Angelica sinensis Polysaccharide nanoparticles can improve myocardial ischemia-reperfusion injury by inhibiting ferritinophagy via the ATF6/NCOA4 pathway

Angelica sinensis Polysaccharide nanoparticles can improve myocardial ischemia-reperfusion injury by inhibiting ferritinophagy via the ATF6/NCOA4 pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Angelica sinensis Polysaccharide nanoparticles can improve myocardial ischemia-reperfusion injury by inhibiting ferritinophagy via the ATF6/NCOA4 pathway Ming Bai, cheng chen, Zhao Jing, Maomao Zhao, Shuwen Hu, Pei Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7142720/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Feb, 2026 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Background Ferroptosis aggravates myocardial ischemia-reperfusion injury (MI/RI) by disrupting iron homeostasis, accelerating lipid peroxidation, and elevating reactive oxygen species (ROS) levels. Although Angelica sinensis polysaccharide (ASP) has shown protective effects against MI/RI, its clinical translation remains limited due to poor bioavailability and low target specificity. Methods To address these limitations, we developed ASP@PLGA-PEG nanoparticles using a solvent evaporation method and characterized their morphology, size distribution, and surface charge by transmission electron microscopy, dynamic light scattering, and zeta potential analysis. The protective effects of ASP@PLGA-PEG were evaluated in vitro using HL-1 cardiomyocytes subjected to oxygen and glucose deprivation/reoxygenation (OGD/R). Cell viability, mitochondrial membrane potential, ROS generation, lipid peroxidation, and antioxidant capacity were assessed using CCK-8 assay, JC-1 staining, ROS fluorescence detection, immunofluorescence, and biochemical analyses. In addition, an in vitro MI/RI model was established using the Langendorff isolated heart perfusion system to assess hemodynamic function, infarct size, histopathological changes, and mitochondrial ultrastructure. Results ASP@PLGA-PEG nanoparticles significantly reduced oxidative stress, improved cardiomyocyte viability, and inhibited ferroptosis in OGD/R-injured HL-1 cells. In the Langendorff model, treatment with ASP@PLGA-PEG effectively decreased myocardial infarct size, preserved cardiac hemodynamics, and alleviated structural damage. Mechanistic studies revealed that ASP@PLGA-PEG nanoparticles activate ATF6 signaling, which suppresses NCOA4-mediated ferritinophagy, thereby limiting iron overload and lipid peroxidation to protect cardiomyocytes against ferroptosis during MI/RI. Conclusions This study demonstrates that ASP@PLGA-PEG nanoparticles exert potent cardioprotective effects through a multi-target mechanism involving ER stress modulation, enhanced antioxidative defense, and inhibition of ferritinophagy-driven ferroptosis. These findings highlight the therapeutic potential of ASP@PLGA-PEG as a promising nanomedicine strategy for the prevention and treatment of myocardial ischemia-reperfusion injury. myocardial ischemia-reperfusion injury Angelica sinensis polysaccharides ROS ATF6 ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Acute myocardial infarction(AMI), one of the leading causes of death worldwide, occurs due to the acute occlusion of coronary arteries, leading to prolonged myocardial ischemia and subsequent cardiomyocyte death[1]. Although restoring myocardial blood flow is crucial in treating AMI, timely reperfusion within a short therapeutic window is often difficult in clinical practice[2, 3]. Furthermore, reperfusion itself can trigger ischemia/reperfusion (I/R) injury, which exacerbates myocardial damage[4, 5]. In AMI, cardiomyocyte injury involves several cell death mechanisms, including necrosis, apoptosis, and ferroptosis. Ferroptosis, a non-apoptotic form of cell death, is induced by iron-dependent lipid peroxidation and has drawn increasing attention in AMI research. The mechanism of ferroptosis in myocardial ischemia-reperfusion injury is primarily driven by dysregulated iron metabolism and lipid peroxidation[6-8]. disturbed iron metabolism leads to iron overload in cardiomyocytes, which produces large amounts of reactive oxygen species (ROS) via the Fenton reaction, worsening oxidative stress[9]. During reperfusion, depletion of glutathione (GSH) and inactivation of glutathione peroxidase 4 (GPX4) impair the cell's ability to clear lipid peroxides, thereby triggering lipid peroxidation and membrane disruption[10, 11]. Mitochondrial dysfunction also contributes to further ROS accumulation and damage[12, 13]. Overall, ferroptosis exacerbates ischemia-reperfusion injury by promoting lipid peroxidation, iron overload, and ROS accumulation, leading to more extensive cardiomyocyte death. Alongside ferroptosis, necrosis and apoptosis also play significant roles in AMI pathology. Angelica sinensis Polysaccharide (ASP), extracted from Angelica sinensis , are known for their wide range of biological activities, including antioxidant, anti-inflammatory, immune-regulating, and even anti-apoptotic effects[14-19]. These properties have been demonstrated to provide significant protection to the cardiovascular system.Ai et al[20]. demonstrated that ASP can mitigate cerebral ischemia/reperfusion injury in rabbits through antioxidant mechanisms. Similarly, Lei et al[21]. reported that ASP protects PC12 neuronal cells from H₂O₂-induced cytotoxicity by reducing apoptosis, lowering intracellular ROS levels, and enhancing mitochondrial membrane potential in cells treated with H₂O₂. Additionally, Niu et al[18]. found that ASP alleviates endoplasmic reticulum stress and oxidative stress by activating the ATF6(Activating Transcription Factor 6) pathway, thereby protecting H9c2 cells from H₂O₂-induced injury.Studies suggest that ASP can reduce oxidative stress and inhibit inflammation, two key drivers of myocardial damage during ischemia-reperfusion injury. Additionally, ASP has shown potential in improving endothelial function, enhancing microcirculation, and promoting angiogenesis[22, 23]. all of which contribute to improving cardiac function after AMI.However, despite its cardioprotective effects, ASP faces several challenges. Its short half-life, poor bioavailability, and lack of tissue-specific targeting limit its efficacy[24-26]. To overcome these limitations, We designed ASP@PLGA-PEG nanoparticles to evaluate the efficacy of ASP@PLGA-PEG on myocardial ischemia/reperfusion injury(MI/RI) and its mechanism in depth by experiment. Materials and Methods Preparation and Characterization of ASP@PLGA-PEG ASP@PLGA-PEG nanoparticles were prepared using the nanoprecipitation method. Briefly, 80 mg of PLGA-PEG (Xarxbio, Xi'an, China) was dissolved in 2 mL of dichloromethane to prepare the oil phase, while 10 mg of Angelica sinensis polysaccharide (ASP; Solarbio, Beijing, China) was dissolved in 200 μL of deionized water as the aqueous phase. The oil phase was slowly added dropwise to the aqueous phase under continuous stirring. The mixture was then emulsified using a probe sonicator (SCIENTZ JY99-IIDN, Ningbo, China) under an ice bath to form a primary emulsion. Subsequently, 4 mL of 1.5% (w/v) polyvinyl alcohol (PVA) aqueous solution was added, and the resulting mixture was further emulsified by sonication to form a double emulsion. The emulsion was stirred at room temperature for 6 hours to allow complete evaporation of dichloromethane. The resulting ASP@PLGA-PEG nanoparticles were collected and transferred to a cuvette for characterization. The particle size and zeta potential were measured using a dynamic light scattering (DLS) instrument (Brookhaven Instruments 90Plus PALS, USA). The morphology of the nanoparticles was examined by transmission electron microscopy (TEM) (FEI Tecnai G2 Spirit Bio-TWIN, USA). Cell Culture and Treatment HL-1 cardiomyocytes were maintained in DMEM medium (Gibco, Waltham, ME, USA) supplemented with 10% fetal bovine serum (FBS, Gibco). The cells were incubated in a humidified atmosphere at 37 °C with 5% CO 2 . To establish an in vitro MI/MR model, we intervened on HL-1 cardiomyocytes for 4 hours using the hypoxia-glucose deprivation (OGD) method. Prior to the experiment, cells were cultured in a conventional incubator. After the cells grew to 80% confluence, the cells were transferred to a HeraCell VIOS 160i incubator (Thermo Fisher Scientific, Waltham, MA, USA) and hypoxia was simulated by releasing a gas mixture containing 1% O₂, 5% CO₂, and 94% N₂ (37°C). During hypoxia, HL-1 cardiomyocytes were cultured in serum-free, glucose-free DMEM medium to cut off the cellular fuel supply. In contrast, control cells were cultured under normoxic conditions with regular DMEM medium in a conventional incubator. Cell Viability Assay Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China). HL-1 cardiomyocytes were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and subjected to the simulated in vitro MI/RI model. Following the treatment, an appropriate volume of CCK-8 solution was added according to the kit instructions, and the cells were incubated for 2 hours. Absorbance (OD) at 450 nm was measured using a microplate reader (Tecan, Infinite M200 PRO, Männedorf, Switzerland). Detection of Reactive Oxygen Species Reactive oxygen species (ROS) levels were measured using a ROS assay kit (Beyotime, Shanghai, China). HL-1 cardiomyocytes were cultured in confocal dishes and treated according to the experimental model. After treatment, the cells were incubated with 1 mL of DCFH-DA reagent at 37°C for 20 minutes, following the kit instructions. After incubation, the cells were washed three times with PBS and then observed under an Olympus IX71 microscope. Fluorescence intensity was quantified using ImageJ software (version 1.51 k; Bethesda, MD, USA). Measurement of Mitochondrial Membrane Potential Mitochondrial membrane potential (MMP) was assessed using the JC-1 Mitochondrial Membrane Potential Assay Kit (Solarbio, Beijing, China). HL-1 cells were seeded into confocal dishes and treated according to the experimental model. Following treatment, the cells were incubated with 1 mL of JC-1 dye solution at 37°C for 20 minutes. After staining, the cells were observed under an Olympus microscope. The images were analyzed using ImageJ software, and MMP was quantified by calculating the red-to-green fluorescence ratio, which reflects the ratio of JC-1 aggregates (red) to monomers (green). Immunofluorescence Detection of 4-HNE Immunofluorescence quantification of 4-HNE was performed on HL-1 cardiomyocytes cultured in confocal petri dishes and processed according to the experimental protocol. After fixing the cells with 4% paraformaldehyde (PFA) for 10 min, permeabilizing the cells with 0.1% Triton X-100 for 10 min, and blocking with goat serum for 30 min, the cells were incubated with 4-HNE primary antibody (Thermo Fisher Scientific, MA5-27570) at 4°C overnight. On the following day, after washing three times with phosphate-buffered saline (PBS), HL-1 cardiomyocytes were incubated with FITC-coupled secondary antibody (Thermo Fisher Scientific) for 1 h under light protection. Then, the cells were washed again three times and restained with DAPI-containing medium to visualize the nuclei. Fluorescence imaging was performed using an Olympus IX71 microscope and quantitative analysis was performed using ImageJ software. Animals and Treatments C57BL/6 mice aged 6-8 weeks, weighing 22-25 g, were selected as experimental animals.The experimental animals were purchased from the Laboratory Animal Center of Lanzhou University (Lanzhou, Gansu, China).The mice were provided with unrestricted access to standard rodent chow and sterile water, and were housed in an environment with an ambient temperature of 22 ± 2 ℃ and a 12-hour light/dark cycle. All procedures involving animals were approved by the Animal Care Committee of the First Hospital of Lanzhou University and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Langendorff Isolated Mouse Heart Perfusion Mice in the Sham and I/R groups were injected with saline via the tail vein for 3 consecutive days. Mice in the ASP group received angelica polysaccharides (20 mg/kg) via tail vein injection for 3 consecutive days. Mice in the Nps group were injected with varying doses of ASP@PLGA-PEG via the tail vein for 3 consecutive days. After the final drug injection, the mice were anesthetized with an intraperitoneal injection of 2% pentobarbital sodium. The mice were then euthanized by cervical dislocation. The hearts were rapidly excised, placed in pre-cooled Krebs-Henseleit (KH) solution(pH 7.4, gases 95% O2 and 5% CO2) in mM: 4.7 KCl, 118 NaCl, 25 NaHCO3, 1.8 CaCl2, 1.4 MgSO4, 1.2 KH2PO4, 11 D-glucose, and ligated with a 22G steel needle. Finally, the hearts were connected to the Langendorff apparatus, and retrograde perfusion was initiated. The hearts were perfused with KH solution. A latex balloon filled with normal saline and connected to a PowerLab system (AD Instruments, Sydney, NSW, Australia) was inserted into the left ventricle through the mitral valve. After placement, the balloon was inflated to maintain a left ventricular end-diastolic pressure (LVEDP) of 5–10 mmHg. Hemodynamic parameters of the isolated hearts were continuously monitored and analyzed using the LabChart 8 data acquisition system (AD Instruments). At the end of the stabilization period, perfusion was completely stopped for 35 minutes to induce global ischemia, followed by 60 minutes of reperfusion. During reperfusion, cardiac function was continuously monitored using LabChart software, and hemodynamic data were recorded every 10 minutes for statistical analysis. According to the experimental protocol, healthy mice were divided into 6 experimental groups: 1. Sham group (saline): perfusion with KH solution for 125 min; 2. Ischemia/reperfusion group (saline): stabilization period for 30 min, global ischemia for 35 min, and reperfusion for 60 min; 3. ASP group (20 mg/kg): stabilization period for 30 min, global ischemia for 35 min, reperfusion for 60 min; 4. NpsL group (5 mg/kg): stabilization period for 30 min, global ischemia for 35 min, reperfusion 60 min; 5. NpsM group (10 mg/kg): stabilization period for 30 min, global ischemia for 35 min, reperfusion 60min; 6.NpsH group (20 mg/kg): stabilization period for 30 min, global ischemia for 35 min, reperfusion 60 min; Biochemical Assessment The content of malondialdehyde (MDA), a lipid peroxidation product, and the activities of antioxidant markers including superoxide dismutase (SOD), catalase (CAT), lactate dehydrogenase (LDH), and reduced glutathione (GSH), as well as the concentration of Fe²⁺ in cell lysates and tissue homogenates were determined using commercial assay kits (Elabscience, Wuhan, China). Hematoxylin and Eosin Staining Histopathological changes in myocardial tissue sections were examined by hematoxylin and eosin (H&E) staining. Following completion of the in vitro experiments on the Langendorff apparatus, the hearts were fixed in 4% paraformaldehyde for 24 hours. The tissue was then dehydrated through a graded ethanol series (70%, 90%, and 100%), cleared in xylene at 60–70 °C for 60 minutes, and embedded in paraffin. Paraffin-embedded tissues were sectioned at a thickness of 4 μm and stained using a commercial H&E staining kit (Servicebio, Wuhan, China). Two authors independently evaluated the sections under an optical microscope (Olympus IX-71) in a blinded manner. The slides were subsequently scanned using a digital slide scanner (3DHISTECH, Budapest, Hungary). Transmission Electron Microscopy Myocardial tissue samples were prepared for transmission electron microscopy (TEM) to assess mitochondrial morphology. Briefly, tissues were initially fixed in 3% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.4) at 4 °C for 2 hours to preserve cellular ultrastructure. Following primary fixation, samples were post-fixed in 1% osmium tetroxide (OsO₄; Sigma-Aldrich) in 0.1 M phosphate buffer (pH 7.4) for 1 hour at room temperature to enhance contrast and stabilize membrane structures. After post-fixation, the tissues were dehydrated through a graded series of acetone solutions (30%, 50%, 70%, 90%, and 100%), 15 minutes for each step, and then infiltrated with a mixture of acetone and Epon 812 resin (SPI Supplies, West Chester, PA, USA). The tissues were subsequently embedded in pure Epon 812 resin, and polymerization was performed at 60 °C for 48 hours. Ultrathin sections (60–90 nm) were obtained using a diamond knife (Leica Microsystems, Wetzlar, Germany) on an ultramicrotome (Leica EM UC7). Sections were mounted on copper grids (200 mesh) and sequentially stained with 2% uranyl acetate (SPI Supplies) for 10 minutes and lead citrate (Electron Microscopy Sciences, Hatfield, PA, USA) for 5 minutes to increase electron density and contrast. The stained sections were examined under a transmission electron microscope (FEI Tecnai G2 Spirit Bio-TWIN, USA) at a magnification of 10,000× to capture high-resolution images of interfibrillar mitochondria. Mitochondrial morphological parameters, including size, shape, and cristae integrity, were quantified by an investigator blinded to the experimental groups. Western Blot Analysis Cardiac tissues or cells were lysed in a mixture of RIPA buffer (Beyotime, Shanghai, China) supplemented with PMSF (Beyotime, Shanghai, China) to extract total protein. Lysates were incubated on ice for 30 minutes with periodic vortexing, and the supernatants were collected for further analysis. Protein concentration was determined using a BCA Protein Assay Kit (Elabscience, Wuhan, China) according to the manufacturer’s instructions. Equal amounts of protein were separated by 10%–15% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA) in transfer buffer for 2 hours. Membranes were blocked with 5% skimmed milk or bovine serum albumin (BSA) for 2 hours at room temperature. After blocking, membranes were incubated overnight at 4 °C with the appropriate primary antibodies (see Additional File 1: Table S2 for details). After washing, the membranes were incubated with HRP-conjugated secondary antibodies (MedChemExpress, New Jersey, USA) for 2 hours at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific, Waltham, MA, USA) and detected by exposure to X-ray film or captured using a digital imaging system. Band intensities were analyzed and quantified using ImageJ software (version 1.51k; NIH, Bethesda, MD, USA). Relative protein expression levels were normalized to the housekeeping protein GAPDH. RNA Extraction and Real-Time Quantitative PCR Analysis (qRT-PCR) Total RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen, NY, USA) according to the manufacturer’s instructions. The RNA quality and concentration were assessed using a spectrophotometer (Thermo Fisher Scientific) by measuring the absorbance ratio at 260/280 nm. For cDNA synthesis, 1 µg of total RNA was reverse-transcribed using the PrimeScript RT Reagent Kit (Takara Biotechnology, Shiga, Japan) following the manufacturer’s protocol. The reverse transcription reaction was performed in a total volume of 20 µL at 37 °C for 15 minutes, followed by inactivation at 85 °C for 5 minutes. The resulting cDNA was used as a template for quantitative real-time PCR (qRT-PCR). qRT-PCR was performed on a LightCycler 480 system (Roche Diagnostics, Burgess Hill, UK) using SYBR Green Master Mix (Roche, Basel, Switzerland). Each reaction was carried out in a final volume of 20 µL containing 10 µL of SYBR Green Master Mix, 1 µL of cDNA, 0.5 µL each of forward and reverse primers, and 8 µL of nuclease-free water. The thermal cycling conditions were as follows: an initial denaturation at 95 °C for 10 minutes, followed by 40 cycles of denaturation at 95 °C for 15 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 30 seconds. Relative gene expression levels were calculated using the 2^−ΔΔCt method, with GAPDH used as the internal reference gene. The primers used for the target genes are listed below: ATF6:5'-CAGCAAAGACCATCATCATTCAG-3'and5'-TTAGTCACACACAGTTTTCCGTTC-3' GAPDH:5'-AGGTCGGTGTGAACGGATTTG-3'and5'-TGTAGACCATGTAGTTGAGGTCA-3'. Statistical Analysis All data are presented as the mean ± standard deviation (SD). For comparisons between two independent groups, an unpaired Student’s t-test was performed. For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was conducted to determine specific pairwise differences. A P-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). Results Characterization of ASP@PLGA-PEG Transmission electron microscopy (TEM) revealed that the nanoparticles were uniformly spherical, well-dispersed, and free from aggregation,As showed in Fig.1B. The nanoparticles exhibited an average particle size of 177.1 ± 12.4 nm(Figure 1 A), and the zeta potential was measured at -28.64 mV(Figure 1 C,D), indicating good colloidal stability and surface charge characteristics, which are favorable for nanoparticle stability and bioavailability in biological systems.We determined the encapsulation efficiency and drug loading capacity of ASP@PLGA-PEG nanoparticles using the phenol-sulfuric acid method, and get encapsulation efficiency of 80.5% and a drug loading capacity of 10.06%. The nanoparticles did not show any significant change in particle size and zeta by 30 days observation. The stability in deionized water is shown in Figure 1C and D. The results show that ASP@PLGA-PEG can be stabilized for more than 30 days. This is caused by the repulsive effect between the negatively charged nanoparticles.After 30 days, the nanoparticles gradually lose their stability, which may be due to the degradation of PLGA-PEG. We used HL-1 mouse cardiomyocytes as a model and co-cultured different concentrations of ASP@PLGA-PEG with HL-1 cells for up to 48 h. As shown in Figure 2, the assay did not reveal any significant toxic effect of ASP@PLGA-PEG on HL-1 cells. The protection of ASP@PLGA-PEG on heart function of MI/RI mouses We established the MI/RI model using the Langendorff isolated heart perfusion system. The perfusion protocols for each group are shown in Figure 2. During the isolated heart perfusion, cardiac function was continuously monitored by inserting a Langendorff balloon into the left ventricle and connecting it to a PowerLab data acquisition system. Indicators of isolated cardiac function are presented in Figures 3 and 4. Figure 3 illustrates the dynamic trends of cardiac function parameters throughout the Langendorff perfusion, while Figure 4 shows the statistical comparisons of these parameters at specific time points. During the stabilization period, no significant differences in cardiac parameters were observed among the groups (Additional File 1: Figure S1), confirming comparable baseline cardiac function.During the Langendorff isolated heart perfusion, changes in hemodynamic parameters are shown in Figures 3. LVDP, defined as the difference between left ventricular systolic and end-diastolic pressures, reflects myocardial contractile function; higher LVDP values indicate stronger contractility. The mean LVDP during equilibration was 81.6 ± 3.3 mmHg. Following 35 minutes of global ischemia, LVDP markedly declined, reaching 14.4 ± 3.3 mmHg at 10 minutes of reperfusion in the I/R group. ASP preconditioning (30 mg/kg) partially improved LVDP to 32.9 ± 4.0 mmHg at 60 minutes of reperfusion, whereas high-dose ASP@PLGA-PEG nanoparticles (20 mg/kg) further enhanced recovery, with LVDP reaching 54.7 ± 2.4 mmHg. Consistently, dp/dt max (an index of systolic function) and dp/dt min (an index of diastolic function) showed similar trends. At 60 minutes of reperfusion, dp/dt max in the I/R group was 1325.7 ± 90.6 mmHg/s, which increased to 2174.4 ± 93.0 mmHg/s in the NpsH group. Meanwhile, dp/dt min improved from –1038.6 ± 84.7 mmHg/s in the I/R group to –1769.4 ± 138.2 mmHg/s in the NpsH group. These findings indicate that high-dose ASP@PLGA-PEG nanoparticles markedly enhance the recovery of both systolic and diastolic function following MI/RI. The infarct size ratio is a key indicator for assessing the severity of myocardial infarction. TTC staining was used to distinguish viable myocardium (red) from infarcted tissue (pale), as shown in Figures 5A and 5B. In the I/R group, the infarct size reached 61%, whereas it was significantly reduced to 22.6% in the NpsH group, indicating a pronounced cardioprotective effect of ASP@PLGA-PEG during MI/RI. Histopathological changes in the ischemic myocardium were evaluated by H&E staining (Figure 5C). Myocardial tissues from the I/R group exhibited extensive necrosis, disorganized myocardial architecture, and marked inflammatory cell infiltration. In contrast, myocardial tissues from the ASP@PLGA-PEG–treated group displayed nearly normal histological features. Mitochondria, as key organelles in MI/RI, are closely involved in ferroptosis—an iron-dependent form of cell death driven by lipid peroxidation and oxidative stress. To further investigate mitochondrial morphology, TEM was performed (Figure 5D). In the I/R group, mitochondria showed marked swelling, disrupted cristae, and vacuolization. By contrast, mitochondria in the ASP@PLGA-PEG–treated group maintained more intact cristae and exhibited fewer signs of vacuolization. ASP@PLGA-PEG nanoparticles suppress oxidative stress in isolated hearts by activating ATF6 and inhibiting ER stress. In our previous studies, ASP was shown to activate ATF6 to suppress endoplasmic reticulum (ER) stress and thereby ameliorate MI/RI injury[17, 18]. In this study, this effect was further validated using the Langendorff isolated heart perfusion model. As shown in Figure 6B, ASP significantly upregulated ATF6 mRNA expression compared with the I/R group, and high-dose ASP@PLGA-PEG nanoparticles further enhanced ATF6 mRNA levels compared with ASP alone. Western blot analysis (Figures 6A, C–E) demonstrated that high-dose ASP@PLGA-PEG treatment markedly increased ATF6 protein expression relative to the I/R group. In addition, GRP78/BIP protein expression, an upstream ER stress sensor, was also significantly upregulated, whereas CHOP, a marker of irreversible terminal ER stress, was significantly downregulated. These results indicate that ASP@PLGA-PEG nanoparticles activate ATF6 expression, thereby enhancing the unfolded protein response and reducing excessive ER stress levels. As oxidative stress is a key regulatory factor involved in cardiomyocyte apoptosis and ferroptosis, we further evaluated oxidative stress status after MI/RI and the antioxidative effects of ASP@PLGA-PEG nanoparticles. Markers of oxidation (MDA) and antioxidation (SOD, CAT, and GSH) were measured (Figure 6F–I). Compared with the Sham group, the concentration of malondialdehyde (MDA), the end product of lipid peroxidation, was significantly increased in the I/R group. Treatment with Angelica sinensis polysaccharide and ASP@PLGA-PEG nanoparticles significantly reduced MDA levels. Moreover, the antioxidant system indicators (SOD, CAT, and GSH) were significantly improved after treatment with ASP and ASP@PLGA-PEG nanoparticles. ASP@PLGA-PEG nanoparticles inhibit ferroptosis in MI/RI by blocking NCOA4-mediated ferritinophagy. TEM analysis demonstrated that ASP@PLGA-PEG nanoparticles improved the mitochondrial morphology of mouse cardiomyocytes. Ferroptosis is an iron-dependent form of programmed cell death driven by lipid peroxidation, primarily caused by the inactivation of glutathione peroxidase 4 (GPX4). Fe²⁺ is a key driver of ferroptosis by triggering the Fenton reaction, which accelerates lipid peroxidation and cardiomyocyte death. Using a microcolorimetric assay, we quantified the intracellular Fe²⁺ content (Figure 7B). Compared with the Sham group, Fe²⁺ levels were significantly increased in the I/R group. Pretreatment with low-dose ASP@PLGA-PEG nanoparticles reduced Fe²⁺ levels, while high-dose ASP@PLGA-PEG nanoparticles significantly suppressed the Fe²⁺ elevation. Western blot analysis was performed to detect GPX4 and SLC7A11 protein levels in mouse myocardial tissues (Figure 7A, C, D). Compared with the Sham group, SLC7A11 and GPX4 protein expression levels were markedly decreased in the I/R group, with significant differences between groups. Treatment with ASP@PLGA-PEG nanoparticles effectively prevented the reduction of SLC7A11 and GPX4 expression, likely due to the strong antioxidative capacity of ASP. To further investigate whether the cardioprotective effects of ASP@PLGA-PEG nanoparticles were associated with inhibition of ferritinophagy, Western blot results showed that NCOA4 protein expression was significantly increased in the I/R group and was suppressed following ASP@PLGA-PEG treatment (Figure 7A, E). NCOA4-mediated ferritinophagy involves the binding of NCOA4 to FTH1 to form an NCOA4–FTH1 complex, which is then transported to autophagosomes for degradation. Therefore, we further examined the protein levels of FTH1 and LC3B by Western blotting (Figure 7A, F, G). Compared with the Sham group, FTH1 protein expression was significantly decreased in the I/R group, while LC3-I was extensively converted to LC3-II, indicating enhanced autophagosome formation. These results were consistent with our hypothesis that MI/RI activates NCOA4-mediated ferritinophagy, leading to FTH1 degradation. Pretreatment with ASP@PLGA-PEG nanoparticles reduced the conversion of LC3-I to LC3-II, indicating that the autophagic flux was suppressed. Protective effect of ASP@PLGA-PEG on oxygen and glucose deprivation and reoxygenation (OGD/R) injured HL-1 cells Cell viability after OGD/R injury in HL-1 cardiomyocytes was assessed using the CCK-8 assay, and LDH release was measured to evaluate the extent of cellular damage. To identify the optimal protective concentration of ASP@PLGA-PEG nanoparticles, various concentrations (10, 20, 50, 100, and 200 μg/mL) were tested. As shown in Figure 8A, OGD/R injury markedly reduced HL-1 cell viability to nearly half of the control level, while pretreatment with ASP@PLGA-PEG nanoparticles significantly restored cell viability in a concentration-dependent manner. Among the tested concentrations, 50 μg/mL provided the greatest protective effect, with cell viability improving close to baseline levels. Consistent with this, LDH activity in the culture supernatant showed that cells pretreated with 50 μg/mL ASP@PLGA-PEG released the lowest amount of LDH, indicating reduced membrane damage and better cellular integrity (Figure 8B). Based on these results, 50 μg/mL was selected as the working concentration for subsequent experiments. To compare the protective effects of ASP@PLGA-PEG nanoparticles with free ASP, HL-1 cardiomyocytes were pretreated with either ASP or ASP@PLGA-PEG nanoparticles at the same concentration. Following OGD/R injury, cells treated with ASP@PLGA-PEG showed higher viability than those treated with free ASP, indicating superior cytoprotection of the nanoparticle formulation (Figure 8C). Likewise, LDH activity was lower in the ASP@PLGA-PEG group than in the ASP group, further confirming the enhanced protective effect(Figure 8D). Regarding the mechanism, the expression level of ATF6 mRNA in HL-1 cardiomyocytes increased after OGD/R injury and was further upregulated in the ASP@PLGA-PEG group compared with the free ASP group (Figure 8F). Western blot analysis (Figure 8E, G–I) showed that ATF6 and GRP78 protein expression levels were also elevated in the ASP@PLGA-PEG group, while CHOP expression, which indicates sustained and irreversible ER stress, was markedly reduced. These findings suggest that ASP@PLGA-PEG nanoparticles activate ATF6 and enhance the unfolded protein response to alleviate excessive ER stress and protect cardiomyocytes from OGD/R-induced injury. ASP@PLGA-PEG nanoparticles alleviate oxidative stress CAT, SOD, MDA, and 4-hydroxy-2-nonenal (4-HNE) are commonly used biomarkers for evaluating oxidative stress in cells and tissues. After OGD/R injury, HL-1 cardiomyocytes showed a significant decrease in CAT activity and an increase in both MDA and SOD levels (Figure 9E, G, H). In addition, the fluorescence intensity of 4-HNE was markedly elevated following OGD/R treatment (Figure 9A, C). Pretreatment with ASP@PLGA-PEG or Ferrostatin-1 effectively reversed these changes, indicating a reduction in oxidative damage. ROS accumulation, a hallmark of both ERS and ferroptosis, was also significantly increased after OGD/R injury but was substantially reduced by the antioxidant effect of ASP@PLGA-PEG (Figure 9B, D). Furthermore, the level of reduced GSH was higher in the treatment groups compared with the OGD/R group(Figure 9F), further supporting the antioxidative effect of ASP@PLGA-PEG nanoparticles. ASP@PLGA-PEG nanoparticles protect mitochondrial function and inhibit ferroptosis by suppressing NCOA4-mediated ferritinophagy Mitochondria, as the center of cellular energy metabolism, play a crucial role in maintaining normal cardiomyocyte function[27]. Oxygen acts as the terminal electron acceptor in the mitochondrial electron transport chain (ETC) and is essential for efficient ATP production. Under hypoxic conditions, ETC activity is impaired due to oxygen deficiency, resulting in decreased ATP generation and disrupted cellular energy metabolism. Consequently, the mitochondrial membrane potential (Δψm) gradually declines, leading to membrane depolarization[28]. JC-1 staining further demonstrated that mitochondrial membrane potential decreased sharply after OGD/R injury, as indicated by a shift from red JC-1 aggregates to green monomers. Pretreatment with ASP@PLGA-PEG nanoparticles restored mitochondrial membrane potential, similar to the effect of Ferrostatin-1 (Figure 10E, F). To evaluate the function of the antioxidant defense system after OGD/R injury, the expression levels of GPX4 and SLC7A11 proteins were assessed. Western blot analysis revealed that OGD/R injury significantly reduced GPX4 and SLC7A11 expression, whereas pretreatment with ASP@PLGA-PEG nanoparticles markedly restored both protein levels (Figure 10A–C). Consistently, ferrous iron (Fe²⁺) content was significantly elevated in the OGD/R group, but was reduced by ASP@PLGA-PEG treatment (Figure 10D). To explore whether this protective effect was related to ferritinophagy, Western blot analysis showed that NCOA4 expression was markedly increased in the OGD/R group and significantly decreased after ASP@PLGA-PEG treatment (Figure 10G, H). Compared with the OGD/R group, FTH1 expression was restored in the ASP@PLGA-PEG group (Figure 10I). Furthermore, the conversion of LC3-I to LC3-II was reduced, indicating suppression of autophagosome formation and inhibition of ferritinophagy (Figure 10J). These in vitro findings suggest that ASP@PLGA-PEG nanoparticles protect HL-1 cardiomyocytes from OGD/R-induced injury by modulating the antioxidant system and inhibiting NCOA4-mediated ferritinophagy. Discussion In this study, we developed ASP@PLGA-PEG nanoparticles with a biphasic drug release profile. The drug adsorbed on the nanoparticle surface enables an initial rapid release, while a second, sustained release phase is provided by ASP encapsulated within the polymeric matrix. This dual-phase release protects the active ingredient and prolongs its therapeutic availability[29, 30]. In addition, PEG modification of PLGA allows the nanoparticles to evade clearance by the mononuclear phagocyte system (MPS), significantly extending their circulation time in vivo[31, 32]. We found no significant cytotoxicity in HL-1 cardiomyocytes exposed to ASP@PLGA-PEG for up to 48 hours, indicating their safety for injection. Compared with free ASP, the nanoparticles demonstrated higher bioavailability and lower toxicity due to their prolonged circulation and controlled release. Our previous studies confirmed the protective effects of ASP against myocardial MI/RI[17, 18], mainly due to its potent antioxidant properties. Here, we further validated this by testing ASP@PLGA-PEG in both an OGD/R cell injury model and a Langendorff isolated heart perfusion model. The results showed that ASP@PLGA-PEG significantly improved cardiomyocyte viability and reduced LDH release after OGD/R, outperforming free ASP. In the Langendorff model, ASP@PLGA-PEG treatment improved hemodynamic parameters and reduced infarct size, confirming that PLGA-PEG encapsulation enhances the protective effects of angelica polysaccharides. ATF6 plays an essential role in MI/RI as a key regulator of the ERS response[33]. Hypoxia disrupts energy metabolism and protein folding in cardiomyocytes, leading to the accumulation of misfolded proteins. When reperfusion restores oxygen supply, excessive ROS production further damages the ER and mitochondria[34]. Under such stress, ATF6 is activated, translocated to the Golgi apparatus, cleaved by proteases, and releases its active fragment, which enters the nucleus to initiate transcription of genes that help restore ER function, such as GRP78/BiP. GRP78/BiP promotes correct protein folding and helps clear misfolded proteins, alleviating ER stress[35-38]. Persistent ER stress can activate CHOP, which triggers apoptosis. In this study, ASP@PLGA-PEG treatment increased the expression of ATF6 and BiP while decreasing CHOP levels, suggesting that the nanoparticles activate the ATF6 pathway to strengthen the unfolded protein response (UPR) and help maintain protein homeostasis under ischemic stress[33, 39, 40]. Notably, ASP@PLGA-PEG also reduced ROS levels, indicating that these nanoparticles not only alleviate ER stress but also mitigate oxidative damage, further supporting cardiomyocyte recovery after MI/RI. These findings support the idea that activating ATF6 and enhancing the UPR is a key mechanism by which ASP@PLGA-PEG protect the myocardium. Ferroptosis, an iron-dependent form of programmed cell death, is characterized by lipid peroxidation triggered by iron accumulation[41]. ROS play a central role in ferroptosis by disturbing cellular redox balance and damaging membrane lipids, ultimately compromising membrane integrity[42]. ER stress can amplify this process by increasing cellular sensitivity to ROS-induced lipid damage. Glutathione (GSH) is an essential antioxidant that counteracts ROS, and its oxidized form maintains the activity of GPX4, an enzyme that detoxifies lipid peroxides and protects against ferroptosis[43]. System xc⁻, a cystine/glutamate antiporter, supports GSH synthesis by importing cystine, the precursor for cysteine. When System xc⁻ function is impaired, GSH levels drop, oxidative stress increases, and ferroptosis is promoted[44]. In our study, ASP@PLGA-PEG regulated antioxidant enzyme activity, including catalase (CAT), GSH, and SOD, thereby enhancing the cell’s antioxidant capacity. This effect appears linked to ATF6 activation, although whether ATF6 directly modulates System xc⁻ remains to be clarified in future research. Ferritin is the main intracellular iron storage protein that sequesters free iron to prevent oxidative damage[45]. Ferritin heavy chain 1 (FTH1) provides ferroxidase activity to convert ferrous iron (Fe²⁺) into ferric iron (Fe³⁺), safely storing it within the ferritin shell. During MI/RI, the protein NCOA4 promotes ferritin degradation by binding to ferritin and delivering it to autophagosomes for lysosomal degradation—a process known as ferritinophagy[46-48]. This process releases stored iron, raising free Fe²⁺ levels in the cytoplasm, where it can drive the Fenton reaction to produce hydroxyl radicals that worsen oxidative damage[8, 49, 50]. Our results showed increased NCOA4 expression, elevated Fe²⁺ levels, and enhanced LC3B-II conversion in the I/R group, indicating active ferritinophagy. Transmission electron microscopy confirmed the presence of autophagosomes in ischemic myocardium. ASP@PLGA-PEG treatment suppressed NCOA4 expression, reduced Fe²⁺ accumulation, and lowered LC3B-II levels, suggesting inhibition of ferritinophagy. By blocking this pathway, ASP@PLGA-PEG limit free iron release and associated lipid peroxidation, ultimately protecting cardiomyocytes against ferroptosis during MI/RI. Taken together, our findings indicate that ASP@PLGA-PEG nanoparticles exert cardioprotective effects through a multi-faceted mechanism involving ATF6-mediated alleviation of endoplasmic reticulum stress, enhancement of the cellular antioxidant system, and suppression of NCOA4-mediated ferritinophagy, which collectively help maintain mitochondrial function and reduce iron-dependent lipid peroxidation during ischemia-reperfusion injury. However, this study has some limitations. Although our results strongly suggest that ATF6 activation plays a central role in modulating oxidative stress and ferroptosis, the direct mechanistic link between ATF6 signaling and NCOA4-mediated ferritinophagy remains unclear and warrants further investigation. Future studies should explore whether ATF6 can directly influence ferritinophagy-related pathways or interacts with the regulation of iron metabolism under MI/RI conditions. Elucidating these connections will help refine the therapeutic potential of ASP@PLGA-PEG nanoparticles as an intervention for myocardial ischemia-reperfusion injury. Conclusion In conclusion, this study successfully developed ASP@PLGA-PEG nanoparticles and demonstrated their potent antioxidant and cardioprotective effects in vitro. We found that ASP@PLGA-PEG activate ATF6 to alleviate endoplasmic reticulum stress in myocardial ischemia-reperfusion injury, thereby reducing oxidative stress and lipid peroxidation. This cascade effect suppresses NCOA4-mediated ferritinophagy, ultimately preventing iron-dependent ferroptosis. These findings reveal new biological functions of ASP@PLGA-PEG and clarify the key mechanisms underlying their protective action against MI/RI. We propose that targeting excessive ferritinophagy could serve as a promising therapeutic strategy for MI/RI, and ASP@PLGA-PEG nanoparticles hold strong potential for future research and clinical translation in myocardial infarction therapy. Declarations Ethics approval and consent to participate The study protocol was reviewed and approved by the Animal Care Committee of The First Hospital of Lanzhou University. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council(ethics approval number: LDYYLL-2024-719). Consent for publication All authors have consented to the publication of the research fndings. Availability of data and materials Data for this paper can be provided by the corresponding author upon request competing interest The authors declare that there are no competing interests associated with the manuscript. Funding This work was supported by grants from the National Natural Science Foundation of China (NO.82060807), the Science and Technology Program of Gansu Province (NO.21JR1RA100), the Scientific Research Project of Health Industry of Gansu Province (NO.GSWSKY2020-64), the Science and Technology Planning Project of Lanzhou City (NO.2020-ZD-72).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Authors' contributions Ming Bai: Writing – original draft, Writing – review & editing, Methodology, Validation, Investigation. Cheng Chen: Writing – original draft, Writing – review & editing, Methodology, Formal analysis, Data curation, Conceptualization. Jing Zhao: Writing – review & editing, Supervision, Conceptualization, Software. Maomao Zhao: Writing – review & editing, Validation. Shuwen Hu: Writing – review & editing, Methodology, Investigation. Pei Wang: Investigation, Data curation. Peng Lei: Writing – review & editing, Supervision, Conceptualization. Yongxiang Wang: Validation, Methodology. Yu Peng: Investigation. Xiaowei Niu: Writing – original draft, Writing – review & editing, Funding acquisition, Conceptualization, Project administration. Zheng Zhang: Writing – original draft, Writing – review & editing, Funding acquisition, Investigation, Project administration. Acknowledgment None. References Mastoor Y, Murphy E, Roman B: Mechanisms of postischemic cardiac death and protection following myocardial injury. J Clin Invest 2025, 135 . Dabravolski SA, Kalmykov VA, Maksaeva AO, Rozhkova UV, Lapshina KO, Orekhov AN: Necroptosis in myocardial ischaemia-reperfusion injury: current update on mechanisms, therapeutic targets, and translational potential. Apoptosis 2025. Wang X, Liu R, Liu D: The Role of the MAPK Signaling Pathway in Cardiovascular Disease: Pathophysiological Mechanisms and Clinical Therapy. Int J Mol Sci 2025, 26 . 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7142720","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":494422124,"identity":"75e63b56-4d46-4012-a16b-0dc585576eb3","order_by":0,"name":"Ming Bai","email":"","orcid":"","institution":"Lanzhou University First Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Bai","suffix":""},{"id":494422125,"identity":"cc7098cd-a78d-43ea-ba04-3291eaf2f1a2","order_by":1,"name":"cheng chen","email":"","orcid":"","institution":"Lanzhou University First Clinical Medical College: Lanzhou University 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(A) \u003c/strong\u003eParticle size distribution of ASP@PLGA-PEG nanoparticles measured by dynamic light scattering (DLS); the average diameter was approximately 177.1 nm. \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative transmission electron microscopy (TEM) images showing the morphology of ASP@PLGA-PEG nanoparticles; scale bars = 200 nm. \u003cstrong\u003e(C) \u003c/strong\u003eStability profile of ASP@PLGA-PEG nanoparticles in terms of particle size and polydispersity index (PDI) over 30 days.\u003cstrong\u003e (D) \u003c/strong\u003eZeta potential of ASP@PLGA-PEG nanoparticles measured over 30 days to assess colloidal stability. \u003cstrong\u003e(E) \u003c/strong\u003eStandard calibration curve for ASP quantification. \u003cstrong\u003e(F) \u003c/strong\u003eCell viability of HL-1 cardiomyocytes treated with various concentrations (0, 10, 20, 50, 100, and 200 μg/mL) of ASP@PLGA-PEG nanoparticles for 6, 12, 24, and 48 hours, measured by CCK-8 assay. All data are presented as mean ± SD (n = X per group).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7142720/v1/e186ca08ac71110a17f2c3fd.jpeg"},{"id":88782446,"identity":"db921f8c-9084-47bd-80b0-7a412dcc5a0b","added_by":"auto","created_at":"2025-08-11 11:03:53","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":288541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of the perfusion protocol used in the Langendorff isolated heart experiment.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7142720/v1/161a3f1682d56d1c7b1f6866.jpeg"},{"id":88782454,"identity":"c9bc6f73-64e0-4996-9a70-ea27cb731209","added_by":"auto","created_at":"2025-08-11 11:03:53","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3468196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASP@PLGA-PEG nanoparticles improve cardiac function parameters in isolated hearts following MI/RI. (A)\u003c/strong\u003e Time-course changes in left ventricular developed pressure (LVDP) during the stabilization period, ischemia, and reperfusion. \u003cstrong\u003e(B) \u003c/strong\u003eTime-course changes in heart rate (HR). \u003cstrong\u003e(C) \u003c/strong\u003eTime-course changes in left ventricular end-diastolic pressure (LVEDP). \u003cstrong\u003e(D)\u003c/strong\u003e Time-course changes in left ventricular systolic pressure (LVSP). \u003cstrong\u003e(E) \u003c/strong\u003eTime-course changes in the maximum rate of pressure rise (dp/dt\u003csub\u003eMax\u003c/sub\u003e). \u003cstrong\u003e(F)\u003c/strong\u003e Time-course changes in the maximum rate of pressure decline (dp/dt\u003csub\u003eMin\u003c/sub\u003e). All data are expressed as mean ± SD (n = X per group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7142720/v1/7e95be490b1ccf139b89885e.jpeg"},{"id":88783571,"identity":"e60b4a30-be6f-46cf-93d4-84ecf1ee0236","added_by":"auto","created_at":"2025-08-11 11:11:53","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1222312,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStatistical comparison of cardiac function parameters at different reperfusion time points in isolated hearts subjected to MI/RI. (A)\u003c/strong\u003e Left ventricular developed pressure (LVDP) at 10, 30, and 60 minutes of reperfusion in each group.\u003cstrong\u003e (B) \u003c/strong\u003eHeart rate (HR) at 10, 30, and 60 minutes of reperfusion. \u003cstrong\u003e(C) \u003c/strong\u003eLeft ventricular end-diastolic pressure (LVEDP) at 10, 30, and 60 minutes of reperfusion. \u003cstrong\u003e(D) \u003c/strong\u003eLeft ventricular systolic pressure (LVSP) at 10, 30, and 60 minutes of reperfusion. \u003cstrong\u003e(E) \u003c/strong\u003eMaximum rate of left ventricular pressure rise (dp/dt\u003csub\u003eMax\u003c/sub\u003e). \u003cstrong\u003e(F) \u003c/strong\u003eMaximum rate of left ventricular pressure decline (dp/dtMin). Blue, brown, and grey bars represent values at reperfusion 10, 30, and 60 minutes, respectively. Data are presented as mean ± SD (n = X per group). Statistical significance: ns, not significant; ns, not significant， *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7142720/v1/5cd5dddef05adeae3b08a963.jpeg"},{"id":88783871,"identity":"6500e424-8cee-40cc-8db0-ae7a34e8d936","added_by":"auto","created_at":"2025-08-11 11:19:54","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":32130868,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASP@PLGA-PEG nanoparticles reduce infarct size, attenuate myocardial tissue damage, and preserve mitochondrial morphology in isolated hearts following MI/RI.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eRepresentative TTC-stained heart slices from each group, showing viable myocardium in red and infarcted tissue in pale white.\u003cstrong\u003e (B) \u003c/strong\u003eQuantification of infarct size ratio (%) in different groups. \u003cstrong\u003e(C)\u003c/strong\u003e Lactate dehydrogenase (LDH) activity as an indicator of myocardial injury. \u003cstrong\u003e(D)\u003c/strong\u003e Representative hematoxylin and eosin (H\u0026amp;E) staining of myocardial tissue sections from each group. The right panels show enlarged regions from the boxed areas, highlighting histopathological changes such as myocardial fiber integrity and inflammatory infiltration. \u003cstrong\u003e(E) \u003c/strong\u003eTransmission electron microscopy (TEM) images showing mitochondrial ultrastructure in myocardial tissues from each group. Red boxed areas indicate regions of interest; scale bars are shown in each image.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7142720/v1/c5c307a85be0f39e6a4e6a7d.jpeg"},{"id":88782452,"identity":"c4a0b511-898b-450f-a31c-88f161b9ffc2","added_by":"auto","created_at":"2025-08-11 11:03:53","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3052634,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRepresentative effects of ASP and ASP@PLGA-PEG nanoparticles on ATF6-mediated ER stress response and oxidative stress in isolated hearts.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eRepresentative Western blot bands showing protein expression levels of ATF6, GRP78/BIP, CHOP, and GAPDH in myocardial tissues from different groups. \u003cstrong\u003e(B)\u003c/strong\u003eRelative mRNA expression levels of ATF6 detected by qRT-PCR. \u003cstrong\u003e(C) \u003c/strong\u003eQuantitative analysis of ATF6 protein expression normalized to GAPDH. \u003cstrong\u003e(D)\u003c/strong\u003eQuantitative analysis of GRP78/BIP protein expression normalized to GAPDH. \u003cstrong\u003e(E)\u003c/strong\u003eQuantitative analysis of CHOP protein expression normalized to GAPDH. \u003cstrong\u003e(F)\u003c/strong\u003eMalondialdehyde (MDA) content as an indicator of lipid peroxidation. \u003cstrong\u003e(G)\u003c/strong\u003eSuperoxide dismutase (SOD) activity. \u003cstrong\u003e(H) \u003c/strong\u003eReduced glutathione (GSH) content. \u003cstrong\u003e(I) \u003c/strong\u003eCatalase (CAT) activity. All data are presented as mean ± SD (n = X per group).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7142720/v1/137b32087515311f67cc1bf3.jpeg"},{"id":88784736,"identity":"4d0ae2c4-fe90-43f2-ae16-3607cdbc3249","added_by":"auto","created_at":"2025-08-11 11:27:53","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":566966,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASP@PLGA-PEG nanoparticles inhibit ferroptosis by blocking NCOA4-mediated ferritinophagy in MI/RI. (A) \u003c/strong\u003eRepresentative Western blot images showing the protein expression of GPX4, SLC7A11, NCOA4, FTH1, LC3B, and GAPDH in myocardial tissues from each group. \u003cstrong\u003e(B)\u003c/strong\u003e Quantification of Fe²⁺ content in myocardial tissues. \u003cstrong\u003e(C) \u003c/strong\u003eDensitometric analysis of GPX4 protein expression normalized to GAPDH. \u003cstrong\u003e(D) \u003c/strong\u003eDensitometric analysis of SLC7A11 protein expression normalized to GAPDH. \u003cstrong\u003e(E) \u003c/strong\u003eDensitometric analysis of NCOA4 protein expression normalized to GAPDH. \u003cstrong\u003e(F) \u003c/strong\u003eDensitometric analysis of FTH1 protein expression normalized to GAPDH. \u003cstrong\u003e(G)\u003c/strong\u003e LC3-II/I ratio indicating autophagic activity related to ferritinophagy. All data are presented as mean ± SD (n = X per group).\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7142720/v1/a380a1ba8084de3567869769.jpeg"},{"id":88783573,"identity":"0ad60d45-54d6-478c-a844-d285849ea3c9","added_by":"auto","created_at":"2025-08-11 11:11:53","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3853839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtective effect of ASP@PLGA-PEG nanoparticles against OGD/R-induced injury in HL-1 cardiomyocytes and its regulation of the ATF6 pathway. (A) \u003c/strong\u003eCell viability of HL-1 cardiomyocytes treated with different concentrations of ASP@PLGA-PEG nanoparticles (10, 20, 50, 100, and 200 μg/mL) under OGD/R conditions, assessed by the CCK-8 assay. \u003cstrong\u003e(B) \u003c/strong\u003eLDH activity in the culture supernatant of HL-1 cardiomyocytes treated with various concentrations of ASP@PLGA-PEG nanoparticles under OGD/R conditions.\u003cstrong\u003e (C) \u003c/strong\u003eComparison of cell viability between the ASP and ASP@PLGA-PEG groups (both at 50 μg/mL) after OGD/R injury. \u003cstrong\u003e(D) \u003c/strong\u003eComparison of LDH activity between the ASP and ASP@PLGA-PEG groups after OGD/R injury.\u003cstrong\u003e (E) \u003c/strong\u003eRepresentative Western blot bands showing the protein expression levels of ATF6, GRP78, and CHOP in HL-1 cardiomyocytes in the Control, OGD/R, and ASP@PLGA-PEG groups.\u003cstrong\u003e (F)\u003c/strong\u003eRelative expression of ATF6 mRNA detected by qRT-PCR. \u003cstrong\u003e(G–I) \u003c/strong\u003eQuantification of ATF6\u003cstrong\u003e (G), \u003c/strong\u003eGRP78\u003cstrong\u003e (H), \u003c/strong\u003eand CHOP\u003cstrong\u003e (I) \u003c/strong\u003eprotein levels normalized to GAPDH.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7142720/v1/6159d2884c853462b3c2f9e4.jpeg"},{"id":88782459,"identity":"d76ec2f5-d55f-4ec3-9420-4c3682c55866","added_by":"auto","created_at":"2025-08-11 11:03:53","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":7208402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASP@PLGA-PEG nanoparticles attenuate oxidative stress in HL-1 cardiomyocytes subjected to OGD/R injury. (A) \u003c/strong\u003eRepresentative immunofluorescence images showing 4-HNE expression (green) and DAPI-stained nuclei (blue) in HL-1 cardiomyocytes under control, OGD/R, ASP@PLGA-PEG, and Ferrostatin-1 (Fer-1) treatment conditions. Scale bar = X μm. \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative DCFH-DA fluorescence images indicating intracellular ROS levels in HL-1 cardiomyocytes under the indicated treatments. Scale bar = X μm. \u003cstrong\u003e(C) \u003c/strong\u003eQuantification of relative 4-HNE protein expression. \u003cstrong\u003e(D) \u003c/strong\u003eQuantification of DCFH fluorescence intensity as a measure of intracellular ROS accumulation. \u003cstrong\u003e(E–H) \u003c/strong\u003eBiochemical analysis of oxidative stress markers: MDA content \u003cstrong\u003e(E), \u003c/strong\u003eGSH content \u003cstrong\u003e(F), \u003c/strong\u003eSOD activity\u003cstrong\u003e (G), \u003c/strong\u003eand CAT activity \u003cstrong\u003e(H) \u003c/strong\u003ein HL-1 cardiomyocytes.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7142720/v1/ebabab42cf38f70f1980a122.jpeg"},{"id":88782457,"identity":"1b0966f7-1680-412d-9216-b5c19065abb0","added_by":"auto","created_at":"2025-08-11 11:03:53","extension":"jpeg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":8431400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eASP@PLGA-PEG nanoparticles protect HL-1 cardiomyocytes against OGD/R-induced ferroptosis and mitochondrial dysfunction by regulating antioxidant proteins and inhibiting ferritinophagy. (A) \u003c/strong\u003eRepresentative Western blot bands showing the protein expression levels of GPX4 and SLC7A11 in HL-1 cardiomyocytes under control, OGD/R, ASP@PLGA-PEG, and Ferrostatin-1 (Fer-1) treatment conditions. \u003cstrong\u003e(B, C) \u003c/strong\u003eQuantification of GPX4 (B) and SLC7A11 (C) protein levels normalized to GAPDH. \u003cstrong\u003e(D) \u003c/strong\u003eIntracellular ferrous iron (Fe²⁺) content measured in HL-1 cardiomyocytes under the indicated conditions. \u003cstrong\u003e(E) \u003c/strong\u003eRepresentative JC-1 staining images showing mitochondrial membrane potential (Δψm) changes; red fluorescence indicates JC-1 aggregates (high Δψm), and green fluorescence indicates JC-1 monomers (low Δψm). \u003cstrong\u003e(F) \u003c/strong\u003eQuantification of the JC-1 polymer-to-monomer fluorescence ratio. \u003cstrong\u003e(G) \u003c/strong\u003eRepresentative Western blot bands showing the expression of NCOA4, FTH1, and LC3B proteins under the indicated treatments. \u003cstrong\u003e(H–J) \u003c/strong\u003eQuantification of NCOA4 (H), FTH1 (I), and the LC3-II/I ratio (J) normalized to GAPDH.\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7142720/v1/9ca7f2e20adb979d62c6982f.jpeg"},{"id":103765601,"identity":"00ea9f82-94f8-44c1-a25f-59f05dcfffd4","added_by":"auto","created_at":"2026-03-02 16:05:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":72894317,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7142720/v1/8f115879-ac85-4d0c-a3e7-03a439696e7e.pdf"},{"id":88783578,"identity":"5a3b4949-4ce2-456f-9a89-ab41fb95076f","added_by":"auto","created_at":"2025-08-11 11:11:53","extension":"doc","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3237888,"visible":true,"origin":"","legend":"","description":"","filename":"additionalfile.doc","url":"https://assets-eu.researchsquare.com/files/rs-7142720/v1/b3dbd0bb6d49d4acd59235f0.doc"}],"financialInterests":"","formattedTitle":"Angelica sinensis Polysaccharide nanoparticles can improve myocardial ischemia-reperfusion injury by inhibiting ferritinophagy via the ATF6/NCOA4 pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcute myocardial infarction(AMI), one of the leading causes of death worldwide, occurs due to the acute occlusion of coronary arteries, leading to prolonged myocardial ischemia and subsequent cardiomyocyte death[1]. Although restoring myocardial blood flow is crucial in treating AMI, timely reperfusion within a short therapeutic window is often difficult in clinical practice[2, 3]. Furthermore, reperfusion itself can trigger ischemia/reperfusion (I/R) injury, which exacerbates myocardial damage[4, 5]. In AMI, cardiomyocyte injury involves several cell death mechanisms, including necrosis, apoptosis, and ferroptosis. Ferroptosis, a non-apoptotic form of cell death, is induced by iron-dependent lipid peroxidation and has drawn increasing attention in AMI research. The mechanism of ferroptosis in myocardial ischemia-reperfusion injury is primarily driven by dysregulated iron metabolism and lipid peroxidation[6-8].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003edisturbed iron metabolism leads to iron overload in cardiomyocytes, which produces large amounts of reactive oxygen species (ROS) via the Fenton reaction, worsening oxidative stress[9]. During reperfusion, depletion of glutathione (GSH) and inactivation of glutathione peroxidase 4 (GPX4) impair the cell\u0026apos;s ability to clear lipid peroxides, thereby triggering lipid peroxidation and membrane disruption[10, 11]. Mitochondrial dysfunction also contributes to further ROS accumulation and damage[12, 13]. Overall, ferroptosis exacerbates ischemia-reperfusion injury by promoting lipid peroxidation, iron overload, and ROS accumulation, leading to more extensive cardiomyocyte death. Alongside ferroptosis, necrosis and apoptosis also play significant roles in AMI pathology.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAngelica sinensis\u003c/em\u003e Polysaccharide (ASP), extracted from \u003cem\u003eAngelica sinensis\u003c/em\u003e, are known for their wide range of biological activities, including antioxidant, anti-inflammatory, immune-regulating, and even anti-apoptotic effects[14-19]. These properties have been demonstrated to provide significant protection to the cardiovascular system.Ai et al[20]. demonstrated that ASP can mitigate cerebral ischemia/reperfusion injury in rabbits through antioxidant mechanisms. Similarly, Lei et al[21]. reported that ASP protects PC12 neuronal cells from H₂O₂-induced cytotoxicity by reducing apoptosis, lowering intracellular ROS levels, and enhancing mitochondrial membrane potential in cells treated with H₂O₂. Additionally, Niu et al[18]. found that ASP alleviates endoplasmic reticulum stress and oxidative stress by activating the ATF6(Activating Transcription Factor 6) pathway, thereby protecting H9c2 cells from H₂O₂-induced injury.Studies suggest that ASP can reduce oxidative stress and inhibit inflammation, two key drivers of myocardial damage during ischemia-reperfusion injury. Additionally, ASP has shown potential in improving endothelial function, enhancing microcirculation, and promoting angiogenesis[22, 23]. all of which contribute to improving cardiac function after AMI.However, despite its cardioprotective effects, ASP faces several challenges. Its short half-life, poor bioavailability, and lack of tissue-specific targeting limit its efficacy[24-26]. To overcome these limitations, We designed ASP@PLGA-PEG nanoparticles to evaluate the efficacy of ASP@PLGA-PEG on myocardial ischemia/reperfusion injury(MI/RI) and its mechanism in depth by experiment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePreparation and Characterization of ASP@PLGA-PEG\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eASP@PLGA-PEG nanoparticles were prepared using the nanoprecipitation method. Briefly, 80 mg of PLGA-PEG (Xarxbio, Xi\u0026apos;an, China) was dissolved in 2 mL of dichloromethane to prepare the oil phase, while 10 mg of Angelica sinensis polysaccharide (ASP; Solarbio, Beijing, China) was dissolved in 200 \u0026mu;L of deionized water as the aqueous phase. The oil phase was slowly added dropwise to the aqueous phase under continuous stirring. The mixture was then emulsified using a probe sonicator (SCIENTZ JY99-IIDN, Ningbo, China) under an ice bath to form a primary emulsion. Subsequently, 4 mL of 1.5% (w/v) polyvinyl alcohol (PVA) aqueous solution was added, and the resulting mixture was further emulsified by sonication to form a double emulsion. The emulsion was stirred at room temperature for 6 hours to allow complete evaporation of dichloromethane. The resulting ASP@PLGA-PEG nanoparticles were collected and transferred to a cuvette for characterization. The particle size and zeta potential were measured using a dynamic light scattering (DLS) instrument (Brookhaven Instruments 90Plus PALS, USA). The morphology of the nanoparticles was examined by transmission electron microscopy (TEM) (FEI Tecnai G2 Spirit Bio-TWIN, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture and Treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHL-1 cardiomyocytes were maintained in DMEM medium (Gibco, Waltham, ME, USA) supplemented with 10% fetal bovine serum (FBS, Gibco). The cells were incubated in a humidified atmosphere at 37\u0026nbsp;\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eTo establish an in vitro MI/MR model, we intervened on HL-1 cardiomyocytes for 4 hours using the hypoxia-glucose deprivation (OGD) method. Prior to the experiment, cells were cultured in a conventional incubator. After the cells grew to 80% confluence, the cells were transferred to a HeraCell VIOS 160i incubator (Thermo Fisher Scientific, Waltham, MA, USA) and hypoxia was simulated by releasing a gas mixture containing 1% O₂, 5% CO₂, and 94% N₂ (37\u0026deg;C). During hypoxia, HL-1 cardiomyocytes were cultured in serum-free, glucose-free DMEM medium to cut off the cellular fuel supply. In contrast, control cells were cultured under normoxic conditions with regular DMEM medium in a conventional incubator.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Viability Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability was assessed using the Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China). HL-1 cardiomyocytes were seeded in 96-well plates at a density of 1 \u0026times; 10⁴ cells per well and subjected to the simulated in vitro MI/RI model. Following the treatment, an appropriate volume of CCK-8 solution was added according to the kit instructions, and the cells were incubated for 2 hours. Absorbance (OD) at 450 nm was measured using a microplate reader (Tecan, Infinite M200 PRO, M\u0026auml;nnedorf, Switzerland).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of Reactive Oxygen Species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReactive oxygen species (ROS) levels were measured using a ROS assay kit (Beyotime, Shanghai, China). HL-1 cardiomyocytes were cultured in confocal dishes and treated according to the experimental model. After treatment, the cells were incubated with 1 mL of DCFH-DA reagent at 37\u0026deg;C for 20 minutes, following the kit instructions. After incubation, the cells were washed three times with PBS and then observed under an Olympus IX71 microscope. Fluorescence intensity was quantified using ImageJ software (version 1.51 k; Bethesda, MD, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of Mitochondrial Membrane Potential\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial membrane potential (MMP) was assessed using the JC-1 Mitochondrial Membrane Potential Assay Kit (Solarbio, Beijing, China). HL-1 cells were seeded into confocal dishes and treated according to the experimental model. Following treatment, the cells were incubated with 1 mL of JC-1 dye solution at 37\u0026deg;C for 20 minutes. After staining, the cells were observed under an Olympus microscope. The images were analyzed using ImageJ software, and MMP was quantified by calculating the red-to-green fluorescence ratio, which reflects the ratio of JC-1 aggregates (red) to monomers (green).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence Detection of 4-HNE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence quantification of 4-HNE was performed on HL-1 cardiomyocytes cultured in confocal petri dishes and processed according to the experimental protocol. After fixing the cells with 4% paraformaldehyde (PFA) for 10 min, permeabilizing the cells with 0.1% Triton X-100 for 10 min, and blocking with goat serum for 30 min, the cells were incubated with 4-HNE primary antibody (Thermo Fisher Scientific, MA5-27570) at 4\u0026deg;C overnight. On the following day, after washing three times with phosphate-buffered saline (PBS), HL-1 cardiomyocytes were incubated with FITC-coupled secondary antibody (Thermo Fisher Scientific) for 1 h under light protection. Then, the cells were washed again three times and restained with DAPI-containing medium to visualize the nuclei. Fluorescence imaging was performed using an Olympus IX71 microscope and quantitative analysis was performed using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals and Treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL/6 mice aged 6-8 weeks, weighing 22-25 g, were selected as experimental animals.The experimental animals were purchased from the Laboratory Animal Center of Lanzhou University (Lanzhou, Gansu, China).The mice were provided with unrestricted access to standard rodent chow and sterile water, and were housed in an environment with an ambient temperature of 22 \u0026plusmn; 2 ℃ and a 12-hour light/dark cycle. All procedures involving animals were approved by the Animal Care Committee of the First Hospital of Lanzhou University and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLangendorff Isolated Mouse Heart Perfusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice in the Sham and I/R groups were injected with saline via the tail vein for 3 consecutive days. Mice in the ASP group received angelica polysaccharides (20 mg/kg) via tail vein injection for 3 consecutive days. Mice in the Nps group were injected with varying doses of ASP@PLGA-PEG via the tail vein for 3 consecutive days. After the final drug injection, the mice were anesthetized with an intraperitoneal injection of 2% pentobarbital sodium. The mice were then euthanized by cervical dislocation. The hearts were rapidly excised, placed in pre-cooled Krebs-Henseleit (KH) solution(pH 7.4, gases 95% O2 and 5% CO2) in mM: 4.7 KCl, 118 NaCl, 25 NaHCO3, 1.8 CaCl2, 1.4 MgSO4, 1.2 KH2PO4, 11 D-glucose, and ligated with a 22G steel needle. Finally, the hearts were connected to the Langendorff apparatus, and retrograde perfusion was initiated.\u003c/p\u003e\n\u003cp\u003eThe hearts were perfused with KH solution. A latex balloon filled with normal saline and connected to a PowerLab system (AD Instruments, Sydney, NSW, Australia) was inserted into the left ventricle through the mitral valve. After placement, the balloon was inflated to maintain a left ventricular end-diastolic pressure (LVEDP) of 5\u0026ndash;10 mmHg. Hemodynamic parameters of the isolated hearts were continuously monitored and analyzed using the LabChart 8 data acquisition system (AD Instruments).\u003c/p\u003e\n\u003cp\u003eAt the end of the stabilization period, perfusion was completely stopped for 35 minutes to induce global ischemia, followed by 60 minutes of reperfusion. During reperfusion, cardiac function was continuously monitored using LabChart software, and hemodynamic data were recorded every 10 minutes for statistical analysis.\u003c/p\u003e\n\u003cp\u003eAccording to the experimental protocol, healthy mice were divided into 6 experimental groups:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1. Sham group (saline):\u003c/strong\u003e perfusion with KH solution for 125 min;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Ischemia/reperfusion group (saline):\u003c/strong\u003e stabilization period for 30 min, global ischemia for 35 min, and reperfusion for 60 min;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. ASP group (20 mg/kg):\u003c/strong\u003e stabilization period for 30 min, global ischemia for 35 min, reperfusion for 60 min;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. NpsL group (5 mg/kg):\u003c/strong\u003e stabilization period for 30 min, global ischemia for 35 min, reperfusion 60 min;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e5. NpsM group (10 mg/kg):\u003c/strong\u003e stabilization period for 30 min, global ischemia for 35 min, reperfusion 60min;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.NpsH group (20 mg/kg):\u003c/strong\u003e stabilization period for 30 min, global ischemia for 35 min, reperfusion 60 min;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical Assessment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe content of malondialdehyde (MDA), a lipid peroxidation product, and the activities of antioxidant markers including superoxide dismutase (SOD), catalase (CAT), lactate dehydrogenase (LDH), and reduced glutathione (GSH), as well as the concentration of Fe\u0026sup2;⁺ in cell lysates and tissue homogenates were determined using commercial assay kits (Elabscience, Wuhan, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHematoxylin and Eosin Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistopathological changes in myocardial tissue sections were examined by hematoxylin and eosin (H\u0026amp;E) staining. Following completion of the in vitro experiments on the Langendorff apparatus, the hearts were fixed in 4% paraformaldehyde for 24 hours. The tissue was then dehydrated through a graded ethanol series (70%, 90%, and 100%), cleared in xylene at 60\u0026ndash;70 \u0026deg;C for 60 minutes, and embedded in paraffin. Paraffin-embedded tissues were sectioned at a thickness of 4 \u0026mu;m and stained using a commercial H\u0026amp;E staining kit (Servicebio, Wuhan, China). Two authors independently evaluated the sections under an optical microscope (Olympus IX-71) in a blinded manner. The slides were subsequently scanned using a digital slide scanner (3DHISTECH, Budapest, Hungary).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMyocardial tissue samples were prepared for transmission electron microscopy (TEM) to assess mitochondrial morphology. Briefly, tissues were initially fixed in 3% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.4) at 4 \u0026deg;C for 2 hours to preserve cellular ultrastructure. Following primary fixation, samples were post-fixed in 1% osmium tetroxide (OsO₄; Sigma-Aldrich) in 0.1 M phosphate buffer (pH 7.4) for 1 hour at room temperature to enhance contrast and stabilize membrane structures. After post-fixation, the tissues were dehydrated through a graded series of acetone solutions (30%, 50%, 70%, 90%, and 100%), 15 minutes for each step, and then infiltrated with a mixture of acetone and Epon 812 resin (SPI Supplies, West Chester, PA, USA). The tissues were subsequently embedded in pure Epon 812 resin, and polymerization was performed at 60 \u0026deg;C for 48 hours. Ultrathin sections (60\u0026ndash;90 nm) were obtained using a diamond knife (Leica Microsystems, Wetzlar, Germany) on an ultramicrotome (Leica EM UC7). Sections were mounted on copper grids (200 mesh) and sequentially stained with 2% uranyl acetate (SPI Supplies) for 10 minutes and lead citrate (Electron Microscopy Sciences, Hatfield, PA, USA) for 5 minutes to increase electron density and contrast. The stained sections were examined under a transmission electron microscope (FEI Tecnai G2 Spirit Bio-TWIN, USA) at a magnification of 10,000\u0026times; to capture high-resolution images of interfibrillar mitochondria. Mitochondrial morphological parameters, including size, shape, and cristae integrity, were quantified by an investigator blinded to the experimental groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCardiac tissues or cells were lysed in a mixture of RIPA buffer (Beyotime, Shanghai, China) supplemented with PMSF (Beyotime, Shanghai, China) to extract total protein. Lysates were incubated on ice for 30 minutes with periodic vortexing, and the supernatants were collected for further analysis. Protein concentration was determined using a BCA Protein Assay Kit (Elabscience, Wuhan, China) according to the manufacturer\u0026rsquo;s instructions. Equal amounts of protein were separated by 10%\u0026ndash;15% SDS-PAGE and transferred onto PVDF membranes (Millipore, USA) in transfer buffer for 2 hours. Membranes were blocked with 5% skimmed milk or bovine serum albumin (BSA) for 2 hours at room temperature. After blocking, membranes were incubated overnight at 4 \u0026deg;C with the appropriate primary antibodies (see Additional File 1: Table S2 for details). After washing, the membranes were incubated with HRP-conjugated secondary antibodies (MedChemExpress, New Jersey, USA) for 2 hours at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Thermo Fisher Scientific, Waltham, MA, USA) and detected by exposure to X-ray film or captured using a digital imaging system. Band intensities were analyzed and quantified using ImageJ software (version 1.51k; NIH, Bethesda, MD, USA). Relative protein expression levels were normalized to the housekeeping protein GAPDH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Extraction and Real-Time Quantitative PCR Analysis (qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from tissues or cells using TRIzol reagent (Invitrogen, NY, USA) according to the manufacturer\u0026rsquo;s instructions. The RNA quality and concentration were assessed using a spectrophotometer (Thermo Fisher Scientific) by measuring the absorbance ratio at 260/280 nm. For cDNA synthesis, 1 \u0026micro;g of total RNA was reverse-transcribed using the PrimeScript RT Reagent Kit (Takara Biotechnology, Shiga, Japan) following the manufacturer\u0026rsquo;s protocol. The reverse transcription reaction was performed in a total volume of 20 \u0026micro;L at 37 \u0026deg;C for 15 minutes, followed by inactivation at 85 \u0026deg;C for 5 minutes. The resulting cDNA was used as a template for quantitative real-time PCR (qRT-PCR). qRT-PCR was performed on a LightCycler 480 system (Roche Diagnostics, Burgess Hill, UK) using SYBR Green Master Mix (Roche, Basel, Switzerland). Each reaction was carried out in a final volume of 20 \u0026micro;L containing 10 \u0026micro;L of SYBR Green Master Mix, 1 \u0026micro;L of cDNA, 0.5 \u0026micro;L each of forward and reverse primers, and 8 \u0026micro;L of nuclease-free water. The thermal cycling conditions were as follows: an initial denaturation at 95 \u0026deg;C for 10 minutes, followed by 40 cycles of denaturation at 95 \u0026deg;C for 15 seconds, annealing at 60 \u0026deg;C for 30 seconds, and extension at 72 \u0026deg;C for 30 seconds. Relative gene expression levels were calculated using the 2^\u0026minus;\u0026Delta;\u0026Delta;Ct method, with GAPDH used as the internal reference gene. The primers used for the target genes are listed below:\u003c/p\u003e\n\u003cp\u003eATF6:5\u0026apos;-CAGCAAAGACCATCATCATTCAG-3\u0026apos;and5\u0026apos;-TTAGTCACACACAGTTTTCCGTTC-3\u0026apos;\u003c/p\u003e\n\u003cp\u003eGAPDH:5\u0026apos;-AGGTCGGTGTGAACGGATTTG-3\u0026apos;and5\u0026apos;-TGTAGACCATGTAGTTGAGGTCA-3\u0026apos;.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are presented as the mean \u0026plusmn; standard deviation (SD). For comparisons between two independent groups, an unpaired Student\u0026rsquo;s t-test was performed. For comparisons among multiple groups, one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test was conducted to determine specific pairwise differences. A P-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eCharacterization of ASP@PLGA-PEG\u003c/h2\u003e\n\u003cp\u003eTransmission electron microscopy (TEM) revealed that the nanoparticles were uniformly spherical, well-dispersed, and free from aggregation,As showed in Fig.1B. The nanoparticles exhibited an average particle size of 177.1 \u0026plusmn; 12.4 nm(Figure 1 A), and the zeta potential was measured at -28.64 mV(Figure 1 C,D), indicating good colloidal stability and surface charge characteristics, which are favorable for nanoparticle stability and bioavailability in biological systems.We determined the encapsulation efficiency and drug loading capacity of ASP@PLGA-PEG nanoparticles using the phenol-sulfuric acid method, and get encapsulation efficiency of 80.5% and a drug loading capacity of 10.06%. The nanoparticles did not show any significant change in particle size and zeta by 30 days observation. The stability in deionized water is shown in Figure 1C and D. The results show that ASP@PLGA-PEG can be stabilized for more than 30 days. This is caused by the repulsive effect between the negatively charged nanoparticles.After 30 days, the nanoparticles gradually lose their stability, which may be due to the degradation of PLGA-PEG. We used HL-1 mouse cardiomyocytes as a model and co-cultured different concentrations of ASP@PLGA-PEG with HL-1 cells for up to 48 h. As shown in Figure 2, the assay did not reveal any significant toxic effect of ASP@PLGA-PEG on HL-1 cells.\u003c/p\u003e\n\u003ch2\u003eThe protection of ASP@PLGA-PEG on heart function of MI/RI mouses\u003c/h2\u003e\n\u003cp\u003eWe established the MI/RI model using the Langendorff isolated heart perfusion system. The perfusion protocols for each group are shown in Figure 2. During the isolated heart perfusion, cardiac function was continuously monitored by inserting a Langendorff balloon into the left ventricle and connecting it to a PowerLab data acquisition system.\u003c/p\u003e\n\u003cp\u003eIndicators of isolated cardiac function are presented in Figures 3 and 4. Figure 3 illustrates the dynamic trends of cardiac function parameters throughout the Langendorff perfusion, while Figure 4 shows the statistical comparisons of these parameters at specific time points. During the stabilization period, no significant differences in cardiac parameters were observed among the groups (Additional File 1: Figure S1), confirming comparable baseline cardiac function.During the Langendorff isolated heart perfusion, changes in hemodynamic parameters are shown in Figures 3. LVDP, defined as the difference between left ventricular systolic and end-diastolic pressures, reflects myocardial contractile function; higher LVDP values indicate stronger contractility. The mean LVDP during equilibration was 81.6 \u0026plusmn; 3.3 mmHg. Following 35 minutes of global ischemia, LVDP markedly declined, reaching 14.4 \u0026plusmn; 3.3 mmHg at 10 minutes of reperfusion in the I/R group. ASP preconditioning (30 mg/kg) partially improved LVDP to 32.9 \u0026plusmn; 4.0 mmHg at 60 minutes of reperfusion, whereas high-dose ASP@PLGA-PEG nanoparticles (20 mg/kg) further enhanced recovery, with LVDP reaching 54.7 \u0026plusmn; 2.4 mmHg. Consistently, dp/dt\u003csub\u003emax\u003c/sub\u003e (an index of systolic function) and dp/dt\u003csub\u003emin\u003c/sub\u003e (an index of diastolic function) showed similar trends. At 60 minutes of reperfusion, dp/dt\u003csub\u003emax\u003c/sub\u003e in the I/R group was 1325.7 \u0026plusmn; 90.6 mmHg/s, which increased to 2174.4 \u0026plusmn; 93.0 mmHg/s in the NpsH group. Meanwhile, dp/dt\u003csub\u003emin\u0026nbsp;\u003c/sub\u003eimproved from \u0026ndash;1038.6 \u0026plusmn; 84.7 mmHg/s in the I/R group to \u0026ndash;1769.4 \u0026plusmn; 138.2 mmHg/s in the NpsH group. These findings indicate that high-dose ASP@PLGA-PEG nanoparticles markedly enhance the recovery of both systolic and diastolic function following MI/RI.\u003c/p\u003e\n\u003cp\u003eThe infarct size ratio is a key indicator for assessing the severity of myocardial infarction. TTC staining was used to distinguish viable myocardium (red) from infarcted tissue (pale), as shown in Figures 5A and 5B. In the I/R group, the infarct size reached 61%, whereas it was significantly reduced to 22.6% in the NpsH group, indicating a pronounced cardioprotective effect of ASP@PLGA-PEG during MI/RI. Histopathological changes in the ischemic myocardium were evaluated by H\u0026amp;E staining (Figure 5C). Myocardial tissues from the I/R group exhibited extensive necrosis, disorganized myocardial architecture, and marked inflammatory cell infiltration. In contrast, myocardial tissues from the ASP@PLGA-PEG\u0026ndash;treated group displayed nearly normal histological features. Mitochondria, as key organelles in MI/RI, are closely involved in ferroptosis\u0026mdash;an iron-dependent form of cell death driven by lipid peroxidation and oxidative stress. To further investigate mitochondrial morphology, TEM was performed (Figure 5D). In the I/R group, mitochondria showed marked swelling, disrupted cristae, and vacuolization. By contrast, mitochondria in the ASP@PLGA-PEG\u0026ndash;treated group maintained more intact cristae and exhibited fewer signs of vacuolization.\u003c/p\u003e\n\u003ch2\u003eASP@PLGA-PEG nanoparticles suppress oxidative stress in isolated hearts by activating ATF6 and inhibiting ER stress.\u003c/h2\u003e\n\u003cp\u003eIn our previous studies, ASP was shown to activate ATF6 to suppress endoplasmic reticulum (ER) stress and thereby ameliorate MI/RI injury[17, 18]. In this study, this effect was further validated using the Langendorff isolated heart perfusion model. As shown in Figure 6B, ASP significantly upregulated ATF6 mRNA expression compared with the I/R group, and high-dose ASP@PLGA-PEG nanoparticles further enhanced ATF6 mRNA levels compared with ASP alone. Western blot analysis (Figures 6A, C\u0026ndash;E) demonstrated that high-dose ASP@PLGA-PEG treatment markedly increased ATF6 protein expression relative to the I/R group. In addition, GRP78/BIP protein expression, an upstream ER stress sensor, was also significantly upregulated, whereas CHOP, a marker of irreversible terminal ER stress, was significantly downregulated. These results indicate that ASP@PLGA-PEG nanoparticles activate ATF6 expression, thereby enhancing the unfolded protein response and reducing excessive ER stress levels.\u003c/p\u003e\n\u003cp\u003eAs oxidative stress is a key regulatory factor involved in cardiomyocyte apoptosis and ferroptosis, we further evaluated oxidative stress status after MI/RI and the antioxidative effects of ASP@PLGA-PEG nanoparticles. Markers of oxidation (MDA) and antioxidation (SOD, CAT, and GSH) were measured (Figure 6F\u0026ndash;I). Compared with the Sham group, the concentration of malondialdehyde (MDA), the end product of lipid peroxidation, was significantly increased in the I/R group. Treatment with Angelica sinensis polysaccharide and ASP@PLGA-PEG nanoparticles significantly reduced MDA levels. Moreover, the antioxidant system indicators (SOD, CAT, and GSH) were significantly improved after treatment with ASP and ASP@PLGA-PEG nanoparticles.\u003c/p\u003e\n\u003ch2\u003eASP@PLGA-PEG nanoparticles inhibit ferroptosis in MI/RI by blocking NCOA4-mediated ferritinophagy.\u003c/h2\u003e\n\u003cp\u003eTEM analysis demonstrated that ASP@PLGA-PEG nanoparticles improved the mitochondrial morphology of mouse cardiomyocytes. Ferroptosis is an iron-dependent form of programmed cell death driven by lipid peroxidation, primarily caused by the inactivation of glutathione peroxidase 4 (GPX4). Fe\u0026sup2;⁺ is a key driver of ferroptosis by triggering the Fenton reaction, which accelerates lipid peroxidation and cardiomyocyte death. Using a microcolorimetric assay, we quantified the intracellular Fe\u0026sup2;⁺ content (Figure 7B). Compared with the Sham group, Fe\u0026sup2;⁺ levels were significantly increased in the I/R group. Pretreatment with low-dose ASP@PLGA-PEG nanoparticles reduced Fe\u0026sup2;⁺ levels, while high-dose ASP@PLGA-PEG nanoparticles significantly suppressed the Fe\u0026sup2;⁺ elevation. Western blot analysis was performed to detect GPX4 and SLC7A11 protein levels in mouse myocardial tissues (Figure 7A, C, D). Compared with the Sham group, SLC7A11 and GPX4 protein expression levels were markedly decreased in the I/R group, with significant differences between groups. Treatment with ASP@PLGA-PEG nanoparticles effectively prevented the reduction of SLC7A11 and GPX4 expression, likely due to the strong antioxidative capacity of ASP.\u003c/p\u003e\n\u003cp\u003eTo further investigate whether the cardioprotective effects of ASP@PLGA-PEG nanoparticles were associated with inhibition of ferritinophagy, Western blot results showed that NCOA4 protein expression was significantly increased in the I/R group and was suppressed following ASP@PLGA-PEG treatment (Figure 7A, E). NCOA4-mediated ferritinophagy involves the binding of NCOA4 to FTH1 to form an NCOA4\u0026ndash;FTH1 complex, which is then transported to autophagosomes for degradation. Therefore, we further examined the protein levels of FTH1 and LC3B by Western blotting (Figure 7A, F, G). Compared with the Sham group, FTH1 protein expression was significantly decreased in the I/R group, while LC3-I was extensively converted to LC3-II, indicating enhanced autophagosome formation. These results were consistent with our hypothesis that MI/RI activates NCOA4-mediated ferritinophagy, leading to FTH1 degradation. Pretreatment with ASP@PLGA-PEG nanoparticles reduced the conversion of LC3-I to LC3-II, indicating that the autophagic flux was suppressed.\u003c/p\u003e\n\u003ch2\u003eProtective effect of ASP@PLGA-PEG on oxygen and glucose deprivation and reoxygenation \u0026nbsp;(OGD/R) injured HL-1 cells\u003c/h2\u003e\n\u003cp\u003eCell viability after OGD/R injury in HL-1 cardiomyocytes was assessed using the CCK-8 assay, and LDH release was measured to evaluate the extent of cellular damage. To identify the optimal protective concentration of ASP@PLGA-PEG nanoparticles, various concentrations (10, 20, 50, 100, and 200 \u0026mu;g/mL) were tested. As shown in Figure 8A, OGD/R injury markedly reduced HL-1 cell viability to nearly half of the control level, while pretreatment with ASP@PLGA-PEG nanoparticles significantly restored cell viability in a concentration-dependent manner. Among the tested concentrations, 50 \u0026mu;g/mL provided the greatest protective effect, with cell viability improving close to baseline levels. Consistent with this, LDH activity in the culture supernatant showed that cells pretreated with 50 \u0026mu;g/mL ASP@PLGA-PEG released the lowest amount of LDH, indicating reduced membrane damage and better cellular integrity (Figure 8B). Based on these results, 50 \u0026mu;g/mL was selected as the working concentration for subsequent experiments.\u003c/p\u003e\n\u003cp\u003eTo compare the protective effects of ASP@PLGA-PEG nanoparticles with free ASP, HL-1 cardiomyocytes were pretreated with either ASP or ASP@PLGA-PEG nanoparticles at the same concentration. Following OGD/R injury, cells treated with ASP@PLGA-PEG showed higher viability than those treated with free ASP, indicating superior cytoprotection of the nanoparticle formulation (Figure 8C). Likewise, LDH activity was lower in the ASP@PLGA-PEG group than in the ASP group, further confirming the enhanced protective effect(Figure 8D).\u003c/p\u003e\n\u003cp\u003eRegarding the mechanism, the expression level of ATF6 mRNA in HL-1 cardiomyocytes increased after OGD/R injury and was further upregulated in the ASP@PLGA-PEG group compared with the free ASP group (Figure 8F). Western blot analysis (Figure 8E, G\u0026ndash;I) showed that ATF6 and GRP78 protein expression levels were also elevated in the ASP@PLGA-PEG group, while CHOP expression, which indicates sustained and irreversible ER stress, was markedly reduced. These findings suggest that ASP@PLGA-PEG nanoparticles activate ATF6 and enhance the unfolded protein response to alleviate excessive ER stress and protect cardiomyocytes from OGD/R-induced injury.\u003c/p\u003e\n\u003ch2\u003eASP@PLGA-PEG nanoparticles alleviate oxidative stress\u003c/h2\u003e\n\u003cp\u003eCAT, SOD, MDA, and 4-hydroxy-2-nonenal (4-HNE) are commonly used biomarkers for evaluating oxidative stress in cells and tissues. After OGD/R injury, HL-1 cardiomyocytes showed a significant decrease in CAT activity and an increase in both MDA and SOD levels (Figure 9E, G, H). In addition, the fluorescence intensity of 4-HNE was markedly elevated following OGD/R treatment (Figure 9A, C). Pretreatment with ASP@PLGA-PEG or Ferrostatin-1 effectively reversed these changes, indicating a reduction in oxidative damage. ROS accumulation, a hallmark of both ERS and ferroptosis, was also significantly increased after OGD/R injury but was substantially reduced by the antioxidant effect of ASP@PLGA-PEG (Figure 9B, D). Furthermore, the level of reduced GSH was higher in the treatment groups compared with the OGD/R group(Figure 9F), further supporting the antioxidative effect of ASP@PLGA-PEG nanoparticles.\u003c/p\u003e\n\u003ch2\u003eASP@PLGA-PEG nanoparticles protect mitochondrial function and inhibit ferroptosis by suppressing NCOA4-mediated ferritinophagy\u003c/h2\u003e\n\u003cp\u003eMitochondria, as the center of cellular energy metabolism, play a crucial role in maintaining normal cardiomyocyte function[27]. Oxygen acts as the terminal electron acceptor in the mitochondrial electron transport chain (ETC) and is essential for efficient ATP production. Under hypoxic conditions, ETC activity is impaired due to oxygen deficiency, resulting in decreased ATP generation and disrupted cellular energy metabolism. Consequently, the mitochondrial membrane potential (\u0026Delta;\u0026psi;m) gradually declines, leading to membrane depolarization[28]. JC-1 staining further demonstrated that mitochondrial membrane potential decreased sharply after OGD/R injury, as indicated by a shift from red JC-1 aggregates to green monomers. Pretreatment with ASP@PLGA-PEG nanoparticles restored mitochondrial membrane potential, similar to the effect of Ferrostatin-1 (Figure 10E, F).\u003c/p\u003e\n\u003cp\u003eTo evaluate the function of the antioxidant defense system after OGD/R injury, the expression levels of GPX4 and SLC7A11 proteins were assessed. Western blot analysis revealed that OGD/R injury significantly reduced GPX4 and SLC7A11 expression, whereas pretreatment with ASP@PLGA-PEG nanoparticles markedly restored both protein levels (Figure 10A\u0026ndash;C). Consistently, ferrous iron (Fe\u0026sup2;⁺) content was significantly elevated in the OGD/R group, but was reduced by ASP@PLGA-PEG treatment (Figure 10D).\u003c/p\u003e\n\u003cp\u003eTo explore whether this protective effect was related to ferritinophagy, Western blot analysis showed that NCOA4 expression was markedly increased in the OGD/R group and significantly decreased after ASP@PLGA-PEG treatment (Figure 10G, H). Compared with the OGD/R group, FTH1 expression was restored in the ASP@PLGA-PEG group (Figure 10I). Furthermore, the conversion of LC3-I to LC3-II was reduced, indicating suppression of autophagosome formation and inhibition of ferritinophagy (Figure 10J).\u003c/p\u003e\n\u003cp\u003eThese in vitro findings suggest that ASP@PLGA-PEG nanoparticles protect HL-1 cardiomyocytes from OGD/R-induced injury by modulating the antioxidant system and inhibiting NCOA4-mediated ferritinophagy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we developed ASP@PLGA-PEG nanoparticles with a biphasic drug release profile. The drug adsorbed on the nanoparticle surface enables an initial rapid release, while a second, sustained release phase is provided by ASP encapsulated within the polymeric matrix. This dual-phase release protects the active ingredient and prolongs its therapeutic availability[29, 30]. In addition, PEG modification of PLGA allows the nanoparticles to evade clearance by the mononuclear phagocyte system (MPS), significantly extending their circulation time in vivo[31, 32]. We found no significant cytotoxicity in HL-1 cardiomyocytes exposed to ASP@PLGA-PEG for up to 48 hours, indicating their safety for injection. Compared with free ASP, the nanoparticles demonstrated higher bioavailability and lower toxicity due to their prolonged circulation and controlled release. Our previous studies confirmed the protective effects of ASP against myocardial MI/RI[17, 18], mainly due to its potent antioxidant properties. Here, we further validated this by testing ASP@PLGA-PEG in both an OGD/R cell injury model and a Langendorff isolated heart perfusion model. The results showed that ASP@PLGA-PEG significantly improved cardiomyocyte viability and reduced LDH release after OGD/R, outperforming free ASP. In the Langendorff model, ASP@PLGA-PEG treatment improved hemodynamic parameters and reduced infarct size, confirming that PLGA-PEG encapsulation enhances the protective effects of angelica polysaccharides.\u003c/p\u003e\n\u003cp\u003eATF6 plays an essential role in MI/RI as a key regulator of the ERS response[33]. Hypoxia disrupts energy metabolism and protein folding in cardiomyocytes, leading to the accumulation of misfolded proteins. When reperfusion restores oxygen supply, excessive ROS production further damages the ER and mitochondria[34]. Under such stress, ATF6 is activated, translocated to the Golgi apparatus, cleaved by proteases, and releases its active fragment, which enters the nucleus to initiate transcription of genes that help restore ER function, such as GRP78/BiP. GRP78/BiP promotes correct protein folding and helps clear misfolded proteins, alleviating ER stress[35-38]. Persistent ER stress can activate CHOP, which triggers apoptosis. In this study, ASP@PLGA-PEG treatment increased the expression of ATF6 and BiP while decreasing CHOP levels, suggesting that the nanoparticles activate the ATF6 pathway to strengthen the unfolded protein response (UPR) and help maintain protein homeostasis under ischemic stress[33, 39, 40]. Notably, ASP@PLGA-PEG also reduced ROS levels, indicating that these nanoparticles not only alleviate ER stress but also mitigate oxidative damage, further supporting cardiomyocyte recovery after MI/RI. These findings support the idea that activating ATF6 and enhancing the UPR is a key mechanism by which ASP@PLGA-PEG protect the myocardium.\u003c/p\u003e\n\u003cp\u003eFerroptosis, an iron-dependent form of programmed cell death, is characterized by lipid peroxidation triggered by iron accumulation[41]. ROS play a central role in ferroptosis by disturbing cellular redox balance and damaging membrane lipids, ultimately compromising membrane integrity[42]. ER stress can amplify this process by increasing cellular sensitivity to ROS-induced lipid damage. Glutathione (GSH) is an essential antioxidant that counteracts ROS, and its oxidized form maintains the activity of GPX4, an enzyme that detoxifies lipid peroxides and protects against ferroptosis[43]. System xc⁻, a cystine/glutamate antiporter, supports GSH synthesis by importing cystine, the precursor for cysteine. When System xc⁻ function is impaired, GSH levels drop, oxidative stress increases, and ferroptosis is promoted[44]. In our study, ASP@PLGA-PEG regulated antioxidant enzyme activity, including catalase (CAT), GSH, and SOD, thereby enhancing the cell\u0026rsquo;s antioxidant capacity. This effect appears linked to ATF6 activation, although whether ATF6 directly modulates System xc⁻ remains to be clarified in future research.\u003c/p\u003e\n\u003cp\u003eFerritin is the main intracellular iron storage protein that sequesters free iron to prevent oxidative damage[45]. Ferritin heavy chain 1 (FTH1) provides ferroxidase activity to convert ferrous iron (Fe\u0026sup2;⁺) into ferric iron (Fe\u0026sup3;⁺), safely storing it within the ferritin shell. During MI/RI, the protein NCOA4 promotes ferritin degradation by binding to ferritin and delivering it to autophagosomes for lysosomal degradation\u0026mdash;a process known as ferritinophagy[46-48]. This process releases stored iron, raising free Fe\u0026sup2;⁺ levels in the cytoplasm, where it can drive the Fenton reaction to produce hydroxyl radicals that worsen oxidative damage[8, 49, 50]. Our results showed increased NCOA4 expression, elevated Fe\u0026sup2;⁺ levels, and enhanced LC3B-II conversion in the I/R group, indicating active ferritinophagy. Transmission electron microscopy confirmed the presence of autophagosomes in ischemic myocardium. ASP@PLGA-PEG treatment suppressed NCOA4 expression, reduced Fe\u0026sup2;⁺ accumulation, and lowered LC3B-II levels, suggesting inhibition of ferritinophagy. By blocking this pathway, ASP@PLGA-PEG limit free iron release and associated lipid peroxidation, ultimately protecting cardiomyocytes against ferroptosis during MI/RI.\u003c/p\u003e\n\u003cp\u003eTaken together, our findings indicate that ASP@PLGA-PEG nanoparticles exert cardioprotective effects through a multi-faceted mechanism involving ATF6-mediated alleviation of endoplasmic reticulum stress, enhancement of the cellular antioxidant system, and suppression of NCOA4-mediated ferritinophagy, which collectively help maintain mitochondrial function and reduce iron-dependent lipid peroxidation during ischemia-reperfusion injury.\u003c/p\u003e\n\u003cp\u003eHowever, this study has some limitations. Although our results strongly suggest that ATF6 activation plays a central role in modulating oxidative stress and ferroptosis, the direct mechanistic link between ATF6 signaling and NCOA4-mediated ferritinophagy remains unclear and warrants further investigation. Future studies should explore whether ATF6 can directly influence ferritinophagy-related pathways or interacts with the regulation of iron metabolism under MI/RI conditions. Elucidating these connections will help refine the therapeutic potential of ASP@PLGA-PEG nanoparticles as an intervention for myocardial ischemia-reperfusion injury.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study successfully developed ASP@PLGA-PEG nanoparticles and demonstrated their potent antioxidant and cardioprotective effects in vitro. We found that ASP@PLGA-PEG activate ATF6 to alleviate endoplasmic reticulum stress in myocardial ischemia-reperfusion injury, thereby reducing oxidative stress and lipid peroxidation. This cascade effect suppresses NCOA4-mediated ferritinophagy, ultimately preventing iron-dependent ferroptosis. These findings reveal new biological functions of ASP@PLGA-PEG and clarify the key mechanisms underlying their protective action against MI/RI. We propose that targeting excessive ferritinophagy could serve as a promising therapeutic strategy for MI/RI, and ASP@PLGA-PEG nanoparticles hold strong potential for future research and clinical translation in myocardial infarction therapy.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent\u0026nbsp;to participate\u003c/h2\u003e\n\u003cp\u003eThe study protocol was reviewed and approved by the Animal Care Committee of The First Hospital of Lanzhou University. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council(ethics approval number: LDYYLL-2024-719).\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eAll authors have consented to the publication of the research fndings.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eData for this paper can be provided by the corresponding author upon request\u003c/p\u003e\n\u003ch2\u003ecompeting interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that there are no competing interests associated with the manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (NO.82060807), the Science and Technology Program of Gansu Province (NO.21JR1RA100), the Scientific Research Project of Health Industry of Gansu Province (NO.GSWSKY2020-64), the Science and Technology Planning Project of Lanzhou City (NO.2020-ZD-72).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026apos; contributions\u003c/h2\u003e\n\u003cp\u003eMing Bai: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Methodology, Validation, Investigation. Cheng Chen: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Methodology, Formal analysis, Data curation, Conceptualization. Jing Zhao: Writing \u0026ndash; review \u0026amp; editing, Supervision, Conceptualization, Software. Maomao Zhao: Writing \u0026ndash; review \u0026amp; editing, Validation. Shuwen Hu: Writing \u0026ndash; review \u0026amp; editing, Methodology, Investigation. Pei Wang: Investigation, Data curation. Peng Lei: Writing \u0026ndash; review \u0026amp; editing, Supervision, Conceptualization. Yongxiang Wang: Validation, Methodology. Yu Peng: Investigation. Xiaowei Niu: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Funding acquisition, Conceptualization, Project administration. Zheng Zhang: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Funding acquisition, Investigation, Project administration.\u003c/p\u003e\n\u003ch2\u003eAcknowledgment\u003c/h2\u003e\n\u003cp\u003eNone.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMastoor Y, Murphy E, Roman B: \u003cstrong\u003eMechanisms of postischemic cardiac death and protection following myocardial injury.\u003c/strong\u003e \u003cem\u003eJ Clin Invest\u003c/em\u003e 2025, \u003cstrong\u003e135\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eDabravolski SA, Kalmykov VA, Maksaeva AO, Rozhkova UV, Lapshina KO, Orekhov AN: \u003cstrong\u003eNecroptosis in myocardial ischaemia-reperfusion injury: current update on mechanisms, therapeutic targets, and translational potential.\u003c/strong\u003e \u003cem\u003eApoptosis\u003c/em\u003e 2025.\u003c/li\u003e\n\u003cli\u003eWang X, Liu R, Liu D: \u003cstrong\u003eThe Role of the MAPK Signaling Pathway in 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\u003cstrong\u003e13:\u003c/strong\u003e1043344.\u003c/li\u003e\n\u003cli\u003eLee J, Roh J: \u003cstrong\u003eFerroptosis: iron release mechanisms in the bioenergetic process.\u003c/strong\u003e \u003cem\u003eCancer Metastasis Rev\u003c/em\u003e 2025, \u003cstrong\u003e44:\u003c/strong\u003e36.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"myocardial ischemia-reperfusion injury, Angelica sinensis polysaccharides, ROS, ATF6, ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-7142720/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7142720/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eFerroptosis aggravates myocardial ischemia-reperfusion injury (MI/RI) by disrupting iron homeostasis, accelerating lipid peroxidation, and elevating reactive oxygen species (ROS) levels. Although Angelica sinensis polysaccharide (ASP) has shown protective effects against MI/RI, its clinical translation remains limited due to poor bioavailability and low target specificity.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eTo address these limitations, we developed ASP@PLGA-PEG nanoparticles using a solvent evaporation method and characterized their morphology, size distribution, and surface charge by transmission electron microscopy, dynamic light scattering, and zeta potential analysis. The protective effects of ASP@PLGA-PEG were evaluated in vitro using HL-1 cardiomyocytes subjected to oxygen and glucose deprivation/reoxygenation (OGD/R). Cell viability, mitochondrial membrane potential, ROS generation, lipid peroxidation, and antioxidant capacity were assessed using CCK-8 assay, JC-1 staining, ROS fluorescence detection, immunofluorescence, and biochemical analyses. In addition, an in vitro MI/RI model was established using the Langendorff isolated heart perfusion system to assess hemodynamic function, infarct size, histopathological changes, and mitochondrial ultrastructure.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eASP@PLGA-PEG nanoparticles significantly reduced oxidative stress, improved cardiomyocyte viability, and inhibited ferroptosis in OGD/R-injured HL-1 cells. In the Langendorff model, treatment with ASP@PLGA-PEG effectively decreased myocardial infarct size, preserved cardiac hemodynamics, and alleviated structural damage. Mechanistic studies revealed that ASP@PLGA-PEG nanoparticles activate ATF6 signaling, which suppresses NCOA4-mediated ferritinophagy, thereby limiting iron overload and lipid peroxidation to protect cardiomyocytes against ferroptosis during MI/RI.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study demonstrates that ASP@PLGA-PEG nanoparticles exert potent cardioprotective effects through a multi-target mechanism involving ER stress modulation, enhanced antioxidative defense, and inhibition of ferritinophagy-driven ferroptosis. These findings highlight the therapeutic potential of ASP@PLGA-PEG as a promising nanomedicine strategy for the prevention and treatment of myocardial ischemia-reperfusion injury.\u003c/p\u003e","manuscriptTitle":"Angelica sinensis Polysaccharide nanoparticles can improve myocardial ischemia-reperfusion injury by inhibiting ferritinophagy via the ATF6/NCOA4 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 11:03:48","doi":"10.21203/rs.3.rs-7142720/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-08-01T16:23:29+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-01T16:21:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-21T15:43:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Translational Medicine","date":"2025-07-18T00:42:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-translational-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtrm","sideBox":"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/jtrm/default.aspx","title":"Journal of Translational Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d34e7b20-70a0-48f1-859c-d3a47e9c7baa","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:01:57+00:00","versionOfRecord":{"articleIdentity":"rs-7142720","link":"https://doi.org/10.1186/s12967-026-07752-8","journal":{"identity":"journal-of-translational-medicine","isVorOnly":false,"title":"Journal of Translational Medicine"},"publishedOn":"2026-02-26 15:58:36","publishedOnDateReadable":"February 26th, 2026"},"versionCreatedAt":"2025-08-11 11:03:48","video":"","vorDoi":"10.1186/s12967-026-07752-8","vorDoiUrl":"https://doi.org/10.1186/s12967-026-07752-8","workflowStages":[]},"version":"v1","identity":"rs-7142720","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7142720","identity":"rs-7142720","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}