Introduction

As is well documented, the anthracycline chemotherapy drug doxorubicin (DOX) has been extensively utilized for the treatment of diverse malignancies1. However, despite its potent efficacy, up to 25% of patients experience DOX-induced cardiac injury2thereby imposing limitations on its utilization. Notably, DOX-induced cardiac injury is dose-dependent and characterized by reduced left ventricular ejection fraction, increased ventricular wall thickness, arrhythmia, heart failure, and mortality3. Although DOX-induced cardiac injury is well known, there is no effective treatment other than routine adjustment of DOX dose4. Currently, Dexrazoxane is the only FDA-approved protective agent for the treatment of DOX-induced cardiac injury5. However, an earlier study reported that the co-administration of dexrazoxane could potentially modulate the reactivity of DOX towards tumors and consequently elevate the susceptibility to secondary malignancies, thereby significantly impacting its clinical utility6. Therefore, there is an urgent need to develop highly effective and safe complementary interventions without adversely impacting the anti-tumor efficacy of doxorubicin, aiming to prevent adverse cardiac events in cancer chemotherapy survivors.

The pathogenesis underlying DOX-induced cardiac injury involves multiple mechanisms, with oxidative stress being one of the major contributors7,8. Numerous studies have established the role of mitochondria as a source and target of oxidative stress9,10,11. Meanwhile, mitochondria are considered the primary organelles affected by DOX, with mitochondrial dysfunction also serving as a significant indicator of its cardiotoxic effects12. Of note, mitochondria continuously undergo fusion and fission, collectively termed mitochondrial dynamics13. Mitochondrial fusion has been described as a defensive response to enhance resistance to stress. In contrast, mitochondrial fission not only supports the high energy demands of cardiomyocytes but also allows the removal of dysfunctional mitochondrial fractions from the mitochondrial network to preserve mitochondrial integrity13. Excessive mitochondrial division or lysis can trigger cell apoptosis and necrosis, resulting in the deterioration of cardiac function14. Dysregulation of mitochondrial dynamics is a hallmark of DOX-induced cardiac injury13,15,16,17. Specifically, DOX disrupts mitochondrial dynamics by promoting mitochondrial fission and suppressing mitochondrial fusion. A recent study reported that mitochondrial fusion 2 (Mfn2) overexpression restored mitochondrial fusion and attenuated DOX-induced apoptosis, oxidative stress, and cardiac dysfunction18. At the same time, mitochondrial fission protein dynamin-related protein 1 (Drp1) heterozygous knockout has been shown to protect against DOX-induced cardiotoxicity19. Therefore, inhibiting fission or promoting fusion may assist in mitigating DOX-induced cardiac injury.

DOX-induced cardiac mitochondrial toxicity induces autophagic (including mitochondrial autophagy) responses20. While moderate levels of mitochondrial autophagy contribute to the maintenance of physiological cellular autophagy by eliminating damaged mitochondria, excessive activation severely impairs mitochondrial function21,22. In cellular models of DOX-induced autophagic overstimulation, cardiac protection was achieved using inhibitors of autophagy initiation20,23,24,25. Therefore, the targeted modulation of mitochondrial autophagy may present a promising therapeutic approach for the management of DOX-induced cardiac injury.

Astaxanthin (ASTA), a member of the carotenoid family, has garnered significant attention owing to its potent antioxidant properties26. ASTA has been investigated in the treatment research of various heart diseases such such as heart failure27 and ischemic cardiomyopathy28. Previous studies concluded that ASTA can enhance cardiac function by attenuating oxidative stress damage and mitochondrial swelling, thereby exerting cardioprotective effects. Moreover, ASTA can enhance mitochondrial function through the activation of the Nuclear factor (erythroid-derived 2)-like 2(Nrf2)/ Heme Oxygenase-1(HO-1)pathway, thereby promoting autophagy and concurrently inhibiting ferroptosis29. Furthermore, it can alleviate the imbalance of mitochondrial dynamics, inhibit mitochondrial fission, and promote mitochondrial fusion30,31. A study had shown that ASTA alleviated oxidative stress and DOX-induced apoptosis and related cardiac development abnormalities in zebrafish embryos32. However, the effects of ASTA on cardiac mitochondria following the administration of doxorubicin, mitochondrial dynamics, and autophagy remain elusive.

Based on the aforementioned evidence, we postulate that ASTA can mitigate the imbalance in mitochondrial fusion and fission and suppress DOX-induced mitophagy, thereby exerting cardioprotective effects. A pooled analysis involving 630 patients with breast cancer and lung cancer demonstrated that the incidences of congestive heart failure were 4.7%, 26%, and 48% when the cumulative dose of doxorubicin reached 400, 550, and 700 mg/m², respectively33. In order to guarantee the success rate of animal modeling, we selected the dose of doxorubicin as 15 mg/kg, which is equivalent to 800 mg/m2 for a 70 kg man. Furthermore, previous studies have demonstrated that significant cardiotoxicity occurs in rats when the cumulative dose reaches 15 mg/kg34.Therefore, a rat model of DOX-induced cardiac injury was established to explore the effects of ASTA on DOX-induced cardiac mitochondrial damage, mitochondrial dynamics, and autophagy.

Methods

Animals and treatments

Healthy male rats (6–8 weeks old) were sourced from the Laboratory Animal Center of Guangxi Medical University. All experiments were conducted in accordance with the Guidelines on the Care and Use of Laboratory Animals established by the National Institutes of Health. This study was approved by the Animal Welfare and Ethics Committee of Guangxi Medical University (approval number: 202404007). Prior to the experiments, the rats were housed in a controlled environment with a 12-hour light/dark cycle and ad libitum access to food and water for one week. Afterward, the rats were randomly assigned to four groups, namely the sham group, DOX group, DOX + ASTA (25 mg/kg) group, and DOX + ASTA (50 mg/kg) group. The rats in the DOX group received intraperitoneal injections of doxorubicinat a dose of 3 mg/kg once every 3 days for a total of five times, the vehicle was prepared in saline with a final volume of 0.5 mL35. The Rat in the treatment group received the same treatment as the DOX group, but on top of that, ASTA was administered orally via intragastric administration 30 min later, at a dose of 25 mg/kg/day or 50 mg/kg/day for 15 consecutive days36,37,Astaxanthin was dissolved in olive oil with a final volume of 1 mL. Rats in the Sham group received saline via intraperitoneal injection at the same volume and frequency as the DOX group, and were administered olive oil by oral gavage matching the volume and frequency of the ASTA (25 mg/kg) group, and ASTA (50 mg/kg) group. Doxorubicin (CSNPHARM, CSN16255, China) and Astaxanthin (CSNPHARM, CSN20493, China) were both acquired from CSNPHARM Company. Each experimental group consisted of at least six rats, and all necessary measures were taken to minimize animal suffering throughout the experiments. After the completion of the experiment, rats were anesthetized and sacrificed by using an excessive dose of pentobarbital sodium for subsequent experiments.

Echocardiography

At the end of the 15-day dosing regimen, cardiac function was assessed using ultra-high-resolution echocardiography. The rats were mildly anesthetized via inhalation of 1.5% isoflurane (RWD, R510-22-10, China) and subsequently placed on a heating table to maintain normal body temperature. A digital animal ultrasound imaging system (Esaote, Italy) equipped with a 12 MHz probe was employed for cardiac imaging. Two-dimensional echocardiography and M-mode echocardiographic assessments were performed in the parasternal long-axis view, and ejection fraction (EF) and fractional shortening (FS) were calculated.

ELISA assay

At the 16th day of the study, after performing ultrasonic measurements, blood samples were collected from each rat via arterial catheterization into syringes and subsequently placed at room temperature for two hours. Following this, the samples were centrifuged at 4℃ and 3000 RPM using a high-speed centrifuge for 10 min. Next, the serum supernatant was extracted for further experimentation. The commercially available troponin I detection kit (NanJing JianCheng Bioengineering Institute, H149-1-1, China) was utilized to determine the concentration of troponin I in rat serum following the manufacturer’s instructions.

Histological staining

The cardiac tissues of the rats were harvested on the 16th day of the study for histological staining .Fresh heart tissue samples were fixed in 4% paraformaldehyde at 4℃ overnight, following which they were embedded in paraffin and sectioned. The resulting sections were subsequently subjected to HE staining, and morphological changes in cardiomyocytes were visualized under a light microscope.

ATP content detection

The measurement of ATP was conducted using the colorimetric method. The principle is that creatine kinase catalyzes the reaction of adenosine triphosphate (ATP) and creatine to form phosphocreatine38. The amount of phosphorus generated was detected by the phosphomolybdate colorimetric method, and the ATP content was calculated accordingly.The specific steps were as follows: 50 mg of fresh myocardial tissue was taken and a 10% homogenate was made in pre-cooled pure water. After homogenization, the supernatant was collected and the protein concentration in the supernatant was simultaneously determined. According to the protocol provided by the ATP detection kit (Nanjing Jiancheng ATP detection kit, A095-1-1, China), blank tubes, standard tubes, measurement tubes, and control tubes were set up. 1 mmol/L ATP standard solution was added to the blank tube and control tube, and the collected supernatant was added to the measurement tube and control tube. 100 µl of final substrate solution I and 200 µl of substrate solution II were added to all tubes. 30 µl of pure water was added to the blank tube and control tube, while 30 µl of accelerator was added to the standard tube and measurement tube. The mixtures were mixed well and incubated at 37 °C for 30 min. After the water bath, 50 µl of precipitating agent was added to all tubes, mixed well, and centrifuged at 4000 rpm for 5 min. 300 µl of the supernatant from each tube was taken for measurement. 500 µl of chromogenic agent was added to the supernatant, mixed well, and left to stand at room temperature for 2 min. Finally, 500 µl of termination agent was added to all tubes, mixed well, and left to stand at 37 °C for 5–10 min. The absorbance values of each tube were measured at 636 nm with a light path of 0.5 cm using double distilled water as the blank. The final ATP content was calculated as: (A measurement - A control) / (A standard - A blank) × Concentration of the standard × sample dilution factor × protein concentration of the homogenate.

Ultrastructural assessment

The cardiac tissues of the rats were harvested on the 16th day of the study for transmission electron microscope Cardiac tissue samples were immersed in a vessel containing a fixative solution for electron microscopy. Thereafter, the tissue was meticulously dissected into 1 mm3 fragments using a scalpel, followed by rinsing with PBS and immersion in fixative solution at room temperature for 24 h. The tissue samples were further fixed with 1% osmic acid. Afterward, the samples were dehydrated, embedded, and prepared into ultrathin sections. The slides were stained and observed under a transmission electron microscope (TEM). Images were captured and preserved for the ensuing analysis.

The specific implementation details of Flameng Score are as follows: Firstly, randomly select mitochondria from the electron microscopy images of each group, a total of 100 mitochondria. The average score of these 100 mitochondria represents the sample score. Next, calculate the average score of mitochondria for each sample. In brief, determine the mitochondrial score for each sample to be a score ranging from 0 to 4, with higher scores indicating more severe damage. The specific scoring rules are detailed in Appendix 1.

Western blot analysis

The cardiac tissues of the rats were harvested on the 16th day of the study for Western blot experiments. Heart tissue samples were homogenized in cold RIPA tissue lysate (Solarbio, P0010, China) supplemented with PMSF protease inhibitor (Solarbio, P0100, China) and phosphatase inhibitor (Solarbio, P1260, Chian) for 10 min. Total proteins were obtained by high-speed centrifugation. Protein concentrations were quantified using the BCA assay (Boeter, AR0146, China). Next, 30 µg of protein was loaded onto a 10% or 12% w/v sulfate-polyacrylamide gel for electrophoresis (SDS-PAGE) for 2 h. Subsequently, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corp, Billerica, MA). The membrane was washed three times with Tris-buffered saline containing Tween 20 (TBST) for 10 min each time. Subsequently, the membrane was incubated with primary antibodies, including DRP1 resistant (1:1000, Cell Signaling Technology, #5391), Mfn2(1:1000, Cell Signaling Technology,# 9482T), LC3 (1:1000, Cell Signaling Technology,#12741T), p62 (1:1000, Bioss antibodies.bs-55207R), ULK1(1:1000,Zenbio, R381887), PINK1 (1:1000, Wanleibio, WL04963), Parkin (1:1000, Servicebi, GB11596),GAPDH (1:5000, Servicebio, GB15004) ,OPA1(1:1000,Proterntech, 27733-1-AP), Beclin-1(1:1000, Cell Signaling Technology, #3495) and Fis-1(1:1000, Cell Signaling Technology, #32525). After washing the membrane three times with TBST for ten minutes each time, it was incubated with goat anti-Rabbit IgG secondary antibody (1:10000, Proteintech, RGAR006) at room temperature for 1.5 h. Finally, after washing with TBST, protein expression was detected and analyzed using an Odyssey two-color infrared scanning fluorescence imaging system(LI-COR, Germany).

Statistical analysis

The experimental data were subjected to normality and homogeneity of variance tests using SPSS (Version 26) software. The results were presented as mean ± standard deviation. One-way ANOVA was employed for comparing experimental data following a normal distribution and met the assumptions of homogeneity of variance across multiple groups. For data not meeting the assumptions of normality or variance homogeneity, non-parametric tests were used to assess differences. GraphPad Prism 8 was utilized to generate plots. P < 0.05 was considered statistically significant.

Results

Astaxanthin prevents DOX-induced left ventricular dysfunction

On the 16th day of the study, echocardiography was performed on each rat to assess cardiac function. As illustrated in Fig. 1, the EF and FS of rats in the DOX group were significantly lower compared with those in the sham group (P < 0.05). Both doses of astaxanthin treatment resulted in a substantial increase in EF and FS compared to the DOX group. However, no significant difference was observed between the two ASTA groups.

Fig. 1
figure 1

ASTA can increase the left ventricular ejection fraction and left ventricular fractional shortening induced by DOX. (A) Representative M-ultrasound images in each group. (B) The results of EF in each group. Dox decreased the EF in rats, and ASTA treatment resulted in a statistically significant increase in EF compared to the DOX group. (C) The result of FS in each group. Dox decreased the FS in rats, and ASTA treatment resulted in a statistically significant increase in FS compared to the DOX group. Values are expressed as the mean ± standard deviation. Sham group: n = 3; other groups: n = 6 each. *P < 0.05. ns: no significance. DOX: Doxorubicin; ASTA: Astaxanthin; EF: Ejection fraction; FS: Fractional shortening.

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Astaxanthin prevents DOX-induced myocardial damage

To evaluate the protective effect of astaxanthin against DOX-induced cardiac injury, rat serum cardiac troponin-I (cTn-I) levels were measured on the 16th day of the study. cTn-I is a specific marker of cardiomyocyte injury that reflects the degree of myocardial injury with higher specificity and sensitivity compared to traditional myocardial enzyme markers. Interestingly, cTn-I levels were significantly elevated in the DOX group, whilst astaxanthin treatment significantly reversed this increase (P < 0.05) (Fig. 2.A).

To investigate the impact of astaxanthin on rat cardiac histomorphological changes, routine HE staining (Fig. 2.B) was performed on rat cardiac tissue on the 16th day of the study. Under light microscopy, myocardial tissues in the sham group displayed clear structural integrity with uniformly colored fibers, well-defined cell boundaries, and no apparent pathological alterations. In the DOX group, occasional myocardial nuclear rupture, cytoplasm disintegration, and minimal lymphocyte infiltration were noted. Conversely, the ASTA-treated group displayed clear heart tissue structure with distinct cell boundaries and absence of noticeable necrosis or inflammatory cell infiltration.

Fig. 2
figure 2

Astaxanthin can attenuate myocardial injury induced by DOX. The above samples were collected on the 16th day of the study. (A) The serum levels of cTn-I in each group. The content of serum cTn-I in the DOX group increased. After treatment with ASTA, the content of serum cTn-I decreased. (B) HE staining of the hearts in each group. ASTA can alleviate the myocardial cell damage and immune cell infiltration resulting from doxorubicin. (left:1×, right:20×) Values are expressed as the mean ± standard deviation. n = 6. *P < 0.05. ns: no significance. DOX: Doxorubicin; ASTA: Astaxanthin; Tn-I: troponin-I.

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Astaxanthin prevents cardiac mitochondrial structural and functional damage caused by DOX

The electron microscope serves as a crucial tool for observing the mitochondrial structure. To further expound on the protective effect of ASTA, transmission electron microscopy was utilized to examine the mitochondrial alterations of myocardial cells in each experimental group on the 16th day (Fig. 3A). In the sham group, cardiomyocytes appeared normal, with minimally swollen mitochondria and visible membranes and ridges. Conversely, cardiomyocytes in the DOX group displayed evident aberrations, characterized by moderate edema and an oval shape, with only a limited number of mitochondria retaining their structural ridges. Additionally, numerous vacuoles and autophagosomes were observed within the cytoplasm. The degree of myocardial cell damage was lower in both ASTA-treated group compared to the DOX groups. Specifically, mild mitochondrial edema and fewer autophagosomes were noted in both ASTA-treated group. Based on the assessment approach outlined by Li et al.38., the Flameng score is employed to assess mitochondrial damage (Fig. 3B). The specific scoring rules are detailed in Appendix 1. Mitochondria are the central site of energy metabolism within cells, and their primary function is to generate ATP. Therefore, the ATP levels in cells can directly reflect mitochondrial function. In our study, astaxanthin increased ATP content in rat cardiac tissue, indicating its ability to alleviate DOX-induced cardiac mitochondrial dysfunction (Fig. 3C).

Fig. 3
figure 3

Transmission electron microscopy images of myocardial cells in each group. The above samples were collected on the 16th day of the study. (A) Electron microscopy images of hearts in each group. ASTA mitigates cardiac mitochondrial edema and rupture mediated by doxorubicin and reduces the quantity of autophagosomes. The scale is 5.0 μm. The white arrow marks normal mitochondria, and the yellow arrow marks autophagosomes. (B) The results of the flameng scorce on mitochondria. A higher flameng score indicates more severe mitochondrial damage. ASTA effectively ameliorated cardiac mitochondrial damage induced by DOX. (C) The results indicate the ATP content in cardiomyocytes. Values are expressed as the mean ± standard deviation. n = 6. *P < 0.05. ns: no significance, DOX: Doxorubicin, ASTA: Astaxanthin.

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Astaxanthin improves the imbalance in cardiac mitochondrial dynamics induced by DOX

Mitochondria are highly dynamic organelles that regulate their size and shape in response to various stress conditions through coordinated division, fusion, and transport functions. To further investigate the protective effect of astaxanthin against DOX-induced cardiac injury, we assessed the expression levels of DRP1, Mfn2,OPA1 and Fis-1 using western blot analysis (Fig. 4). In the DOX group, compared to the sham group, there was an increase in DRP1 and Fis-1 expression accompanied by a decrease in Mfn2 and OPA1 expression. However, after treatment with astaxanthin, there was a significant decrease in DRP1 and Fis-1 expression along with an increase in Mfn2 and OPA1 expression. These findings suggest that astaxanthin can ameliorate the imbalance of cardiac mitochondrial fusion and division induced by DOX.

Fig. 4
figure 4

ASTA can reduce the expression of DRP1 and Fis-1 and increase the expression of Mfn2 and OPA1 in DOX-induced cardiomyocytes. (A) Western blot (WB) stripes of DRP1,Mfn2, OPA1 and Fis-1. (B-E) Statistical analysis of the WB strip of DRP1, Mfn 2, OPA1 and Fis-1. ASTA decreased the expression of DRP1 and Fis-1, inhibited cardiac excessive mitochondrial fission induced by DOX. And ASTA augmented the expression of Mfn2 and OPA1, facilitated cardiac mitochondrial fusion. The measurement data were presented as mean ± standard deviation. n = 5, *P < 0.05. ns: no significance. DOX: Doxorubicin; ASTA: Astaxanthin; DRP1: Dynamin-related protein 1; Mfn2: Mitochondrial fusion protein 2. OPA1: Optic Atrophy 1; Fis-1:Fission 1 Protein.

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Astaxanthin attenuates DOX-induced aberrant activation of cardiac mitochondrial autophagy through the PINK1/Parkin pathway

The results of TEM demonstrated mitochondrial autophagy played a pivotal role in doxorubicin-induced cardiac injury. To further examine the relationship between mitochondrial autophagy and the protective effect of astaxanthin, the expression levels of key proteins closely associated with mitochondrial autophagy, including Unc-51 Like Autophagy Activating Kinase 1 (ULK1), PTEN-Induced Putative Kinase 1 (PINK1), Parkin RBR E3 Ubiquitin Protein Ligase (Parkin), Beclin-1, Sequestosome 1(P62), and Microtubule-Associated Proteins 1 A/1B Light Chain 3 (LC3), were detected. As depicted in Fig. 5, compared to the sham group, the expression levels of ULK1, Parkin, Beclin-1 and LC3 were significantly higher in the DOX group, whereas those of P62 were lower (P < 0.05), indicating an active state of mitochondrial autophagy. Compared to the DOX group, the expression levels of ULK1, Parkin, Beclin-1 and PINK1 were significantly lower in both ASTA groups (P < 0.05), accompanied by a lower LC3II/LC3I ratio (P < 0.05) and higher expression levels of P62 (P < 0.05), signaling that ASTA inhibited mitochondrial autophagy (Fig. 5).

Fig. 5
figure 5

Astaxanthin exhibits inhibitory effects on DOX-induced cardiac mitochondrial autophagy. (A) WB bands of mitochondrial autophagy-associated proteins ULK1, Parkin, Beclin-1,PINK1, P62, and LC3. (B-G) WB analysis data of ULK1, Parkin and PINK1, P62, LC3 and Beclin-1 were obtained. ASTA reduces the expression of ULK1, Parkin, Beclin-1and PINK1, lowers the ratio of LC3II to LC3I, and increases the expression of P62, suggesting that astaxanthin can alleviate cardiac mitochondrial autophagy induced by DOX. The measurement data were presented as mean ± standard deviation, n = 6, *P value < 0.05. ns: no significance. DOX: Doxorubicin; ASTA: Astaxanthin; ULK1: Unc-51 Like Autophagy Activating Kinase 1; PINK1: PTEN-Induced Putative Kinase 1; Parkin: Parkin RBR E3 Ubiquitin Protein Ligase; P62: Sequestosome 1; LC3: Microtubule-Associated Proteins 1 A/1B Light Chain 3.

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Discussion

Currently, certain anticancer medications (e.g., anthracyclines such as DOX) frequently induce severe cardiac dysfunction while exerting their anti-tumorigenic effects39. Mitochondria are regarded as the primary target organelles of DOX-induced cardiac injury12. ASTA can penetrate into mitochondria in the cytoplasm32. In various cardiac diseases, ASTA can improve cardiac function by inhibiting mitochondrial swelling40,41,42. In the rat model of DOX-induced cardiac injury generated in this study, ASTA lowered serum cTn-I levels, thereby attenuating the decline in cardiac function induced by DOX, the imbalance in mitochondrial fusion and fission, and mitochondrial autophagy. We posit that ASTA mitigates DOX-induced cardiac mitochondrial damage and exerts cardioprotective effects by affecting mitochondrial dynamics and mitochondrial autophagy.

In order to detect cardiac damage induced by DOX, regular cardiac function monitoring is recommended. However, this method is associated with a delayed response43. Troponin is an early organ-specific biomarker for myocardial injury. Moreover, studies have identified cTn-I as a risk marker for a significant future reduction in LVEF44. More importantly, existing evidence suggests that patients treated with anthracycline drugs or trastuzumab have elevated cTn-I levels45,46. Furthermore, in the 2022 European Society of Cardiology clinical guidelines on cardio-oncology, cardiac troponin I is recommended for diagnosing anthracycline-induced myocardial injury and guiding cardioprotective therapy2. Therefore, we propose that cTn-I may serve as a valuable marker for assessing the cardiotoxicity of DOX.

According to an earlier study, mitochondrial dysfunction is an early indicator of DOX-induced myocardial cell injury47. Mitochondria are crucial organelles responsible for cellular energy metabolism and adapt to cellular and environmental fluctuations through fusion and fission12,27,48. It is worthwhile emphasizing that their morphology is governed by fusion proteins and fission proteins. Studies have concluded that DOX decreases ΔΨm, thereby driving the opening of the mitochondrial Permeability Transition Pore (mPTP) and inhibiting cardiac mitochondrial fusion48,49. Mitochondrial function exhibits progressive impairment alongside increased mitochondrial fission50. In our study, the intracellular ATP content in the doxorubicin-treated group was significantly lower than in the Sham group. Notably, ASTA treatment effectively restored mitochondrial ATP content within cells. In rat hearts, DOX facilitates the phosphorylation of Drp1 at serine 616, which initiates mitochondrial fission51. Noteworthily, ASTA improves the respiratory control index, enhances mitochondrial fusion, and inhibits mitochondrial fission in the mitochondria in rat hearts after isoproterenol-induced injury52. Herein, DOX upregulated the expression of Drp1 and Fis-1, downregulated that of Mfn2 and OPA1, shifting the mitochondrial dynamic balance towards fission. On the other hand, ASTA treatment inhibited mitochondrial fission by down-regulating the expression of DRP1 and Fis-1, up-regulating that of Mfn2 and OPA1, thereby restoring mitochondrial fusion. This indicates that in the DOX-induced cardiac injury model, ASTA alleviates cardiac mitochondrial damage by inhibiting mitochondrial fission and promoting mitochondrial fusion.

After fission via Drp1, the fate of fragmented mitochondria is contingent upon their capacity to restore mitochondrial membrane potential; healthy mitochondria can restore mitochondrial membrane potential and re-engage in the fusion-fission cycle, while those unable to recover are marked and eliminated via mitophagy53,54. Mitochondrial autophagy is a ubiquitous mitochondrial self-renewal mechanism in eukaryotic cells55. To sustain physiological activities, mitochondria eliminate damaged mitochondria via the mitophagy pathway56. Previous studies indicated that heightened levels of mitochondrial autophagy exacerbate mitochondrial damage and compromise overall mitochondrial function in DOX-induced cardiomyocyte injury57,58. Consistent with the observations of earlier studies, this study unveiled the presence of numerous scattered autophagosomes in the DOX group. The results of western blot analysis uncovered an elevated Beclin-1 and LC3-II/LC3-I ratio, down-regulated p62 expression, suggesting that DOX activated mitochondrial autophagy and disrupted mitochondrial function. However, studies investigating the role of mitochondrial autophagy in DOX-induced cardiomyocyte injury have yielded conflicting findings. Some studies pointed out that increased mitochondrial autophagy facilitates mitochondrial rejuvenation, thereby preserving mitochondrial function in Dox-induced cardiotoxicity59,60. This controversial perspective may arise from variations in dosage, duration, and drug interactions61. In the present study, treatment with ASTA decreased the Beclin-1 and LC3-II/LC3-I ratio, up-regulated the expression of p62, indicating inhibition of autophagy. These results suggest that ASTA can alleviate doxorubicin-induced cardiac mitochondrial damage by suppressing mitochondrial autophagy, thereby protecting cardiac function. Some studies indicated that ASTA can suppress autophagy and concomitantly enhance mitochondrial function, in line with the results of this study29,62.

Mitophagy typically occurs through two pathways, namely the ubiquitin-dependent pathway and the ubiquitin-independent pathway63. Among them, mitophagy mediated by the PINK1/Parkin pathway is the most extensively investigated. Under physiological conditions, PINK1 is degraded at the inner mitochondrial membrane, whilst Parkin remains in an inactive state64. Nevertheless, following ΔΨm disruptions, the channel for PINK1 to enter the inner mitochondrial membrane is obstructed, resulting in its accumulation on the outer mitochondrial membrane65. On the outer mitochondrial membrane, PINK1 and Parkin interact to ubiquitinate proteins at the outer mitochondrial membrane66,67. The accumulation of these ubiquitinated proteins contributes to the recruitment of the autophagy initiation factor ULK1, thereby stimulating mitophagy66. DOX-induced mitophagy and mitochondrial damage in cardiomyocytes are partially mediated by the dysregulation of the PINK1/parkin pathway61. Studies have exposed that the administration of ASTA can confer protective effects by regulating mitochondrial autophagy and the PINK1/Parkin pathway68. Herein, the expression levels of the PINK and Parkin proteins were significantly increased in the DOX group, suggesting that DOX activates mitochondrial autophagy by mediating the PINK1/parkin pathway. After treatment with ASTA, the expression levels of the PINK and Parkin proteins decreased, indicating that astaxanthin exerted cardioprotective effects by regulating mitochondrial autophagy via the PINK1/Parkin pathway in DOX-induced cardiac injury.

Astaxanthin not only has demonstrated therapeutic potential in doxorubicin-induced cardiac injury, but also has shown efficacy in various cancer types, including neuroblastoma, breast cancer and gastrointestinal cancer, through its ability to induce apoptosis, inhibit metastasis and disrupt cell growth69,70,71. Glioblastoma is one of the most aggressive primary brain cancers, with a very low survival rate72. Researchers found that when pretreating two human GBM cell lines, U251-MG and T98-MG, with ASTA, their sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was significantly enhanced, thereby increasing the apoptotic response69. In addition, Shao et al. demonstrated that AXT inhibits the proliferation of H22 liver cancer cells both in vitro and in vivo, and induces apoptosis and necrosis of the cells73. Moreover, ASTA treatment can reduce the ROS level in HepG2 cells, thereby significantly inhibiting cell growth. Notably, this cytotoxicity is selective, that is, it does not cause damage to normal liver cells (THLE-2 cells), highlighting the potential of ASTA for targeted cancer therapy without harming healthy cells74. As a common health care products, astaxanthin, when used in combination with doxorubicin during chemotherapy, may not only alleviate the cardiotoxicity caused by doxorubicin but also assist doxorubicin in exerting its anti-tumor effect, providing a laboratory basis for the treatment of doxorubicin-induced cardiac injury.

In our research, we established a rat model of DOX-induced cardiac injury and observed whether ASTA improves DOX-induced cardiac mitochondrial damage and exerts cardioprotective effects by affecting mitochondrial dynamics and autophagy. Several limitations exist in our current research. The research was limited to investigate astaxanthin’s protective effects against doxorubicin-induced cardiac injury at the animal model level, and did not extend to explore its underlying mechanisms through in vitro experimental.

Conclusion

In summary, ASTA ameliorated myocardial damage and cardiac dysfunction in a rat model of DOX-induced cardiac injury. The potential mechanism may involve ASTA down-regulating the expression of DRP1 and Fis-1 and up-regulating that of Mfn2 and OPA1 to induce mitochondrial fusion, limit mitochondrial fission, and suppress mitochondrial autophagy mediated by the PINK1/Parkin pathway.