麦冬皂苷D通过激活β-catenin/FUNDC1/线粒体自噬轴减轻阿霉素诱导的小鼠心肌肥厚

doi:10.12122/j.issn.1673-4254.2026.04.09

摘要/Abstract

摘要：

目的探讨麦冬皂苷D（OD）是否通过调控β-catenin/FUNDC1/线粒体自噬信号轴减轻阿霉素（Dox）诱导的心肌肥厚。方法通过Dox诱导心肌肥厚，分组如下：对照组，Dox刺激组，Dox刺激+OD治疗组，Dox刺激+感染AAV-β-catenin组，Dox刺激+感染AAV（腺相关病毒载体）组。细胞RNA测序分析Dox刺激组与对照组基因表达差异；Western blotting检测β-catenin，活化β-catenin，FUNDC1，LC3，p62，β-MHC（β肌球蛋白重链），α-actin；免疫组化/荧光检测β-catenin，FUNDC1；透射电镜检测线粒体损伤；染色质免疫共沉淀（ChIP）及双荧光素酶报告基因检测β-catenin对FUNDC1的转录调节作用。结果相比对照组，Dox刺激显著抑制β-catenin信号（P<0.001）和FUNDC1介导的线粒体自噬（P<0.001），导致线粒体损伤和心肌细胞肥大（P<0.001）。相比Dox组，OD处理可逆转上述效应，恢复β-catenin信号（P<0.001），增强FUNDC1转录与表达（P<0.001），并促进线粒体自噬，减轻心肌肥厚（P<0.001）。过表达β-catenin或FUNDC1均模拟了OD的心肌保护作用，而敲低β-catenin则加重心肌肥厚，且FUNDC1过表达可逆转该表型。机制上，β-catenin直接结合FUNDC1启动子并激活其转录。结论 OD通过激活β-catenin/FUNDC1/线粒体自噬轴，增强线粒体质量控制，从而缓解Dox诱导的心肌肥厚。

关键词: 麦冬皂苷D, 心肌肥厚, β-catenin, 线粒体自噬, 阿霉素

Abstract:

Objective To investigate whether ophiopogonin D (OD) alleviates doxorubicin (Dox)‑induced myocardial hypertrophy in mice by regulating the β‑catenin/FUNDC1/mitophagy signaling axis. Methods Thirty C57BL/6J mice were randomized equally into control group, Dox treatment group, Dox with OD treatment group, Dox treatment group with injection of adeno-associated virus (AAV) vector carrying β‑catenin, and Dox treatment group with injection of AAV vector. RNA sequencing analysis was used to identify differentially expressed genes in cultured mouse cardiac cells following Dox treatment. Western blotting was performed to examine the protein levels of β‑catenin, active β‑catenin, FUNDC1, LC3, p62, β‑myosin heavy chain (β-MHC), and α‑actin; immunohistochemistry and immunofluorescence staining were used to assess the localization and expression of β-catenin and FUNDC1. Transmission electron microscopy was employed to evaluate mitochondrial damage in the cardiac myocytes. Chromatin immunoprecipitation and dual-luciferase reporter gene assays were used to investigate the transcriptional regulation of FUNDC1 by β‑catenin. Results Dox treatment significantly inhibited β‑catenin signaling and FUNDC1-mediated mitophagy, leading to cardiomyocyte hypertrophy and mitochondrial damage. OD treatment obviously reversed these effects, restored β‑catenin signaling, enhanced FUNDC1 transcription and expression, and promoted mitophagy. Overexpression of β‑catenin or FUNDC1 mimicked the cardioprotective effect of OD, while knockdown of β‑catenin aggravated myocardial hypertrophy, which was reversed by FUNDC1 overexpression. Mechanistically, β‑catenin directly bound to the FUNDC1 promoter and activated its transcription. Conclusion OD alleviates Dox-induced myocardial hypertrophy in mice by activating the β‑catenin/FUNDC1/mitophagy axis and enhancing mitochondrial quality control.

Key words: ophiopogonin D, myocardial hypertrophy, β-catenin, mitophagy, doxorubicin

雷艳萍, 宋嘉晟, 徐乐吾, 刘睿, 赵岳. 麦冬皂苷D通过激活β-catenin/FUNDC1/线粒体自噬轴减轻阿霉素诱导的小鼠心肌肥厚[J]. 南方医科大学学报, 2026, 46(4): 803-815.

Yanping LEI, Jiasheng SONG, Lewu XU, Rui LIU, Yue ZHAO. Ophiopogonin D alleviates doxorubicin-induced myocardial hypertrophy in mice by activating the β-catenin/FUNDC1/mitophagy axis[J]. Journal of Southern Medical University, 2026, 46(4): 803-815.

图/表 9

图1 Dox致心肌损伤与线粒体自噬及心肌肥大相关

Fig.1 Dox-induced myocardial injury is associated with mitophagy and myocardial hypertrophy. A: Heatmap of gene expression profiles in doxorubicin (Dox)-treated cardiomyocytes as compared with control (CTL) group. B: Bubble plot of Gene Ontology (GO) enrichment analysis for mitophagy-related genes. C: Bubble plot of GO enrichment analysis for myocardial hypertrophy-related genes. D: Bubble plot of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for mitophagy-related pathways. E: Bubble plot of KEGG enrichment analysis for myocardial hypertrophy-related pathways. F: Gene Set Enrichment Analysis (GSEA) plot for myocardial hypertrophy-related gene set (GO:0003300), showing enrichment scores and distribution of genes in Dox and CTL groups. G: Volcano plot showing differential mRNA expression profile in Dox and CTL groups.

图2 Dox抑制心肌细胞中β-catenin信号及线粒体自噬, 促进心肌肥大

Fig.2 Dox inhibits β-catenin activation and mitochondrial autophagy in cardiomyocytes and promotes cardiomyocyte hypertrophy. A: Western blot bands of active β‑catenin, total β‑catenin, FUNDC1, LC3II/LC3I and p62 protein levels of CTL (control) and Dox (doxorubicin)-treated primary cardiomyocytes. Lanes 1 and 2 on the Western blotting indicated sample 1 and sample 2, respectively, within the group. B-F: Quantitative analysis of the protein levels (Mean±SD, n=6; ***P<0.001 vs CTL group). G-I: Western blot bands and quantitative analysis of α-actin (H) and β-MHC (I) protein levels in CTL and Dox-treated groups. Data are presented as Mean±SD (n=6). ***P<0.001 vs CTL group.

图3 OD激活β-catenin信号通路并增强线粒体自噬,缓解Dox诱导的心肌肥大

Fig.3 Ophiopogonin D (OD) activates β-catenin signaling and enhances mitophagy to improve Dox-induced cardiac hypertrophy. A: Western blotting bands of active β-catenin, total β-catenin, FUNDC1, LC3II/LC3I and p62 in the heart tissues of mice treated with Dox and OD. B-F: Quantitative analysis of the protein levels. G: Immunohistochemical staining for β‑catenin and FUNDC1 in the myocardial sections (Scale bar=20 μm). H-J: Western blotting bands and quantitative analysis of α-actin and β-MHC protein levels in the heart tissues of the mice in different groups. Data are presented as Mean±SD (n=6). ***P<0.001 vs CTL group; ###P<0.001 vs Dox group.

图4 OD激活β-catenin信号并增强线粒体自噬,对抗Dox诱导的心肌细胞肥大

Fig.4 OD activates β-catenin signaling and enhances mitophagy to counteract Dox-induced cardiomyocyte hypertrophy. A-F: Western blot bands of active β-catenin, total β-catenin, FUNDC1, LC3II/LC3I and p62 proteins and quantitative analysis of their expression levels in primary cardiomyocytes from different treatment groups. G: Immunofluorescence staining for β-catenin (red) and FUNDC1 (green) in the cells from each group (Scale bar=25 μm). The cell nuclei were stained with DAPI (blue). H-J: Western blotting bands of α-actin and β-MHC protein and their expression levels in the cells from each group. Data are presented as Mean±SD (n=6). ***P<0.001 vs CTL group; #P<0.05, ###P<0.001 vs Dox group.

图5 体内实验显示心脏过表达β-catenin恢复线粒体自噬, 并改善Dox诱导的心肌肥厚

Fig.5 Overexpression of β-catenin in the heart restores mitophagy and alleviates Dox-induced myocardial hypertrophy in mice. A-F: Western blotting bands of activated β-catenin, total β-catenin, FUNDC1, LC3II/LC3I and p62 proteins and their expression levels in cardiac tissues of the mice in each group. G: Immunohistochemistry for β-catenin and FUNDC1 in the myocardial tissues in each group (Scale bar=20 μm). H: Transmission electron microscopy of the myocardial tissues in each group. Yellow arrows indicates damaged mitochondria (Scale bar=300 nm). I-K: Western blotting bands of α-actin and β-MHC proteins and their expression levels in each group. Data are presented as Mean±SD (n=6). *P<0.05, ***P<0.001 vs CTL group; #P<0.05, ###P<0.001 vs Dox group.

图6 体外实验显示, 心肌细胞内过表达β-catenin恢复线粒体自噬, 并改善Dox诱导的心肌细胞肥大

Fig.6 Cardiomyocyte-specific overexpression of β-catenin restores mitophagy and ameliorates Dox-induced hypertrophy in H9c2 cells. A-F: Western blotting bands of active β-catenin, total β-catenin, FUNDC1, LC3II/LC3I and p62 proteins and their expression levels in H9c2 cells in each group. G: Immunofluorescence staining for β-catenin and FUNDC1 in H9c2 cells (Scale bar=25 μm). H: Transmission electron microscopy of H9c2 cells in each group. Yellow arrows indicate damaged mitochondria (Scale bar=300 nm). I: Detection of autophagic flux in each group of cells using the mRFP-GFP-LC3 adenovirus probe (Scale bar=25 μm). Yellow spots represent autophagosomes (GFP+/mRFP+), and red spots represent autolysosomes (GFP-/mRFP+). J: Green and red fluorescent points in the indicated groups. K-M: Western blotting bands of α-actin and β-MHC proteins and their expression levels in each group of cells. N: Rhodamine staining of H9c2 cells and cross-sectional area of the cells in each group (×40). O: Statistical analysis of cross-sectional area of myocardial cells. Data are presented as Mean±SD (n=6).**P<0.01, ***P<0.001 vs CTL group; ##P<0.01, ###P<0.001 vs Dox group. pcD-β-cat: pcDNA-β-catenin; pcD: pcDNA.

图7 过表达FUNDC1促进线粒体自噬,逆转Dox诱导的心肌细胞肥厚

Fig.7 Overexpression of FUNDC1 promotes mitophagy and reverses Dox-induced cardiomyocyte hypertrophy. A-F: Western blotting bands of active β-catenin, total β-catenin, FUNDC1, LC3II/LC3I and p62 proteins and their expression levels in H9c2 cells in different treatment groups. G-I: Western blotting bands of α-actin and β-MHC proteins and their expression levels in each group. Data are presented as Mean±SD (n=6). **P<0.01, ***P<0.001 vs CTL group; ##P<0.01, ###P<0.001 vs Dox group. pcD-FUNDC1: pcDNA-FUNDC1; pcD: pcDNA.

图8 β-catenin作为转录因子直接调控 FUNDC1 介导的线粒体自噬

Fig.8 β‑catenin, as a transcription factor, directly regulates FUNDC1-mediated mitophagy. A-D: Western blotting of active β-catenin, β‑catenin and FUNDC1 proteins in H9c2 transfected with β‑catenin-siRNA and pcDNA‑β‑catenin. E, F: RT-PCR for detection of FUNDC1 mRNA levels in each group of cells. G: Chromatin immunoprecipitation (ChIP) analysis showing the binding of β‑catenin protein to the promoter region of the FUNDC1 gene. H: Dual-luciferase reporter gene assay analysis showing that overexpression of β‑catenin significantly enhances transcriptional activity of the FUNDC1 promoter. I-N: Results of Western blotting of active β‑catenin, β‑catenin, FUNDC1, LC3II/LC3I and p62 protein levels in the cardiomyocytes with β‑catenin knockdown after overexpression of FUNDC1. O, P: RT-PCR for detection of FUNDC1 mRNA levels in each group. Q, R: RT-PCR for detecting LC3 mRNA levels in each group. S, T: RT-PCR for detecting p62 mRNA levels in each group. Data are presented as Mean±SD (n=6). **P<0.01, ***P<0.001 vs CTL group; ##P<0.01, ###P<0.001 vs β-catenin-siRNA group. β-cat-siR: β-catenin-siRNA; Sc-siR: Scramble-siRNA; pcD-β-cat: pcDNA-β-catenin; pcD: pcDNA; FUNDC1: pcDNA-FUNDC1.

图9 β-catenin/FUNDC1 轴调控心肌细胞肥大

Fig.9 The β-catenin/FUNDC1 axis regulates cardiomyocyte hypertrophy. A-C: Western blotting of α-actin and β-MHC proteins in cardiomyocytes after overexpression of FUNDC1 on the basis of β-catenin knockdown. D, E: RT-PCR for detecting α-actin mRNA levels in each group of the cells. F, G: RT-PCR for detecting β-MHC mRNA levels in each group. Data are presented as Mean±SD (n=6).*P<0.05, **P<0.01, ***P<0.001 vs CTL group; #P<0.01, ###P<0.001 vs β-catenin-siRNA group.

参考文献 30

[1]	Nsairat H, Lafi Z, Abualsoud BM, et al. Vitamin C as a cardioprotective agent against doxorubicin-induced cardiotoxicity[J]. J Am Heart Assoc, 2025, 14(16): e042534. doi：10.1161/jaha.125.042534
[2]	Zhang S, Xu HF, Zhang ZX, et al. Research on doxorubicin-induced cardiotoxicity mechanism and its forensic application[J]. Fa Yi Xue Za Zhi, 2025, 41(2): 120-6.
[3]	Goje ID, Goje GI, Ordodi VL, et al. Doxorubicin-induced cardiotoxicity and the emerging role of SGLT2 inhibitors: from glycemic control to cardio-oncology[J]. Pharmaceuticals (Basel), 2025, 18(5): 681. doi：10.3390/ph18050681
[4]	Ito-Hagiwara K, Hagiwara J, Endo Y, et al. Cardioprotective strategies against doxorubicin-induced cardiotoxicity: a review from standard therapies to emerging mitochondrial transplantation[J]. Biomed Pharmacother, 2025, 189: 118315. doi：10.1016/j.biopha.2025.118315
[5]	Yeh ETH, Chang HM. Anthracycline-induced cardiotoxicity: mechanism and prevention[J]. Trans Am Clin Climatol Assoc, 2025, 135: 184-95.
[6]	Chang ZS, Wang L, Zhou JX, et al. FoxO3 activation alleviates doxorubicin-induced cardiomyopathy by enhancing autophagic flux and suppressing mTOR/ROS signalling[J]. J Cell Mol Med, 2025, 29(15): e70775. doi：10.1111/jcmm.70775
[7]	Liu PH, Xu TH, Luo YJ, et al. SIRT3 attenuates sepsis-induced EndMT and cardiac remodeling by facilitating mitophagy process via PINK1/Parkin signaling[J]. Int Immunopharmacol, 2025, 164: 115377. doi：10.1016/j.intimp.2025.115377
[8]	Li SJ, Li XY. Mitophagy in hypertensive cardiac hypertrophy: mechanisms and therapeutic implications[J]. J Clin Hypertens (Greenwich), 2025, 27(8): e70127. doi：10.1111/jch.70127
[9]	Shen Y, Gao X, Xiang Y, et al. Exploiting mitochondria by triggering a faulty unfolded protein response leads to effective cardioprotection[J]. Int J Med Sci, 2025, 22(1): 188-96. doi：10.7150/ijms.100523
[10]	Wang J, Zhuang H, Jia L, et al. Nuclear receptor subfamily 4 group A member 1 promotes myocardial ischemia/reperfusion injury through inducing mitochondrial fission factor-mediated mitochondrial fragmentation and inhibiting FUN14 domain containing 1-depedent mitophagy[J]. Int J Biol Sci, 2024, 20(11): 4458-75. doi：10.7150/ijbs.95853
[11]	Liu JQ, Xiao Q, Xiao JN, et al. Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities[J]. Signal Transduct Target Ther, 2022, 7(1): 3. doi：10.1038/s41392-021-00762-6
[12]	Lei YP, Hu HJ, Tang HF, et al. Homocysteine promotes cardiomyocyte hypertrophy through inhibiting β-catenin/FUNDC1 mediated mitophagy[J]. Sci Rep, 2025, 15(1): 22207. doi：10.1038/s41598-025-06772-6
[13]	Xiang SL, Tang XW. Interfering with AQP1 alleviates ferroptosis, improves mitochondrial function and energy metabolic disorder in hypoxia/reoxygenation-induced H9c2 cardiomyocytes via Wnt/β-catenin pathway[J]. Microvasc Res, 2025, 160: 104821. doi：10.1016/j.mvr.2025.104821
[14]	Deng YG, Hou MZ, Wu YB, et al. SIRT3-PINK1-PKM2 axis prevents osteoarthritis via mitochondrial renewal and metabolic switch[J]. Bone Res, 2025, 13(1): 36. doi：10.1038/s41413-025-00413-4
[15]	Li HY, Leung JCK, Yiu WH, et al. Tubular β-catenin alleviates mitochondrial dysfunction and cell death in acute kidney injury[J]. Cell Death Dis, 2022, 13(12): 1061. doi：10.1038/s41419-022-05395-3
[16]	Zhang YQ, Chen B, Zhang H, et al. Extraction, purification, structural characterization, bioactivities, modifications and structure-activity relationship of polysaccharides from Ophiopogon japonicus: a review[J]. Front Nutr, 2024, 11: 1484865. doi：10.3389/fnut.2024.1484865
[17]	Chen YT, Ma LL, Yan YZ, et al. Ophiopogon japonicus polysaccharide reduces doxorubicin-induced myocardial ferroptosis injury by activating Nrf2/GPX4 signaling and alleviating iron accumulation[J]. Mol Med Rep, 2025, 31(2): 36. doi：10.3892/mmr.2024.13401
[18]	Lei YP, Xu LW, Liu R, et al. Ophiopogonin D mitigates doxorubicin-induced cardiomyocyte ferroptosis through the β‑catenin/GPX4 pathway[J]. Front Pharmacol, 2025, 16: 1586937. doi：10.3389/fphar.2025.1586937
[19]	Li XP, Luo WB, Tang Y, et al. Semaglutide attenuates doxorubicin-induced cardiotoxicity by ameliorating BNIP3-Mediated mitocho-ndrial dysfunction[J]. Redox Biol, 2024, 72: 103129. doi：10.1016/j.redox.2024.103129
[20]	Wallace KB, Sardão VA, Oliveira PJ. Mitochondrial determinants of doxorubicin-induced cardiomyopathy[J]. Circ Res, 2020, 126(7): 926-41. doi：10.1161/circresaha.119.314681
[21]	Liu XQ, Hussain R, Mehmood K, et al. Mitochondrial-endoplasmic reticulum communication-mediated oxidative stress and autophagy[J]. Biomed Res Int, 2022, 2022: 6459585. doi：10.1155/2022/6459585
[22]	Liu RX, Xu CL, Zhang WL, et al. FUNDC1-mediated mitophagy and HIF1α activation drives pulmonary hypertension during hypoxia[J]. Cell Death Dis, 2022, 13(7): 634. doi：10.1038/s41419-022-05091-2
[23]	He WB, Sun ZC, Tong G, et al. FUNDC1 alleviates doxorubicin-induced cardiotoxicity by restoring mitochondrial-endoplasmic reticulum contacts and blocked autophagic flux[J]. Theranostics, 2024, 14(9): 3719-38. doi：10.7150/thno.92771
[24]	Haybar H, Khodadi E, Shahrabi S. Wnt/β‑catenin in ischemic myocardium: interactions and signaling pathways as a therapeutic target[J]. Heart Fail Rev, 2019, 24(3): 411-9. doi：10.1007/s10741-018-9759-z
[25]	Tao H, Yang JJ, Shi KH, et al. Wnt signaling pathway in cardiac fibrosis: New insights and directions[J]. Metabolism, 2016, 65(2): 30-40. doi：10.1016/j.metabol.2015.10.013
[26]	Wang XY, He K, Ma LL, et al. Puerarin attenuates isoproterenol-induced myocardial hypertrophy via inhibition of the Wnt/β-catenin signaling pathway[J]. Mol Med Rep, 2022, 26(4): 306. doi：10.3892/mmr.2022.12822
[27]	Huo PP, Wang SJ, Li ZN, et al. ACSS3 protein macromolecule regulates glycolysis in keloid through Wnt/β‑catenin signaling pathway: Bioinformatics, machine learning, and experimental validation[J]. Cell Signal, 2025, 135: 112056. doi：10.1016/j.cellsig.2025.112056
[28]	Zhang HY, Kang XZ, Ruan J, et al. Ophiopogonin D improves oxidative stress and mitochondrial dysfunction in pancreatic β cells induced by hydrogen peroxide through Keap1/Nrf2/ARE pathway in diabetes mellitus[J]. Chin J Physiol, 2023, 66(6): 494-502. doi：10.4103/cjop.cjop-d-23-00069
[29]	Huang XY, Wang YG, Wang Y, et al. Ophiopogonin D reduces myocardial ischemia-reperfusion injury via upregulating CYP2J3/EETs in rats[J]. Cell Physiol Biochem, 2018, 49(4): 1646-58. doi：10.1159/000493500
[30]	Zhang YY, Meng C, Zhang XM, et al. Ophiopogonin D attenuates doxorubicin-induced autophagic cell death by relieving mitochondrial damage in vitro and in vivo [J]. J Pharmacol Exp Ther, 2015, 352(1): 166-74. doi：10.1124/jpet.114.219261