定白细胞介素-1β（interleukin-1β，IL-1β）、IL-18、三磷酸腺苷（adenosine triphosphate，ATP）水平。结果 UHPLC-MS鉴定出DHI中8种活性成分（丹酚酸、紫草酸B、丹参素、咖啡酸、丹参素钠等）；分子对接结果显示，各靶点和活性成分均具有较强亲和力。体外实验结果显示，与模型组比较，DHI组线粒体形态明显改善，外膜连续，嵴结构相对完整，数量增加，基质密度恢复接近正常，MMP和ATP升高（P＜0.01），mtROS释放量减少（P＜0.01），p-AMPK/AMPK值升高（P＜0.01），p-Drp1/Drp1值下降（P＜0.01），MFN2、OPA1蛋白表达上调（P＜0.01），FIS1、NLRP3、ASC、Caspase-1和GSDMD-NT蛋白表达下调（P＜0.01），IL-1β、IL-18水平显著降低（P＜0.01），与AICAR、MCC950、Mdivi-1作用一致。Compound C能部分抑制DHI的上述作用。结论 DHI通过激活AMPK/Drp1通路，抑制线粒体过度分裂以及促进线粒体融合，降低NLRP3炎性小体活化与焦亡，从而减轻缺氧/缺糖诱导的H9c2细胞损伤。

Objective To elucidate the mechanism by which Danhong Injection (丹红注射液, DHI) ameliorates mitochondrial dynamics imbalance and inhibits NOD-like receptor family pyrin domain containing 3 (NLRP3)-mediated pyroptosis in H9c2 cells subjected to oxygen-glucose deprivation based on AMP-activated protein kinase (AMPK)/dynamin-related protein 1 (Drp1) pathway. Methods Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was employed to identify the core active components of DHI. Molecular docking was performed using AutoDock Vina software to assess the affinity between DHI core components and target proteins, including AMPK, Drp1, optic atrophy 1 (OPA1) and cystein-asparate protease-1 (Caspase-1). An H9c2 cell oxygen-glucose deprivation model was established, control group, model group, DHI group, DHI + Compound C (AMPK inhibitor) group, AICAR (AMPK agonist) group, MCC950 (NLRP3 inhibitor) group and Mdivi-1 (Drp1 inhibitor) group were set up. The optimal intervention concentration of DHI was screened using CCK-8 assay. Mitochondrial membrane potential (MMP) was measured using JC-1 probe, and mitochondrial reactive oxygen species (mtROS) level was determined using MitoSOX^TM. Mitochondrial ultrastructure was observed via transmission electron microscopy. Immunofluorescence was used to detect the protein expressions of p-AMPK and p-Drp1. Western blotting was performed to detect the expressions of AMPK/Drp1 pathway-related proteins, mitochondrial dynamics-related proteins [mitochondrial fission 1 protein (FIS1), mitofusin 2 (MFN2) and OPA1] and pyroptosis-related proteins [NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), Caspase-1 and N-terminal fragment of gasdermin D (GSDMD-NT)]. ELISA was used to determine the levels of interleukin-1β (IL-1β), IL-18 and adenosine triphosphate (ATP). Results UHPLC-MS identified eight active components of DHI, including salvianolic acid, lithospermic acid B, danshensu, caffeic acid, sodium danshensu, etc. The molecular docking results showed that each target and active ingredient had strong affinity. The in vitro experimental results showed that compared with model group, mitochondrial morphology in DHI group was significantly improved, with continuous outer membrane and relatively intact cristae structure, increased quantity, and matrix density restored to near normal. MMP and ATP levels were increased (P < 0.01), mtROS release was decreased (P < 0.01), p-AMPK/AMPK value was increased (P < 0.01), p-Drp1/Drp1 value was decreased (P < 0.01), MFN2 and OPA1 protein expressions were upregulated (P < 0.01), FIS1, NLRP3, ASC, Caspase-1 and GSDMD-NT protein expressions were downregulated (P < 0.01), and IL-1β and IL-18 levels were significantly decreased (P < 0.01), consistent with the effects of AICAR, MCC950 and Mdivi-1. Compound C could partially inhibit the above-mentioned effects of DHI. Conclusion DHI activates AMPK/Drp1 pathway, inhibits mitochondrial excessive division and promotes mitochondrial fusion, reduces NLRP3 inflammasome activation and pyroptosis, thereby alleviating hypoxia/hypoglycemia induced H9c2 cell damage.

R285.5

国家自然科学基金面上项目（82274412）