中草药

丹红注射液通过AMPK/Drp1通路改善H9c2细胞线粒体动力学失衡并抑制细胞焦亡

PDF下载

叶嘉豪, 吴子政, 张垚, 孟骊冲, 胡志希

中草药 | 药理与临床 2026,57(11): 4196-4211

收起

中草药 |药理与临床 2026 , 57 (11) : 4196 -4211

丹红注射液通过AMPK/Drp1通路改善H9c2细胞线粒体动力学失衡并抑制细胞焦亡

全屏

叶嘉豪, 吴子政, 张垚, 孟骊冲, 胡志希

作者信息

通讯作者:

胡志希

作者简介:

叶嘉豪: 叶嘉豪（1996—），男，博士研究生，研究方向为心血管疾病证本质与诊治规律。E-mail:1062940906@qq.com

Danhong Injection ameliorates mitochondrial dynamics imbalance and inhibits pyroptosis in H9c2 cells via AMPK/Drp1 pathway

YE Jiahao, WU Zizheng, ZHANG Yao, MENG Lichong, HU Zhixi

Affiliations

doi: 10.7501/j.issn.0253-2670.2026.11.011

文章导航

摘要

收起

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MitoSOXTM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。

关键词

丹红注射液 / AMPK/Drp1通路 / 线粒体动力学 / 细胞焦亡 / 线粒体融合 / 线粒体分裂 / 丹酚酸 / 紫草酸B / 丹参素 / 咖啡酸 / 丹参素钠 / Boc-D-谷氨酰胺 / D-半乳糖1-[2-(2-叠氮乙氧基)乙氧基乙基]-2,3,4,6-四-O-乙酸酯 / 鸟苷

Abstract

收起

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.

Key words

Danhong Injection / AMPK/Drp1 pathway / mitochondrial dynamics / pyroptosis / mitochondrial fusion / mitochondrial fission / salvianolic acid / lithospermic acid B / danshensu / caffeic acid / sodium danshensu / Boc-D-glutamine / D-galactose 1-[2-(2-azidoethoxy)ethoxyethyl]-2,3,4,6-tetra-O-acetate / guanosine

引用本文

叶嘉豪, 吴子政, 张垚, 孟骊冲, 胡志希. 丹红注射液通过AMPK/Drp1通路改善H9c2细胞线粒体动力学失衡并抑制细胞焦亡. 中草药, 2026 , 57 (11) : 4196 -4211 . DOI: 10.7501/j.issn.0253-2670.2026.11.011

YE Jiahao, WU Zizheng, ZHANG Yao, MENG Lichong, HU Zhixi. Danhong Injection ameliorates mitochondrial dynamics imbalance and inhibits pyroptosis in H9c2 cells via AMPK/Drp1 pathway[J]. Chinese Traditional and Herbal Drugs, 2026 , 57 (11) : 4196 -4211 . DOI: 10.7501/j.issn.0253-2670.2026.11.011

基金

收起

国家自然科学基金面上项目 (82274412)

参考文献

收起

文献

收起

参考文献引证文献

排序方式：

Ahmed R, Botezatu B, Nanthakumar M, et al. Surgery for heart failure: Treatment options and implications [J]. J Card Surg, 2021, 36(4): 1511-1519.
McDonagh T, Metra M. 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure [J]. Russ J Cardiol, 2023, 28(1): 5168.
Mosterd A, Hoes A W. Clinical epidemiology of heart failure [J]. Heart, 2007, 93(9): 1137-1146.
Ostrominski J W, DeFilippis E M, Bansal K, et al. Contemporary American and European guidelines for heart failure management JACC: Heart failure guideline comparison [J]. JACC Heart Fail, 2024, 12(5): 810-825.
Savarese G, Becher P M, Lund L H, et al. Global burden of heart failure: A comprehensive and updated review of epidemiology [J]. Cardiovasc Res, 2023, 118(17): 3272-3287.
Brown D A, Perry J B, Allen M E, et al. Mitochondrial function as a therapeutic target in heart failure [J]. Nat Rev Cardiol, 2017, 14(4): 238-250.
Du J, Li H, Song J, et al. AMPK activation alleviates myocardial ischemia-reperfusion injury by regulating Drp1-mediated mitochondrial dynamics [J]. Front Pharmacol, 2022, 13: 862204.
Danial N N, Korsmeyer S J. Cell death: Critical control points [J]. Cell, 2004, 116(2): 205-219.
Ong S B, Kalkhoran S B, Cabrera-Fuentes H A, et al. Mitochondrial fusion and fission proteins as novel therapeutic targets for treating cardiovascular disease [J]. Eur J Pharmacol, 2015, 763: 104-114.
Grel H, Woznica D, Ratajczak K, et al. Mitochondrial dynamics in neurodegenerative diseases: Unraveling the role of fusion and fission processes [J]. Int J Mol Sci, 2023, 24(17): 13033.
Westermann B. Mitochondrial fusion and fission in cell life and death [J]. Nat Rev Mol Cell Biol, 2010, 11(12): 872-884.
G W D. Mitochondrial fission/fusion and cardiomyopathy [J]. Curr Opin Genet Dev, 2016, 38: 38-44.
Rosdah A A, K Holien J, Delbridge L M D, et al. Mitochondrial fission - a drug target for cytoprotection or cytodestruction? [J]. Pharmacol Res Perspect, 2016, 4(3): e00235.
Zhou H, Wang J, Zhu P J, et al. Retraction note to: NR4A1 aggravates the cardiac microvascular ischemia reperfusion injury through suppressing FUNDC1-mediated mitophagy and promoting Mff-required mitochondrial fission by CK2α [J]. Basic Res Cardiol, 2023, 118(1): 24.
Qi J, Wang F, Yang P, et al. Mitochondrial fission is required for angiotensin II-induced cardiomyocyte apoptosis mediated by a Sirt1-p53 signaling pathway [J]. Front Pharmacol, 2018, 9: 176.
Hasan P, Saotome M, Ikoma T, et al. Mitochondrial fission protein, dynamin-related protein 1, contributes to the promotion of hypertensive cardiac hypertrophy and fibrosis in Dahl-salt sensitive rats [J]. J Mol Cell Cardiol, 2018, 121: 103-106.
王波, 沈松林, 李丹. Drp1在心肌梗死后不良重塑中的变化及作用[J]. 西部医学, 2019, 31(10): 1517-1521.
Chang Y W, Chang Y T, Wang Q C, et al. Quantitative phosphoproteomic study of pressure-overloaded mouse heart reveals dynamin-related protein 1 as a modulator of cardiac hypertrophy [J]. Mol Cell Proteom, 2013, 12(11): 3094-3107.
Sharp W W. Dynamin-related protein 1 as a therapeutic target in cardiac arrest [J]. J Mol Med, 2015, 93(3): 243-252.
赵强, 刘芬, 杨毅宁. 心力衰竭中心肌细胞线粒体融合与分裂[J]. 中国组织化学与细胞化学杂志, 2018, 27(3): 273-279.
Hoffman N J, Parker B L, Chaudhuri R, et al. Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates [J]. Cell Metab, 2015, 22(5): 922-935.
Wang D D, Jin S, Liu H Y, et al. Celastrol alleviates atopic dermatitis by regulating Ezrin-mediated mitochondrial fission and fusion [J]. J Cell Mol Med, 2024, 28(14): e18375.
Sakamuru S, Zhao J H, Attene-Ramos M S, et al. Mitochondrial membrane potential assay [J]. Methods Mol Biol, 2022, 2474: 11-19.
Ding P, Song Y M, Yang Y, et al. NLRP3 inflammasome and pyroptosis in cardiovascular diseases and exercise intervention [J]. Front Pharmacol, 2024, 15: 1368835.
Zheng Y D, Xu L, Dong N G, et al. NLRP3 inflammasome: The rising star in cardiovascular diseases [J]. Front Cardiovasc Med, 2022, 9: 927061.
Forte M, Schirone L, Ameri P, et al. The role of mitochondrial dynamics in cardiovascular diseases [J]. Br J Pharmacol, 2021, 178(10): 2060-2076.
叶嘉豪, 吴子政, 张垚, 等. 基于AMPK/Drp1通路探讨丹红注射液对慢性心力衰竭大鼠线粒体动力学的作用机制[J]. 中国实验方剂学杂志, 2025, 31(16): 126-135.
Trott O, Olson A J. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading [J]. J Comput Chem, 2010, 31(2): 455-461.
孙聪, 郭帅, 宁娜, 等. 脂联素抑制β1-肾上腺素受体自身抗体诱导的大鼠H9c2心肌细胞自噬流下降[J]. 中国病理生理杂志, 2020, 36(5): 796-802.
郑君毅, 张莹莹, 刘园园, 等. FUNDC1通过调控线粒体分裂影响高糖损伤H9c2心肌细胞凋亡的机制研究[J]. 天津医药, 2022, 50(8): 791-795.
常红波, 王振涛, 吴鸿, 等. 抗纤益心方通过调节Drp1活性抑制去甲肾上腺素诱导大鼠H9c2细胞肥大[J]. 中药药理与临床, 2022, 38(5): 53-57.
王佳慧, 梁欢, 方典, 等. 抑制线粒体活性氧自由基可减轻高糖诱导的心肌细胞焦亡和铁死亡[J]. 南方医科大学学报, 2021, 41(7): 980-987.
顾宇重, 周晓兰, 常波. 丹红注射液联合环磷腺苷葡胺治疗老老年人心力衰竭的临床观察[J]. 中西医结合心脑血管病杂志, 2014, 12(12): 1481-1483.
熊霞军, 胡思远, 钟森杰, 等. 基于miR-378探讨丹红注射液干预压力超负荷心力衰竭大鼠心肌纤维化机制[J]. 湖南中医药大学学报, 2023, 43(3): 383-390.
黄成龙, 张红. 丹红注射液治疗不稳定型心绞痛的疗效及对超敏C反应蛋白的影响[J]. 现代中西医结合杂志, 2012, 21(25): 2778-2779.
马红, 冯一平, 薛素芳, 等. 丹红注射液联合去乙酰毛花苷治疗急性左心衰竭的临床研究[J]. 现代药物与临床, 2025, 40(12): 3095-3099.
杨丽敏, 肖耀龙, 欧阳家惠. 紫草酸镁B抑制缺血/再灌注心肌细胞c-Jun N-末端激酶3 mRNA的表达[J]. 药学学报, 2003, 38(7): 487-491.
侯林华. 紫草酸和原儿茶酸抗心肌损伤作用机制研究[D]. 保定: 河北大学, 2024.
Fan C M, Qin K L, Iroegbu C D, et al. Magnesium lithospermate B enhances the potential of human-induced pluripotent stem cell-derived cardiomyocytes for myocardial repair [J]. Chin Med J, 2024, 137(15): 1857-1869.
Luo Z J, Zang M W, Guo W. AMPK as a metabolic tumor suppressor: Control of metabolism and cell growth [J]. Future Oncol, 2010, 6(3): 457-470.
苏岩, 季宇彬, 郭鹏. AMPK与心血管疾病[J]. 黑龙江医药, 2013, 26(1): 20-24.
Sakamoto K, Göransson O, Hardie D G, et al. Activity of LKB1 and AMPK-related kinases in skeletal muscle: Effects of contraction, phenformin, and AICAR [J]. Am J Physiol Endocrinol Metab, 2004, 287(2): E310-E317.
Woods A, Johnstone S R, Dickerson K, et al. LKB1 is the upstream kinase in the AMP-activated protein kinase cascade [J]. Curr Biol, 2003, 13(22): 2004-2008.
Li C Y, Keaney J F. AMP-activated protein kinase: A stress-responsive kinase with implications for cardiovascular disease [J]. Curr Opin Pharmacol, 2010, 10(2): 111-115.
毛治尉, 张涛, 武永新, 等. 基于AMPK/SIRT1/PGC1α信号通路探究灯盏花素联合有氧运动对冠心病大鼠心功能障碍的改善作用及机制研究[J]. 中国康复医学杂志, 2023, 38(9): 1185-1191.
王江侠, 杨丽霞, 米登海, 等. 当归多糖对糖尿病肾病KK-Ay小鼠肾脏AMPK信号通路及线粒体自噬的影响[J]. 中草药, 2023, 54(10): 3189-3196.
方舟. 丹蛭降糖胶囊对糖尿病心肌病患者临床疗效及大鼠心肌AMPK/SIRT1通路的影响[D]. 合肥: 安徽中医药大学, 2023.
余思仪. 苓桂术甘汤通过干预线粒体改善心肌梗死后慢性心衰大鼠心功能[D]. 十堰: 湖北医药学院, 2022.
Wikstrom J D, Israeli T, Bachar-Wikstrom E, et al. AMPK regulates ER morphology and function in stressed pancreatic β-cells via phosphorylation of DRP1[J]. Mol Endocrinol, 2013, 27(10): 1706-1723.
Zhang Y, Wang Y, Xu J N, et al. Melatonin attenuates myocardial ischemia-reperfusion injury via improving mitochondrial fusion/mitophagy and activating the AMPK-OPA1 signaling pathways [J]. J Pineal Res, 2019, 66(2): e12542.
Tábara L C, Burr S P, Frison M, et al. MTFP1 controls mitochondrial fusion to regulate inner membrane quality control and maintain mtDNA levels [J]. Cell, 2024, 187(14): 3619-3637.e27.
Sakamuru S, Zhao J H, Attene-Ramos M S, et al. Mitochondrial membrane potential assay [A] // High-Throughput Screening Assays in Toxicology [M]. New York: Springer US, 2022: 11-19.
Yu J W, Lee M S. Mitochondria and the NLRP3 inflammasome: Physiological and pathological relevance [J]. Arch Pharm Res, 2016, 39(11): 1503-1518.
Bu W J, Li S S, Liu C, et al. Nepetin limits NLRP3 inflammasome activation and alleviates NLRP3-driven inflammatory diseases via PINK1-dependent mitophagy [J]. Free Radic Biol Med, 2025, 227: 420-433.
Zhang Y F, Zhou L, Mao H Q, et al. Mitochondrial DNA leakage exacerbates odontoblast inflammation through gasdermin D-mediated pyroptosis [J]. Cell Death Discov, 2021, 7: 381.
Huang Y, Xu W, Zhou R B. NLRP3 inflammasome activation and cell death [J]. Cell Mol Immunol, 2021, 18(9): 2114-2127.
Xu Y J, Zheng L, Hu Y W, et al. Pyroptosis and its relationship to atherosclerosis [J]. Clin Chim Acta, 2018, 476: 28-37.
Xu B, Jiang M Z, Chu Y, et al. Gasdermin D plays a key role as a pyroptosis executor of non-alcoholic steatohepatitis in humans and mice [J]. J Hepatol, 2018, 68(4): 773-782.
Ding J J, Shao F. Growing a gasdermin pore in membranes of pyroptotic cells [J]. EMBO J, 2018, 37(15): EMBJ2018100067.
Ding J J, Wang K, Liu W, et al. Pore-forming activity and structural autoinhibition of the gasdermin family [J]. Nature, 2016, 535(7610): 111-116.
Zeng C, Duan F Q, Hu J, et al. NLRP3 inflammasome-mediated pyroptosis contributes to the pathogenesis of non-ischemic dilated cardiomyopathy [J]. Redox Biol, 2020, 34: 101523.

2026年第57卷第11期

PDF下载

引用本文

BibTeX

文章信息

doi: 10.7501/j.issn.0253-2670.2026.11.011

接收时间：2025-11-02
首发时间：2026-09-09

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2025-11-02

基金

作者信息

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/zcy/CN/10.7501/j.issn.0253-2670.2026.11.011

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

关闭全屏

BibTeX
EndNote
RefWorks
TxT