Dracocephalum moldavica L. Flavonoids Alleviate Doxorubicin-Induced Cardiotoxicity by Activating the AMPK/PGC1αPathway to Preserve Mitochondrial Homeostasis

Figures

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  Figure 1. TFDM alleviated cardiac dysfunction and myocardial injury in mice subjected to DOX injection. (a) Representative images of M-mode echocardiograms from each group. (b–e) Ejection fraction (EF, (b)), fractional shortening (FS, (c)), left ventricular internal diameter at end-systole (LVIDs, (d)) and left ventricular internal diameter at end-diastole (LVIDd, (e)) were analyzed to assess their cardiac dysfunction (n = 6). (f,g) Activities of LDH (f) and CK-MB (g) in sera as cardiac damage markers. (n = 6). (h–j) Levels of MDA and activities of SOD and GSH-Px to reveal the oxidative stress in myocardial oxidative stress (n = 6). (k–m) Representative mitochondrial images from left ventricle myocardial tissue by TEM (The lower panel is the magnification from the white rectangle in the upper panel. magnification: upper panel 5000×; lower panel 30,000×), (k) and their analysis, including mitochondria number per cell (l) and the percentage of abnormal mitochondria (m) (n = 6). Data were shown as mean ± SD, # p < 0.05, ## p < 0.01 vs. the control group; * p < 0.05, ** p < 0.01 vs. the DOX group.

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  Figure 2. TFDM attenuates DOX-induced injury in H9c2 cardiomyocytes. (a) Cell viability was measured by CCK-8 assay. TFDM treatment concentration-dependently improved the viability of H9c2 cells exposed to DOX (n = 6). (b,c) Intracellular ROS levels were detected using the DCFH-DA probe and analyzed by flow cytometry. (b) Representative flow cytometry histograms; (c) Quantitative analysis of ROS fluorescence intensity (n = 3). (d,e) Apoptosis was evaluated by TUNEL staining. (d) Representative fluorescent images (scale bar: 100 μm); (e) Quantification of TUNEL-positive cells (n = 3). (f–h) Representative blots of Cyto-c, Bax and Bcl2 (f) and their analysis, including the levels of Cyto-c (g) and the ratio between Bax/Bcl-2 (h) (n = 3). Data were shown as mean ± SD. ## p < 0.01, ### p < 0.001 vs. control group; * p < 0.05, ** p < 0.01 vs. DOX group.

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  Figure 3. TFDM relieved DOX-caused mitochondrial abnormities in H9c2 cells. (a) mPTP opening was assessed using the BBcellProbe M61 kit. Fluorescence intensity is inversely proportional to the extent of mPTP opening (n = 3). (b) Intracellular ATP content was measured (n = 5). (c,d) Intracellular Ca2+ levels were determined by flow cytometry with Fluo-4 AM staining. (c) Representative histogram plots. (d) Quantitative analysis of fluorescence intensity (n = 3). (e,f) Mitochondrial membrane potential (ΔΨm) was evaluated using MitoTracker Red CMXRos. (e) Quantitative analysis of red fluorescence intensity. (f) Representative fluorescent images (scale bar: 10 μm) (n = 3). (g,h) Mitochondrial respiratory function was analyzed by high-resolution respirometry. (g) Representative oxygen consumption traces (* denotes replications of the U (FCCP) application). (h) Quantitative assessment of key mitochondrial respiratory parameters. The oxygen consumption rates were normalized to the total cell count (1 × 106 cells per chamber) to ensure cross-sample comparison (n = 7). All data are expressed as mean ± SD. # p < 0.05, ## p < 0.01 vs. control group; * p < 0.05, ** p < 0.01 vs. DOX group.

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  Figure 4. TFDM activates the AMPK/PGC1α signaling pathway suppressed by DOX. (a) Representative immunoblots of AMPKα, p-AMPKα and NRF1 in whole-cell lysates, and PGC1α, TFAM in mitochondrial fractions isolated from myocardial tissue. (b–e) Quantitative analysis of (b) the p-AMPKα/AMPKα ratio, (c) PGC1α, (d) NRF1, and (e) TFAM protein levels in myocardial tissue (n = 3). (f) Representative immunoblots of AMPKα, p-AMPKα and NRF1 in whole-cell lysates, and PGC1α, TFAM in mitochondrial fractions from H9c2 cells. (g–j) Quantitative analysis of (g) the p-AMPKα/AMPKα ratio, (h) PGC-1α, (i) NRF1, and (j) TFAM protein levels in H9c2 cells (n = 3). All data are expressed as mean ± SD. # p < 0.05, ## p < 0.01 vs. control group; * p < 0.05, ** p < 0.01 vs. DOX group.

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  Figure 5. Knockdown of AMPKα abolishes the protective effects of TFDM in DOX-insulted H9c2 cells. (a) Representative immunoblots of AMPKα, p-AMPKα and PGC1αin whole-cell lysates, and NRF1 in mitochondrial fractions from H9c2 cells. (b–e) Quantitative analysis of (b) p-AMPKα, (c) AMPKα, (d) PGC-1α, (e) NRF1 protein levels (n = 3). (f,g) Activities of LDH (f) and SOD (g). (n = 3). (h) Intracellular ATP levels measured by Elisa. (n = 3). (i) mPTP opening was assessed using the BBcellProbe M61 kit. Fluorescence intensity is inversely proportional to the extent of mPTP opening (n = 3). (j,k) Mitochondrial membrane potential was evaluated using MitoTracker Red CMXRos. (j) Representative fluorescent images (scale bar: 8 μm). (k) Quantitative analysis of red fluorescence intensity. (n = 3). Data were shown as mean ± SD, # p < 0.05, ## p < 0.01 vs. control group; * p < 0.05, ** p < 0.01 vs. DOX group; @ p < 0.01 vs. TFDM group.

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  Figure 6. Pharmacological inhibition of AMPK abolishes the protective effects of TFDM in DOX-treated H9c2 cells. (a) Representative immunoblots of AMPKα, p-AMPKα, and NRF1 (with GAPDH as the cytosol/total loading control), and PGC-1α (with COX IV as the mitochondrial loading control) in H9c2 cells. (b–e) Quantitative analysis of (b) p-AMPKα/AMPKα, (c) AMPKα/GAPDH, (d) NRF1/GAPDH, and (e) PGC-1α/COX IV protein levels (n = 3). (f,g) Quantitative analysis of (f) CS activity and (g) intracellular ATP content (n = 3). (h,i) Intracellular SOD activity (h) and extracellular LDH release (i) in H9c2 cells (n = 3). Data are presented as mean ± SD. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. DOX group; @ p < 0.01, @@@ p < 0.001 vs. TFDM group.

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  Figure 7. This Elevated ROS induced by DOX contribute to mitochondrial dysfunction by depolarizing the MMP, inducing mPTP opening, and impairing respiratory function, collectively leading to reduced ATP synthesis. Meanwhile, increased ROS, along with cytosolic Ca2+ overload, induce feedback inhibition of AMPKα, a key kinase normally activated under energy deficit, thus disrupting its role in promoting mitochondrial biogenesis. This suppression further compromises the mitochondrial repair capacity, creating a vicious cycle of deterioration. TFDM counteracts this process by restoring AMPK activity and enhancing the expression of mitochondrial biogenesis-related genes (e.g., PGC-1α, NRF1, TFAM). Consequently, TFDM alleviates DOX-induced mitochondrial injury by inhibiting mPTP opening, stabilizing MMP, improving respiratory function, and ultimately increasing ATP production, thereby preserving cardiomyocyte contractility and mitigating cardiotoxicity.