Background

Cancer is a life-threatening disease with increasing incidence and mortality rates worldwide [1]. Doxorubicin (DOX), an effective anthracycline chemotherapeutic, is widely used to treat different solid tumors and hematologic malignancies [2]. However, DOX therapy frequently causes multiorgan damage, with cardiotoxicity as its primary adverse effect. This typically manifests as arrhythmias, impaired ventricular function, and reduced ejection fraction [3, 4]. A medical survey revealed that nearly one-third of cancer survivors die of chronic heart failure [5]. Moreover, anthracycline-based chemotherapy induces cardiac injury or ejection dysfunction in up to 10% of cancer patients aged > 65 years [6]. It exacerbates chemotherapy-related myelosuppression and increases the risk of secondary malignancies [7]. Therefore, it is clinically crucial to identify targeted therapeutic interventions for these conditions [8].

Mitochondrial quality homeostasis (MQH) encompasses four core processes, namely, mitochondrial dynamics regulating organelle structure through morphological remodeling and intracellular positioning, mitophagy mediating selective degradation, biogenesis for new organelle generation, and mtUPR for maintaining proteostasis [9]. Specifically, mitochondrial dynamics coordinate structural adaptations via three-dimensional morphological changes, spatial redistribution within cells, and microtubule-guided motility along cytoskeletal networks [10]. Mitochondrial abnormalities are a primary contributor to DIC [11, 12]. However, the mechanisms underlying DIC remain elusive [13]. Excessive production of free radicals or ROS causes mitochondrial damage and disrupts calcium homeostasis, which are associated with distinct processes of DOX metabolism. These processes accelerate the development and progression of myocardial injury [14].

DUSP1 mediates the dephosphorylation of both tyrosine and threonine residues in target proteins and blocks downstream biological effects by hydrolyzing phosphate groups within the activation loop of MAPK family members [15]. We have previously reported differential regulatory functions of DUSP1 using different myocardial injury models [16], particularly its involvement in MQH modulation during CRS-3-mediated cardiac injury [17]. Significant suppression of DUSP1 expression following CRS-3-associated cardiac inhibition has been experimentally demonstrated in the myocardial tissues of CRS-3 mouse models [18]. Conversely, DUSP1 transgenic mice exhibited substantially improved cardiac function and myocardial architecture [19]. PHB2, a key mitochondrial inner membrane protein, regulates MQH by stabilizing the mitochondrial cristae structure and interacting with autophagy receptors such as LC3 to facilitate the clearance of damaged mitochondria [20]. The overexpression of PHB2 enhances mitochondrial bioenergetics through NDUFV2 stabilization, thus providing a potential therapeutic target for DIC. These findings align intricately with those of our current investigation of PHB2’s function in cardiovascular diseases [21].

In recent years, nanodrug delivery systems targeting the mitochondrial pathway for drug therapy have shown distinct advantages. Among them, mitochondrial autophagy, mitochondrial dynamics, and mitochondrial biosynthesis serve as core regulatory phenotypes in regulating cellular energy metabolism [22]. However, due to the complex bilayer membrane structure of mitochondria and the dynamic morphological/structural changes of mitochondrial dynamics, traditional therapeutic drugs struggle to achieve efficient targeting and effective accumulation of drug efficacy [23]. New research-oriented nanodrug delivery systems can precisely regulate drug particle size and utilize surface modifications with drugs such as Ligustrazine (LIG), tanshinone, quercetin, as well as mitochondrial-targeting peptide sequences and other targeting regulatory molecules. The nanodrug system can effectively overcome the mitochondrial biological barrier, enter target cells through endocytosis after drug intervention, and achieve targeted and precise drug delivery and enrichment through the personalized regulation mechanism of mitochondrial membrane potential [24]. In mitochondrial-targeted therapy, nanodrug delivery systems can encapsulate drugs using nanocarriers such as polymer micelles and mesoporous silica. This approach significantly enhances the stability of hydrophobic drugs or nucleic acid-based drugs, thereby controlling drug release and extending the duration of drug action targeting the mitochondria [25]. In terms of drug delivery to the mitochondrial membrane, its nanoparticle drug delivery system can enhance the interaction between drug nanoparticles and the mitochondrial membrane through surface modifications such as cationic polymers or mitochondrial-penetrating peptides, thereby overcoming the mitochondrial membrane and significantly improving the drug delivery efficiency. In recent years, studies have also found that nanoparticle carriers can encapsulate self-assembled structures integrating natural drug active ingredients. Through synergistic actions such as reducing membrane potential, inducing mitochondrial DNA damage, or inhibiting respiratory chain complexes, these carriers can enhance efficacy while reducing toxicity [26].

LIG, the primary active compound derived from the traditional Chinese herb Ligusticum chuanxiong, possesses unique cardioprotective and vasoprotective properties [27, 28]. Our prior studies confirm its mitochondrial protection via enhanced energy metabolism, calcium overload suppression, and inhibition of mitochondrial pathway-mediated cell death [29]. Clinical translation of natural compounds remains challenging due to poor water solubility, rapid degradation, and low bioavailability. The liposome–LIG complex significantly enhanced drug solubility, prolonged systemic circulation, and improved free LIG stability. Genetic and cellular models of DIC were used to elucidate the therapeutic mechanisms of LIG nanodrug (LIG–Na) delivery systems.

Integrated analysis of spatial transcriptomics (ST), scRNA-seq, and bulk RNA sequencing data provide novel insights into disease progression by identifying novel cellular subpopulations, uncovering potential biomarkers, and resolving cellular population heterogeneity [30]. We used bioinformatic approaches to further investigate the involvement of the DUSP1–PHB2 axis in DIC, screen potential therapeutic agents, and construct transgenic animal and cellular models. We have previously demonstrated that DOX provides targeted mitochondrial protection by enhancing mitochondrial energy metabolism, suppressing calcium overload, and inhibiting mitochondrial pathway-mediated programmed cell death [29]. We further elucidated the potential mechanism of DUSP1–PHB2 in DIC using bioinformatics and evaluated the therapeutic effect of LIG–Na in modulating this pathway and its ability to alleviate mitochondrial damage phenotypes associated with DIC.

Methods

LIG liposome synthesis

Phospholipids, LIG, and cholesterol were precisely weighed according to a predetermined stoichiometric ratio and transferred to a 100 mL round-bottom flask. Subsequently, 30 mL of chloroform was added to the flask, followed by sonication (100 W for 10 min) for complete dissolution. The organic solvent was evaporated under reduced pressure using a rotary evaporator at 37 °C, with a condensation reflux for 150 min, yielding a homogeneous nanoparticle suspension. The solution was hydrated by adding 4 mL of triple-distilled water to the dried lipid film, and the mixture was incubated at 65 °C for 2 h with gentle agitation. The hydrated suspension was rapidly cooled in an ice bath and subjected to pulsed sonication (100 W, 5 min; 2 s on/3 s off intervals) using a probe-type cell disruptor. Finally, the colloidal dispersion was extruded through a polycarbonate membrane-equipped liposome extruder for 10 cycles to refine liposome size and uniformity. The resultant LIG liposome solution was stored at 4 °C and appropriately diluted before subsequent experimental applications.

Animal treatment and drug intervention

SIRT5f/f mice, cardiomyocyte-specific SIRT5 knockout (SIRT5CKO), DUSP1f/f mice, cardiomyocyte-specific DUSP1 knockout (DUSP1CKO), DUSP1 transgenic (DUSP1tg), and PHB2CKO/S91A/S91D mice. Mice were generated and maintained per established protocols. All procedures complied with the U.S. National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Experimental Animal Ethics Committee (no. GZTCMF-20230097) of Guangzhou University of Chinese Medicine. C57BL/6J mice from the same institution were housed under 12-h light/dark cycles with free access to food/water. The investigators remained genotype-blinded during data analysis to ensure unbiased outcomes. Six animals were used in each group.

C57BL/6J mice were acclimatized for 2 weeks under standardized housing. The DIC model group received a single intraperitoneal injection of DOX (12.5 mg/kg; D1515, Sigma-Aldrich in saline), while controls received equivalent saline volumes [31]. Therapeutic interventions compared free LIG versus nanoparticle-encapsulated formulations at escalated doses (25, 50, or 100 mg/kg/day) for 14 days.

Dataset Preparation and analysis

The datasets utilized were derived from the sequencing data generated from the samples in this study and publicly available resources in the GEO database. Bulk RNA sequencing data included GSE157282 [32], GSE17800 [33], GSE120895 [34], GSE226114, and GSE97642, whereas scRNA-seq data were obtained from the dataset GSE145154 [35]. Spatial transcriptomic (ST) data were sourced from the dataset GSE214611. GSE157282 contains six human cardiac cell samples, including three DOX-treated samples. GSE17800 comprises 40 myocardial samples from patients, including 32 cases of dilated cardiomyopathy (DCM) and 8 normal myocardial tissues from healthy controls. GSE120895 comprised 55 myocardial samples from patients, including 47 DCM cases and 8 myocardial tissues from healthy controls. GSE226114 comprised six murine cardiac endothelial cell samples, three of which were induced with DOX. GSE97642 contains 10 murine cardiac samples, 5 of which represent DOX-induced models. GSE145154 is a scRNA-seq dataset containing eight DCM samples and four myocardial tissue samples from healthy controls. GSE214611 contains the ST data from patients with heart failure. Mitochondria-associated genes were obtained from the MitoCarta3.0 database. Batch effects between GSE17800 and GSE120895 were corrected with the SVA package.

The WGCNA algorithm classified genes and identified module-trait associations using the variable genes from GSE157282. Modules were merged via dynamic tree cutting (threshold = 0.25), with the strongest-correlated modules mapped across both classification methods.

ScRNA seq analysis

The single-cell dataset, GSE145154, was obtained from the GEO database. We used R for single-cell transcriptomic analysis, and the samples were processed using the Seurat package.

Spatial transcriptomic data analysis

ST data were processed and visualized via the Seurat R package, including normalization with SCTransform (SCT) and integration using PrepSCTIntegration, FindIntegrationAnchors, SelectIntegrationFeatures, and IntegrateData functions. Subsequently, unsupervised clustering was performed to group spatially adjacent regions with similar expression profiles. Cell population annotation was achieved by integrating H&E-stained histological sections and HVGs specific to each cluster. Spatial expression patterns were resolved using the Spatial DimPlot and spatial feature plot functions to map cellular activity across tissue coordinates.

Enrichment and gene set characterization

We performed enrichment analysis for DEGs with statistical significance. Enriched terms were visualized using bubble plots displaying the adjusted p-values and gene ratios. Complementary GSEA was performed using ClusterProfiler in R to identify pathway-level alterations in distinct biological states, with ridge plots illustrating normalized enrichment scores. All statistical comparisons were performed using Wilcoxon rank-sum tests for DEG identification and Benjamini–Hochberg correction for multiple testing adjustments.

Protein–ligand Docking

Target protein sequences were modeled using AlphaFold3 for structural prediction. Compound structures from PubChem were energy-minimized in ChemBio3D and converted to mol2 format. Protein structures from UniProt were processed using AutoDock Tools (hydrogenation and charge assignment). Molecular docking was performed using AutoDock Vina, followed by interaction visualization using PyMOL.

Echocardiographic analysis

Following myocardial infarction modeling and pharmacological interventions, mice underwent transthoracic echocardiography using 1.0–2.5% isoflurane anesthesia. Left ventricular functional parameters, including FS% and LVEF, and E/A ratio, were measured at the papillary muscle level using the short-axis M-mode imaging (Toshiba SSA-700 A ultrasound system).

RNA-sequencing

Murine cardiac tissue RNA was extracted via standard protocol. Subsequently, rigorous quality control was performed on RNA samples, primarily using an Agilent 2100 Bioanalyzer, to precisely assess RNA integrity. Sequencing libraries were constructed from high-quality RNA samples. Finally, paired-end sequencing (PE150) with a 2 × 150 bp read length was conducted on a NovaSeq 6000 platform (Illumina).

qRT-PCR

Cardiac tissue RNA was isolated via TRIzol reagent (Yeasen), reverse-transcribed to cDNA, and quantified by qPCR using Hieff™ SYBR Green Master Mix (Yeasen) on a CFX96™ system (Bio-Rad).

Cell culture and drug treatment

Primary cardiomyocytes were isolated from WT mice and SIRT5/DUSP1/PHB2 gene-modified mice. HL-1 cardiomyocytes were maintained in DMEM (Gibco) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 5-bromo-2-deoxyuridine to inhibit fibroblast growth. We established the DIC model by pretreating cardiomyocytes for 24 h with DOX dissolved in dimethyl sulfoxide (DMSO) at specified concentrations, using the DMEM complete medium and filtered through 0.2 μm syringe filters.

Cardiomyocytes from si/ad-DUSP1 and si/ad-PHB2 groups were divided into DUSP1TG/CKO subgroups (control, DOX model, DOX + LIG-Na groups) and DOX + LIG-Na + PHB2CKO/PHB2TG groups to investigate LIG–Na-mediated regulatory effects using the DUSP1–PHB2 pathway. LIG–Na concentrations were 25 µmol/L (low), 50 µmol/L (mid), and 100 µmol/L (high). Adenovirus-transfected cells (5–10 × 10⁴ cells/well) were infected at 30–40% confluence using MOI-based viral titers. Medium replacement occurred after 12 to 14 h and polymerase chain reaction (PCR)/western blot validation was performed at 72 h.

Lipofectamine 3000-siRNA complexes (prepared in Opti-MEM) were applied to 70–90% confluent cells in 24-well plates for siRNA transfection. Functional analyses were conducted at 24 to 48 h post-transfection.

Immunofluorescence and laser confocal

DIC tissue was fixed with 4% paraformaldehyde and washed three times with PBS under standardized procedures. Non-fixed groups received direct MitoTracker/ROS/JC-1/F-actin staining (≥ 30 min) with DAPI counterstaining. All samples were mounted in 95% glycerol for fluorescence microscopy analysis, with frozen tissue sections processed identically.

ELISA

The ELISA was conducted in sequential phases: First, antigen coating was performed by diluting the target protein to 5 µg/mL in a carbonate–bicarbonate buffer, followed by 50 µL/well plate coating either at 4 °C overnight or 37 °C for 2 h. After three PBST (PBS with Tween-20) washes, non-specific binding sites were blocked using 250 µL/well of 1% BSA at 37 °C for 2 h. This was followed by PBST washes and subsequently the addition of horseradish-conjugated (HRP)-conjugated secondary antibody at 37 °C for 1 h. This was again followed by PBST washes; afterward, wells were incubated with the 3,3′,5,5′-tetramethylbenzidine substrate in the dark at RT, and measure serum levels.

Mitochondrial permeability transition pore assay

Mitochondrial permeability transition pore opening was assessed using a commercial detection kit. Specifically, the calcein-based staining solutions and fluorescence detection working solutions were freshly prepared according to the manufacturer’s recommended concentrations and volume ratios. Next, a 6-well plate containing the treated cells was incubated with the working solution at 37 °C (5% CO₂) for a specified duration. Afterward, fluorescence imaging was conducted using an Olympus BX53 microscope equipped with excitation/emission filters, and images were subsequently subjected to quantitative fluorescence intensity analysis using the ImageJ software (v1.53) with background subtraction and threshold normalization routines.

Mitochondrial correlation analysis

Cellular bioenergetic profiling was conducted using the Seahorse XFe24 Analyzer (Agilent Technologies) with standardized procedures. Briefly, cardiomyocytes were plated in XF24 cell culture microplates (Agilent 100777-004) at 3–4 × 10⁴ cells/well to reach approximately 95% confluency. The cells were maintained in Seahorse XF DMEM supplemented with 10 mM pyruvate and 2 mM glutamine. The instrument’s sensor cartridge was loaded with ATP rate assay modulators (oligomycin/rotenone–antimycin A) according to the manufacturer’s loading concentrations. Real-time metabolic data acquisition was performed under controlled conditions at 37 °C, with subsequent quantification of basal/maximal ATP production rates using the Wave Desktop Software (v2.6.1).

Western blotting (WB)

Cardiac tissue lysates were prepared using commercial extraction reagents (Invent Biotechnologies SD-001/SN-002), and protein concentrations were quantified using the bicinchoninic acid assay (Beyotime P0012). Denatured protein samples (20 µg/lane) were separated using SDS–PAGE gels under 80–120 V constant voltage, followed by semi-dry transfer onto polyvinylidene fluoride membranes at 300 mA for 90 min. The membranes were blocked with 5% non-fat milk in TBST before overnight incubation at 4 °C with specific primary antibodies against PHB2 (1:10,000; ab182139), SIRT5 (1:2,000; ab236501), DUSP1 (1:2,000; ab236501), and GAPDH (1:2,000; ab8245). Subsequently, HRP-conjugated secondary antibodies (CST 7074/7076; 1:5,000) were added for 1 h at RT, followed by three TBST washes (10 min each) and chemiluminescent visualization using a Bio-Rad ChemiDoc MP system.

Cellular thermal shift assay

Three-time-passaged HL-1 cardiomyocytes were seeded into 25 cm² flasks at a density of 5 × 10⁵ cells per flask and cultured until confluence. Next, the cells were washed twice with PBS, and the total protein was extracted using the radioimmunoprecipitation assay lysis buffer. The protein concentration was quantified using the Bradford assay and adjusted to 1 mg/mL. Subsequently, 600 µg of the total protein was allocated to two treatment groups: the LIG–Na group (supplemented with 100 µM LIG–Na) and the DMSO control group (supplemented with an equivalent volume of DMSO). After incubation at 4 °C for 2 h on a shaker, the protein mixtures were aliquoted into six equal aliquots. These aliquots were subjected to heat stress for 5 min at precisely controlled temperatures (42, 47, 52, 57, 62, or 67 °C) in a metal bath. Post-heating, the samples were equilibrated at RT for 3 min, chilled on ice for 10 min, mixed with 10 µL of 5× SDS–PAGE loading buffer, and denatured at 100 °C for 5 min before storage at − 20 °C. Protein samples were resolved on 10% SDS–PAGE gels and transferred for WB. The membranes were probed with an anti-SIRT5 antibody to assess the thermal stability of SIRT5 under different treatments and temperatures. The resulting immunoblot bands were quantified using the ImageJ software to evaluate comparative protein stability.

Co-immunoprecipitation (Co-IP) assay

Protein–protein interactions were analyzed by Co-IP using cultured HL-1 cardiomyocytes lysed in cold radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (Roche #04693132001) and 1 mM phenylmethylsulfonyl fluoride. The lysate was centrifuged at 12,000 × g to remove the debris, following which 500 µg of the pre-cleared lysate per reaction was incubated with either 2 µg (Primary Antibody Name) antibody or species-matched IgG control, followed by a 4-h incubation with protein A/G Plus agarose beads. The beads were washed four times with cold lysis buffer, resuspended in 2× Laemmli buffer, and boiled at 95 °C for 10 min to elute immunoprecipitated complexes for subsequent detection via SDS–PAGE/WB using antibodies against SIRT5, PHB2, and DUSP1.

Statistical analysis

Quantitative data are expressed as mean ± standard and analyzed using complementary statistical platforms (GraphPad Prism 9.0, IBM SPSS 26.0). Intergroup comparisons were studied using the Shapiro–Wilk normality test (α = 0.05) with Levene’s variance homogeneity verification, selecting either Student’s t-test for normally distributed data with equal variances or nonparametric Mann–Whitney U test for non-Gaussian distributions. Multivariate analyses were performed using one-way ANOVA with Tukey’s post hoc test (equal variance confirmation) or two-way ANOVA with Fisher’s least significant difference adjustments. Statistical significance was defined as a two-tailed p < 0.05, with differential magnitudes annotated using an asterisk hierarchy. All analytical outcomes were cross-validated using dual-software verification to ensure methodological robustness.

Results

Design, synthesis, and characterization of LIG nanoparticles

The film-dispersion method was used to synthesize LIG nanoparticles. Briefly, phospholipids, LIG, and cholesterol were mixed in a set ratio and dissolved in chloroform via sonication. The solution was subjected to rotary evaporation to remove the solvent, resulting in a uniform nanoparticle suspension. The synthesis and physicochemical characteristics of LIG nanoencapsulation were systematically investigated. As illustrated in Fig. 1A, the microencapsulation technology was used to encapsulate phospholipids, cholesterol, chloroform, and LIG in nanoparticles, which significantly improved the solubility and bioavailability of LIG (Fig. S1 A, Fig. S1B). TEM images (Fig. 1B–D) revealed uniformly dispersed spherical nanoparticles with smooth surfaces, as confirmed at varying magnifications (scale bars: 500, 200, and 100 nm). Dynamic light scattering analysis demonstrated a narrow size distribution of nanoparticles, with an average hydrodynamic diameter of 81.4 ± 0.41 nm and a low polydispersity index of 0.23 ± 0.03 (Fig. 1E), indicating excellent colloidal homogeneity. The surface charge quantified by zeta potential was measured at − 0.373 ± 0.032 mV (Fig. 1F), suggesting moderate stability of the formulation. Volume-based size distribution analysis (Fig. 1G) further corroborated this uniformity, with the majority of the nanoparticles residing within the 60–120 nm range. In addition, a strong correlation was observed between nanoparticle characteristics and their size distribution profiles (Fig. 1H), highlighting the reproducibility of the synthesis process, and the LIG nanoparticles exerted the primary pharmacological effects (Fig. S2). Thus, LIG-loaded nanoparticles with favorable physicochemical properties for potential therapeutic applications were successfully developed. The synthetic materials and related information (including liposome drug loading and encapsulation efficiency data) are provided in Supplementary Material 1.

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.
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The Synthesis and Characterization of LIG Nanoencapsulation. (A)The schematic illustration of ligustrazine nanoencapsulation delineates a formulation process utilizing microencapsulation technology to integrate four essential components (phospholipids, cholesterol, chloroform and ligustrazine). And representative transmission electron microscope images showing the morphology of Ligustrazine Nanoencapsulation. (B)Scale bar: 500 nm, (C) 200 nm, (D) 100 nm. (E)The size distribution of Ligustrazine nanoparticles was 81.4 ± 0.41 nm, and the aggregation index(PDI) was 0.23 ± 0.03. (F)The average zeta potential of Ligustrazine nanoparticles was − 0.373 ± 0.032. (G)Size distribution by volume refers to the volume-based distribution of ligustrazine nanoparticles across different size ranges, describing the proportion of contribution of particles of various sizes to the total volume of the sample.(H)Measure the correlation strength between ligustrazine nanoparticles and their size distribution characteristics

SIRT5 May represent a potential therapeutic target for DIC

The normalization process for cardiac sequencing is shown in Fig. 2A, comprising three DIC models and three normal control groups. Differential analysis revealed that the mitochondria-associated gene Sirt5 was significantly downregulated in the DOX group, exhibiting the most substantial fold-change. The corresponding volcano plots and cluster heat maps are shown in Fig. 2B and C, respectively. Enrichment analysis of DEGs revealed a significant role of inflammatory response in DIC (Fig. 2D). We analyzed SIRT5 because it is a crucial mitochondria-associated gene. The WGCNA clustered the genes into eight co-expression modules (Fig. 2E), with Sirt5 identified in the yellow module, which was highlighted as a key module associated with DIC (Fig. 2F). The GSEA of the dataset revealed that mitophagy, inflammatory responses, oxidative stress, and mitochondrial fission have been implicated in DIC (Fig. 2G and H).

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Sequencing data processing and analysis (A) Data Standardization (B) Volcano plot (C) Heatmap (D) Enrichment analysis of differentially expressed genes (E) TOM heatmap of 8 modules (F) Heatmap of correlation analysis of module eigengenes with clinical features (G, H) Gene set enrichment analysis (GSEA)

Differential expression of Sirt5 in our sequencing dataset is shown in Fig. 3A. Validation of Sirt5 expression was further supported by mouse sequencing datasets GSE226114 (Fig. 3B) and GSE97642 (Fig. 3C) from the GEO database. The human cardiac GEO dataset GSE157282 (Fig. 3D) was normalized; the volcano plot and cluster heatmap from differential analysis are shown in Fig. 3E and F, respectively. In addition, SIRT5 expression was validated using this dataset (Fig. 3G). Mitochondria-related GSEA further demonstrated a strong association between DIC and mitophagy (Fig. 3H–J).

Fig. 3
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Validation and Analysis of Multiple Datasets (A) Expression of Sirt5 in the sequencing dataset (B) Expression of Sirt5 in GSE226114 (C) Expression of Sirt5 in GSE97642 (D) Data Standardization of GSE157282 (E) Volcano plot (F) Heatmap (G) Expression of SIRT5 in GSE157282 (H-J) Gene set enrichment analysis (GSEA)

SIRT5 May also represent a critical therapeutic target in DIC progression

DIC is a distinct subtype of secondary DCM characterized by myocardial damage resulting from the cardiotoxic effects of DOX [36]. We integrated two DCM datasets, namely, GSE17800 and GSE120895, comprising 16 normal heart samples and 87 DCM samples, respectively. As shown in Fig. 3, the gene expression and PCA for each sample are displayed before (Fig. 4A) and after (Fig. 4B) batch-effect removal. We validated SIRT5 expression using an integrated dataset (Fig. 4C). The volcano plot and heat map of the differential analysis of the integrated dataset are shown in Fig. 4D and E, respectively. An enrichment analysis of DEGs revealed the significant role of inflammatory response in DIC (Fig. 4D). We used the high and low expression of SIRT5 to perform differential analysis of genes in the integrated dataset. The results are visualized as heatmaps (Fig. 4I) and correlation heatmaps (Fig. 4J). GO and Kyoto Encyclopedia of Genes and Genomes analyses of associated DEGs are shown in Fig. 4K and L, respectively.

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Integration and Analysis of GSE226114 and GSE97642 Datasets (A) Principal component analysis (PCA) analysis before batch effect removal; (B) PCA analysis after batch effect removal; (C) Expression of SIRT5; (D) Volcano plot of differential analysis between DCM group and control group.(E) Heatmap of differential analysis between DCM group and control group (F) Enrichment analysis (G, H) GSEA analysis. (I) Differential analysis heatmap (SIRT5); (J) Correlation analysis heatmap (SIRT5) (K) Differential gene GO analysis (SIRT5); (L) Differential gene KEGG analysis (SIRT5)

Expression profile of SIRT5–DUSP1–Prohibitin 2 in cardiac tissue cells

Our analysis of the single-cell dataset GSE145154 revealed a strong positive correlation between the sequencing depth and the number of detected genes per cell, as shown in Fig. 5A. Our quality control measures excluded cells that failed to meet the predefined criteria (Fig. 5B). The cell distribution per sample across clusters was identified (Fig. 5C). ElbowPlot and principal components (PC) heat maps were used to identify 20 PCs most suitable for downstream analysis (Fig. 5D). The clustering tree results were used to select a resolution parameter of three for downstream clustering analysis (Fig. 5E). Figure 5F shows 42 distinct cell clusters identified using the UMAP algorithm. Marker genes were acquired from the CellMarker database, and cluster annotation was performed to classify 10 cell types based on their characteristic gene expression patterns (Fig. 5G). The cell–cell communication trajectory is shown in Fig. 5H. The subcellular localization patterns of SIRT5, DUSP1, and Prohibitin 2 are shown in Fig. 5J, and the sample-specific cellular distribution profiles are displayed in Fig. 5L. Cell trajectory analysis is shown in Fig. 5M, and the expression trajectories of SIRT5, DUSP1, and Prohibitin 2 are displayed in Fig. 5N.

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Single-cell sequencing analysis (A) Correlation analysis between sequencing depth and mitochondrial genes. (B) Quality control of single-cell data. (C) Cells in each sample before batching. (D) Single-cell expression profiles were analyzed using principal component analysis (PCA) to determine the optimal (E) Elbowplot and heatmap for PC; (F) Subcellular subgroup (G) Cell subpopulation annotation (H) Intercellular communication. (I) Cell scoring system (J) Expression of SIRT5, DUSP1, and PHB2 in cells (K) Cell trajectory

Spatial expression distribution of SIRT5, DUSP1, and prohibitin 2

We used the SCT method to correct for spatial sequencing depth variations. We performed a series of normalization procedures (Fig. 6A) and identified five distinct cellular subpopulations in spatial dimensions through dimensionality reduction and clustering processes (Fig. 6B). The distribution of cell subpopulations in the cardiac tissue is shown in Fig. 6C. Distributions of SIRT5, DUSP1, and PHB2 are shown in Fig. 6D and E, and 6F, respectively.

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Spatial transcriptomics analysis (A, B) Quality control of Spatial transcriptomics data (C) Subcellular subgroup (D) Spatial distribution of cell subpopulations (E) Spatial distribution of SIRT5 (F) Spatial distribution of DUSP1 (G) Spatial distribution of PHB2 (H) Heatmap (I) Volcano plot

LIG-Na May serve as a potential therapeutic targeting the SIRT5–DUSP1–PHB2 regulatory axis

A comparative analysis between LIG–Na-treated and DIC models suggests that LIG–Na may exert therapeutic effects on DIC by modulating the SIRT5 pathway. The corresponding clustering heatmap and volcano plot are shown in Fig. 6G and H, respectively.

SIRT5-mediated mitophagy impairment in inflammatory injury within the DOX model

We next evaluated the effect of LIG–Na on DIC by conducting immunofluorescence staining, echocardiography, and biochemical assays in the DIC mouse model, and subsequently validated SIRT5 and related indicators. Immunofluorescence staining demonstrated that SIRT5 expression was significantly lower in the DOX-treated group than in the control group (Fig. 7A). We further elucidated the role of SIRT5 in DIC and the underlying mechanisms by generating transgenic mice overexpressing SIRT5 and established a DIC model. DIC significantly reduced cardiac EF (Fig. 7B) and FS% (Fig. 7C), indicating impaired left ventricular systolic function. An analysis of serum inflammatory markers demonstrated that DOX administration increased the expression of inflammatory cytokines, including IL-17 (Fig. 7D), IL-10 (Fig. 7E), and MMP-9 (Fig. 7F). Immunofluorescence results demonstrated elevated Cas-9 expression in SIRT5cko mice. Furthermore, DOX administration significantly enhanced Cas-9 expression, which was markedly reduced in the SIRT5tg group (Fig. 7G). The immunofluorescence results demonstrated that DOX inhibited myocardial mitophagy, which was restored in the SIRT5tg group (Fig. 7H). SIRT5 transgenic intervention effectively reversed these alterations, causing improved cardiac ejection function and reduced myocardial inflammatory injury following DIC SIRT5TG effectively alleviated cardiac fibrosis in SIRT5f/f and SIRT5Cko DIC mice and restored mitochondrial integrity (Fig. 7I-N).

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SIRT5 mediates mitochondrial homeostasis-related doxorubicin-induced myocardial inflammatory injury. (A) Tissue fluorescence expression levels of SIRT5; (B-C) Cardiac function was assessed by echocardiography in mice treated with or without DOX. Parameters included left ventricular ejection fraction (LVEF), fractional shortening (FS); (D-F) Expression levels of inflammatory factors IL-17, IL-18, and MMP-9;(G) Tissue expression level of Caspase-9; (H) Histological double fluorescence staining of TOM20 and LC3;(I) Cardiac function was assessed by echocardiography in mice treated with or without DOX. Parameters included LVEF and FS; (J) Transmission electron microscopy (TEM) of myocardial ultrastructure; (K-M) Expression levels of inflammatory factors IL-17, IL-18, and MMP-9 (N) Trichrome staining according to the Masson method staining reveal DOX-induced myocardial fibrosis and histopathological damage. Experiments were repeated at least three times, and the data are shown as means ± SEM (n = 6 mice or three independent cell isolations per group). *P < 0.05

Previous studies demonstrated that mitophagy, a key mechanism initiating MQH, is suppressed by DIC [31]. The MTT assay (Fig. 8A) demonstrated a significant reduction in the proliferative capacity and cellular damage to cytoskeletal proteins in the DOX-treated group (Fig. 8B). However, overexpression of SIRT5 effectively reversed this detrimental effect. DIC significantly increased the levels of IL-17, IL-18, and MMP-9 in cardiomyocytes and activated the NLRP3 inflammasome (Fig. 8C-G). Thus, mitochondria-mediated inflammatory factors are further activated under conditions of impaired mitophagy and mitochondrial energy metabolism, possibly leading to programmed cell death in cardiomyocytes. Experimental analyses of mitochondrial oxidative stress and energy homeostasis revealed compromised antioxidant defenses (reduced superoxide dismutase [SOD]/glutathione peroxidase [GPX] activity) concurrent with impaired bioenergetic profiles, manifested as diminished ATP production, disrupted metabolic flux, and membrane potential collapse. In addition, SIRT5 overexpression demonstrated therapeutic efficacy by mitigating redox imbalance, reactivating electron transport chain activity, and restoring organelle-level bioenergetic parameters, including transmembrane potential regeneration (Fig. 8H–K).

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SIRT5 mediates mitochondrial homeostasis-related doxorubicin-induced myocardial cytoskeletal damage.(A) MTT assay for cell viability; (B) Expression levels of cytoskeletal proteins; (C-E) Expression levels of inflammatory factors IL-17, IL-18, and MMP-9; (F) Fluorescence expression level of NLRP3; (G) Fluorescence expression level of Caspase-9; (H-I) Expression levels of antioxidant enzymes SOD/GPX; (J) Mitochondrial membrane potential level; (K) ATP synthesis level. Experiments were repeated at least three times, and the data are shown as means ± SEM (n = 6 mice or three independent cell isolations per group). *P < 0.05

LIG–Na treatment restores SIRT5 expression in DIC

Mitochondrial ultrastructure- and dynamics-related proteins were analyzed in cardiomyocytes to investigate whether LIG–Na exerts cardioprotective effects in DIC through SIRT5-dependent mitophagy regulation. LIG–Na treatment restored SIRT5 expression in DIC and ameliorated pathological manifestations, including vascular injury, inflammatory response, fibrosis, and mitochondrial oxidative stress. LIG–Na significantly upregulated SIRT5 expression and reduced inflammatory responses through SIRT5-mediated pathways (Fig. 9A–C) in murine models of DIC. LIG–Na ameliorated DOX-induced vascular injury by suppressing mitochondrial oxidative stress markers and restoring antioxidant enzyme activity. Furthermore, genetic ablation of SIRT5 completely abrogated these regulatory effects (Fig. 9D–M).

Fig. 9
Fig. 9The alternative text for this image may have been generated using AI.
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LIG-Na ameliorates doxorubicin-induced myocardial inflammatory injury via SIRT5-mediated oxidative stress. (A-C) Tissue fluorescence expression levels of SIRT5 and Caspase-9; (B-E) Cardiac function assessed by echocardiography (LVEF, FS) in DOX-treated mice; (F) Expression level of MMP-9; (G-I) Expression levels of oxidative stress marker MDA and antioxidant enzymes SOD/GPX; (J) Tissue fluorescence expression of α-SMA; (K-M) Expression levels of MMP-9, MDA, and SOD/GPX. Experiments were repeated at least three times, and the data are shown as means ± SEM (n = 6 mice or three independent cell isolations per group). *P < 0.05

The MTT assay identified the optimal therapeutic concentration of LIG–Na in DIC models, which subsequently demonstrated significant suppression of NLRP3 inflammasome-mediated inflammatory responses (Fig. 10A–E). Thus, DIC significantly reduced TOM20/LC3 fluorescence intensity (indicating mitochondrial loss), downregulated the transcription of PINK1/Parkin and ATG5, and upregulated the expression of pro-inflammatory cytokines (Caspase-1/8). Treatment with LIG–Na effectively reversed these pathological changes. Notably, this therapeutic efficacy was completely abolished in the SIRT5-knockout and 3-MA (autophagy inhibitor) co-treated groups (Fig. 10F–M). Energy metabolism analysis revealed that DOX treatment significantly reduced ATP synthesis, induced mitochondrial bioenergetic dysfunction, and caused loss of mitochondrial membrane potential (ΔΨm). The administration of LIG–Na reversed these alterations by restoring respiratory chain complex activities (CI-CIV) and stabilizing ΔΨm. In addition, SIRT5 knockout abolished the therapeutic effects of LIG–Na on mitochondrial homeostasis (MQH) and ΔΨm maintenance (Fig. 10N–R).

Fig. 10
Fig. 10The alternative text for this image may have been generated using AI.
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LIG-Na improves doxorubicin-induced myocardial pyroptosis via SIRT5-mediated mitochondrial homeostasis. (A-C) MTT assay for cell viability and NLRP3 expression; (D-E) Expression levels of IL-18 and MMP-9; (F) Cellular fluorescence expression of TOM20 (G) Mitochondria and lysosomes colocalization fluorescence; (H-J) Expression levels of mitophagy-related genes PINK/Parkin and ATG5; (K-N) Mitochondrial membrane potential and Caspase-1/Caspase-8 expression; (O-R) Seahorse assay for mitochondrial energy metabolism.(S) Related mechanism diagram. Experiments were repeated at least three times, and the data are shown as means ± SEM (n = 6 mice or three independent cell isolations per group). *P < 0.05

LIG–Na orchestrates a multi-target regulatory network with SIRT5, DUSP1, and PHB2 in DIC

Gene expression analysis revealed significantly downregulated expression of DUSP1 (Fig. 11A) and PHB2 (Fig. 11B) in cardiomyocytes after DOX treatment. DIC significantly decreased EF and FS. Ventricular systolic function was restored following LIG–Na treatment, whereas the therapeutic effects of LIG–Na were abolished upon PHB2 and DUSP1 knockout, as well as OMY intervention (Fig. 11C–D). The immunofluorescence results demonstrated that DOX inhibited myocardial mitophagy, which was restored after LIG–Na treatment. The therapeutic effects of LIG–Na were abolished following PHB2/DUSP1 knockout and OMY treatment are shown in Fig. 11E–G. Serum inflammatory factor analysis revealed that LIG–Na treatment significantly reduced inflammatory factor levels (Caspase-1/9 and MMP-9), whereas the therapeutic effects of LIG–Na were abolished following PHB2/DUSP1 knockout and OMY intervention (Fig. 11H–J). Thus, DOX induction significantly decreased SIRT5 expression, whereas LIG–Na intervention restored SIRT5 levels. However, the overexpression of DUSP1 or the dephosphorylation-mimetic PHB2 mutant (PHB2S91D) exerted no significant effect on SIRT5 expression. The phosphomimetic PHB2 mutant (PHB2S91A) abolished the therapeutic effects of LIG–Na (Fig. 11K–L). The overexpression of DUSP1 or the dephosphorylation-mimetic PHB2 mutant (PHB2S91D) combined with LIG-Na treatment demonstrated no significant effects on ventricular function for EF and FS. However, the phosphomimetic PHB2 mutant (PHB2S91A) abolished the therapeutic effects of LIG–Na (Fig. 11M-N). An analysis of serum samples revealed significantly elevated malondialdehyde (MDA) levels following DIC, whereas SOD and catalase activities were markedly reduced. The administration of LIG–Na reversed these effects. However, the overexpression of DUSP1 or the dephosphorylation-mimetic PHB2 mutant (PHB2S91D) exerted no significant effect on these parameters. The phosphomimetic PHB2 mutant (PHB2S91A) abolished the therapeutic effects of LIG–Na (Fig. 11O–Q). DOX induction disrupts mitochondrial integrity and exacerbates fibrosis. The dephosphorylation-mimicking PHB2 mutant (PHB2S91D) reversed this effect, whereas the phosphorylation-mimicking PHB2 mutant (PHB2S91A) did not (Fig. 11R).

Fig. 11
Fig. 11The alternative text for this image may have been generated using AI.
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LIG-Na improves doxorubicin-induced myocardial pyroptosis via DUSP1-PHB2-mediated mitochondrial homeostasis. (A-B) Expression levels of DUSP1-PHB2; (C-D) Echocardiographic assessment (LVEF, FS) in DOX-treated mice; (E-G) Histological double fluorescence staining of TOM20 and LC3; (H-J) Transcriptional and protein levels of Caspase-9, Caspase-1, and MMP-9; (K-L) Tissue fluorescence expression of SIRT5; (M-N) Echocardiographic parameters (LVEF, FS); (O-Q) Expression levels of MDA, SOD, and CAT; (R) Transmission electron microscopy and Masson myocardial tissue staining. Experiments were repeated at least three times, and the data are shown as means ± SEM (n = 6 mice or three independent cell isolations per group). *P < 0.05

We next examined the mitochondrial structure in cardiomyocytes. DOX-induced disruption of mitochondrial morphology caused increased mitochondrial fragmentation, suppressed mitochondrial biogenesis, and impaired cardiomyocyte function (Fig. 12A–D). Thus, DOX upregulated the transcription of mitochondrial fission-related proteins (Drp1, Fis1, and Mff) and downregulated the transcription of lysosomal and mitochondrial fusion-related gene Opa1 (Fig. 12E–J, L-N). Co-IP experiments revealed an interaction between SIRT5, DUSP1, and PHB2 (Fig. 12K). Mitochondrial membrane potential (ΔΨm) was maintained in SIRT5 overexpression models, whereas DUSP1 silencing induced complete collapse of ΔΨm (Fig. 12O). DOX induction significantly increased the transcription of pro-inflammatory mediators (Caspase-1, Caspase-3, Caspase-8, and Gasdermin D), whereas LIG–Na treatment reduced the transcript levels of these cytokines. This effect was abolished in SIRT5 knockout models. The silencing of DUSP1 in SIRT5 transgenic mice eliminated the therapeutic effects of LIG–Na (Fig. 12P–T). DOX significantly increased MDA and ROS levels and reduced SOD and GPX4 activities; these changes were reversed by LIG–Na treatment. This effect was abolished in the SIRT5 knockout model. The silencing of DUSP1 in SIRT5 transgenic mice eliminated the therapeutic effect of LIG–Na (Fig. 12U–Y).

Fig. 12
Fig. 12The alternative text for this image may have been generated using AI.
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LIG-Na inhibits doxorubicin-induced myocardial pyroptosis via SIRT5-regulated DUSP1-PHB2-mediated mitochondrial homeostasis. (A-C) Mitochondrial morphology and structural integrity; (D) MTT assay for cell viability; (E) Transcript level of mitochondrial fusion protein OPA1; (F-H) Transcript levels of mitochondrial fission proteins Drp-1/Fis-1/Mff; (I-J) Cellular fluorescence expression of lysosome; (K) Co-immunoprecipitation (Co-IP) (O-P) Mitochondrial membrane potential; (L-N) Transcript levels of mitophagy-related genes PINK/Parkin/ATG5; (Q-T) Expression levels of pyroptosis-related genes Caspase-1, −8, IL-1β, and GSDMD (U-Y) Expression levels of ROS. Experiments were repeated at least three times, and the data are shown as means ± SEM (n = 6 mice or three independent cell isolations per group). *P < 0.05

The binding energies of SIRT5, DUSP1, and PHB2 are shown in Fig. 13A, suggesting the existence of a SIRT5–DUSP1–PHB2 regulatory axis. The molecular docking diagrams are shown in Fig. 13B (SIRT5–PHB2), Fig. 13C (SIRT5–DUSP), and Fig. 13D (DUSP1–PHB2). H&E and Masson staining demonstrated that the overexpression of DUSP1 can reverse myocardial injury, whereas further knockout of SIRT5 or an increase in 3MA based on DUSP1 expression induced myocardial injury (Fig. 13D). DUSP1 transgenic treatment improved EF and FS (%), whereas either SIRT5 or an increase in 3MA based on DUSP1 expression induced myocardial injury (Fig. 13E–F). DUSP1 transgenic treatment attenuated the inflammatory response, whereas either SIRT5 or an increase in 3MA, based on DUSP1 expression, caused inflammation-induced injury (Fig. 13G–K). The molecular docking results and corresponding binding energies are presented in Fig. 13L–N.

Fig. 13
Fig. 13The alternative text for this image may have been generated using AI.
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The interaction relationship between SIRT5, DUSP1, and PHB2 (A) SIRT5-PHB2 protein docking (B) SIRT5-DUSP1 protein docking (C) DUSP1-PHB2 protein docking (D) Transmission electron microscopy and Masson myocardial tissue staining (E-F) Cardiac function was assessed by echocardiography in mice (G-K) Expression levels of inflammatory factors; (L) LIG-SIRT5 molecular docking; (M) LIG-DUSP1 molecular docking; (N) LIG-PHB2 molecular docking. Experiments were repeated at least three times, and the data are shown as means ± SEM (n = 6 mice or three independent cell isolations per group). *P < 0.05

We next conducted histopathological examinations and biochemical assays using heart, liver, and kidney tissues of mice treated with high and low doses of LIG–Na. No significant signs of toxicity or damage to these organs at the therapeutic doses were noted, confirming that LIG–Na did not induce substantial organ-specific toxicity (Supplementary Material 2). CETSA confirmed the targeted interaction between LIG–Na and SIRT5 (Supplementary Material 3).

Discussion

The use of DOX, a cornerstone therapeutic for different malignancies, including solid tumors and leukemias, is restricted by dose limitations owing to cardiotoxicity risks. Among all anthracyclines, cumulative doses exceeding 300 mg/m² may induce irreversible myocardial damage and heart failure [36]. Therefore, there exists an urgent need to develop adjuvant therapeutic strategies offering cardioprotective benefits without compromising antitumor efficacy. A combination of dose optimization and cardiomyopathy-preventive agents can provide dual therapeutic advantages in cancer patients [37]. We used an integrated multidisciplinary strategy, combining innovative molecular targeting, natural product pharmacology, nanodrug delivery, genetic engineering, and bioinformatics, to systematically elucidate the dysregulatory mechanisms of the MQH network in DIC.

Bioenergetic dysfunction induced by mitochondrial ultrastructural and morphological damage constitutes the core pathological mechanism of DIC [10]. DOX exposure disrupts the MQH, causing autophagic flux abnormalities and myocardial energy homeostasis imbalances [38]. Clinical studies have demonstrated that the restoration of autophagic flux and energy metabolism effectively alleviates DIC. The current study proposes multiple mechanistic hypotheses for DOX cardiotoxicity, particularly emphasizing ROS accumulation and impaired mitochondrial biogenesis [38]. Integrative analysis of the sequencing data and GEO datasets (GSE157282, GSE17800, GSE120895, GSE226114, and GSE97642) identified SIRT5 as a novel biomarker for DOX-induced mitochondrial dysfunction (DIMD), exhibiting significant differential expression patterns in cardiomyopathy progression models. In addition, SIRT5 exhibits a strong functional association with autophagy regulation. Both in vitro and in vivo experimental models have confirmed that oxidative stress and inflammatory cascade are key mediators of DIMD in cardiomyocytes. In vivo studies have shown that DIMD in cardiomyopathy is associated with reduced cardiac output, increased oxidative stress, and inflammatory damage, while SIRT5 transgenic intervention effectively alleviates the oxidative/inflammatory cascade reaction induced by DOX. Autophagy inhibitors have no efficacy in reversing DOX mediated decrease in ejection fraction and related bioenergy damage.

SIRT5, as an NAD⁺-dependent mitochondrial deacylase, is primarily located in the mitochondria. SIRT5 can regulate the mitochondrial quality control network through extensive post-translational modifications of proteins. The disruption of the above mechanisms can further mediate the activation of mitochondrial pathway-induced cell death [39, 40]. In terms of energy metabolism, SIRT5 dynamically regulates the activity of the tricarboxylic acid cycle and mitochondrial respiratory chain complexes through modifications such as desuccinylation and demalonylation, further optimizing ATP production efficiency [41, 42]. At the same time, SIRT5 can regulate ketone body production and ammonia metabolism by modulating HMGCS2 and CPS1, thereby adjusting and responding to energy substrates. In the process of oxidative stress damage regulation, SIRT5 can activate the activities of SOD2 and IDH2 through post-translational modifications, further enhancing the ROS scavenging ability. It can also restore mitochondrial energy exchange by improving mitochondrial respiratory chain function. Through these dual pathways, SIRT5 ensures the homeostatic mechanism of mitochondrial redox balance [43, 44]. In the process of stress damage mediated by redox imbalance, the disruption of the mitochondrial quality control network further drives SIRT5 to modulate the dynamic balance of mitochondrial fusion/fission by modifying DRP1/Fis1. This process collaborates with the PINK1/Parkin pathway, the FUNDC1 pathway, and the BNIP3 pathway to promote mitochondrial autophagy, facilitating the self-clearance of damaged mitochondria. Additionally, it inhibits the excessive stress of the UPR and suppression [45]. This is consistent with our research findings. Experimental results suggest that the interaction mechanism between SIRT5 and DUSP1 (dual-specificity phosphatase 1) forms a refined metabolic-signal transduction regulatory network. The molecular interaction mainly manifests in mitochondrial nano-drug delivery and mitochondrial homeostasis regulation. The study found that SIRT5 can directly modify the DUSP1 protein through its NAD⁺-dependent deacylase activity. Research further indicates that, after stress damage, SIRT5 can de-succinylate key sites on DUSP1, a post-translational modification that significantly enhances DUSP1 stability, thereby maintaining its regulatory capacity over the PHB2 mitochondrial membrane protein [17]. Moreover, the SIRT5-DUSP1 axis can form a positive regulatory feedback loop in the mechanism of programmed injury in cardiomyocytes. Activated DUSP1 can regulate the JNK/p38 pathway through mitochondrial circular feedback, indirectly reducing the phosphorylation of the Drp1 at the S616 site. This, in turn, improves mitochondrial dynamics balance, maintains mitochondrial morphology and structural stability, and enhances NAD⁺ level activity [15]. In addition, SIRT5 integrates intracellular environmental signals and gene expression through mitochondrial energy metabolism sensing and epigenetic regulation, forming a multi-level intracellular homeostasis regulatory network. Its functional dysregulation is closely associated with myocardial ischemia, endothelial injury, mitochondrial metabolic reprogramming, and various myocardial damage-related diseases.

We built upon the established DUSP1–PHB2 axis research framework and further explored the functional synergy mechanisms among SIRT5, DUSP1, and PHB2 in MQH. Integrated sequencing analysis revealed LIG–Na-mediated upregulation of SIRT5. Furthermore, molecular docking analysis demonstrated the binding potential between LIG and SIRT5/DUSP1/PHB2, whereas protein interaction modeling suggested functional cooperativity among targets, supporting the hypothesis of a SIRT5–DUSP1–PHB2 regulatory axis in MQH. LIG was encapsulated within biocompatible nanoparticles to achieve an increased surface-area-to-volume ratio that enhanced dissolution kinetics. We used the integrative analysis of single-cell and spatial transcriptomics datasets to systematically map the cellular and spatial distribution patterns of SIRT5, DUSP1, and PHB2 in myocardial tissue microenvironments, providing spatial omics evidence for their functional interaction networks in DOX cardiotoxicity. Significant cardiac dysfunction, elevated inflammatory cytokines, and coordinated low expression of SIRT5/DUSP1/PHB2 were noted in mice with DIC, whereas LIG–Na treatment upregulated their expression in a dose-dependent manner. Cardiac-specific triple-gene knockout completely abolished the cardioprotective effects of LIG–Na Therapeutically, LIG–Na alleviated DIC inflammatory injury and oxidative stress through SIRT5–DUSP1–PHB2 molecular interactions, enhanced cardiomyocyte bioenergetics, corrected cardiac structural abnormalities, and preserved cardiac functions. Integrative single-cell and spatial multiomics analyses highlighted the presence of MQH-related genes in DIC. Experiments using SIRT5–DUSP1–PHB2-modified cellular systems confirmed that LIG–Na activates mitophagy via DUSP1-mediated suppression of pathological mitochondrial fission, inhibits aberrant mtUPR activation, and enhances mitochondrial biogenesis. In addition, LIG–Na preserved cytoskeletal protein integrity, maintained the mitochondrial membrane potential, and prevented apoptosis via the MQH pathway. It targeted the SIRT5–DUSP1 axis to activate PHB2 phosphorylation and demonstrated cardioprotective efficacy superior to that of free LIG through enhanced mitochondrial protection.

Although low- and medium-dose LIG–Na demonstrated no therapeutic efficacy in treating DIC and cardiomyocyte damage, high-dose LIG–Na demonstrated significant therapeutic effects against DOX-induced myocarditis and mitochondrial impairment [46,47,48]. This further confirmed the existence of “dose–response” interactions among active components in traditional Chinese medicine (TCM). TCM compounds exhibited optimal cardioprotective effects on cardiomyocytes and mitochondria within specific dose gradients. Distinct dose-dependent therapeutic responses have been observed with TCM formulations and bioactive monomers. Previous studies have revealed that low-to-medium doses of quercetin and ginsenosides lack robust therapeutic effects, whereas high-dose gradients display marked cardioprotection, a finding completely consistent with our experimental results, reinforcing that high-dose therapeutic concentrations achieve superior treatment efficacy.

Although some researchs have reported the regulatory function of MQH in DIC, the specific upstream regulatory mechanisms remain unclear [49]. Previous investigations on the SIRT family members have revealed significantly reduced SIRT5 expression in transverse aortic constriction-induced heart failure models. This reduction was correlated with diminished SIRT5-mediated isocitrate dehydrogenase desuccinylation, elevated myocardial ROS levels, and impaired mitochondrial functional parameters [50]. SIRT5 overexpression further suppresses DNA–PKcs activation, inhibits Drp1/Fis1 overexpression, maintains mitochondrial integrity, and prevents apoptosis [51].

Conversely, DUSP1 transgenic intervention suppresses Mff activation and Bnip3-mediated mitophagy initiation of mitophagy. DUSP1 alleviates pathological mitochondrial fragmentation by inactivating the JNK pathway [52], enhancing cardiomyocyte functional recovery after ischemic injury, which is consistent with our findings of mitochondrial dysfunction and bioenergetic disturbances in DOX-related myocardial injury. Although DUSP1 localizes outside the mitochondria, it indirectly exerts its mitochondrial regulatory effects through membrane-associated proteins [45, 46].

PHB2, a highly conserved eukaryotic protein localized to the mitochondrial inner membrane, maintains cardiomyocyte mitochondrial architecture and function [53,54,55] while regulating mitophagy and preserving inner membrane protein organization [21, 56]. This study demonstrated that dynamic phosphorylation of PHB2-Ser91 coordinates mitophagic flux and metabolic homeostasis via ATP synthesis [57]. Protein interactome analyses revealed LIG–Na modulates the SIRT5–DUSP1 axis—an upstream regulator of PHB2—preserving contractility and mitigating DOX-induced structural remodeling [58].

These findings corroborated our observations and underscored the critical function of mitochondrial upstream protein interactions in DIC [59]. Our study highlights the mitochondria-targeting mechanisms of LIG–Na and delineates the involvement of the SIRT5–DUSP1–PHB2-mediated regulatory axis. However, our study has some limitations. Although our pharmacological screening identified the action pathways and protein targets of LIG–Na in post-DOX myocardial injury, future studies should explore the mechanisms of action of other drugs or bioactive components on post-DIC and vascular damage using multi-omics approaches. We validated the therapeutic components and mechanisms of LIG–Na in DOX cardiomyopathy mouse models; however, additional animal and cellular models are required to verify its interaction with other TCM bioactive compounds. We plan to conduct pharmacological experiments to compare the components of Chuanxiong (Ligusticum) and Astragalus to elucidate their mechanisms of action in DIC.

Conclusion

DIC activated the SIRT5 pathway, subsequently suppressing DUSP1 expression and disrupting PHB2 phosphorylation at Ser91. Activated SIRT5 directly interacts with DUSP1 and PHB2, inducing pathological phosphorylation cascades that disrupt MQH, while amplifying NLRP3 inflammasome-mediated pyroptosis. LIG–Na mitigates DIC by suppressing SIRT5 activation, restoring physiological phosphorylation patterns at PHB2-Ser91, and re-establishing coordinated MQH. This therapeutic intervention inhibits cardiomyocyte apoptosis, maintains mitochondrial homeostasis, normalizes mitophagy, and protects injured myocardium.