Open Access

Attenuation of the Na/K‑ATPase/Src/ROS amplification signaling pathway by astaxanthin ameliorates myocardial cell oxidative stress injury

  • Authors:
    • Xuefeng Qu
    • Zhouyi Zhang
    • Wenli Hu
    • Minhan Lou
    • Bingzhong Zhai
    • Song Mei
    • Zhihang Hu
    • Lijing Zhang
    • Dongying Liu
    • Zhen Liu
    • Jianguo Chen
    • Yin Wang
  • View Affiliations

  • Published online on: October 19, 2020
  • Pages: 5125-5134
  • Copyright: © Qu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


The 3S, 3'S‑ASTaxanthin (3S, 3'S‑AST) isomer has strong antioxidant activity; however, its protective roles and potential mechanisms against oxidative stress damage in cardiomyocytes have not been investigated. Na+/K+‑ATPase (NKA)/Src signal activation has an important role in increasing reactive oxygen species (ROS) production. The aim of the present study was to investigate the protective effects and mechanism of 3S, 3'S‑AST on hydrogen peroxide (H2O2)‑induced oxidative stress injury in H9c2 myocardial cells. The protective effects of 3S, 3'S‑AST on H2O2‑induced H9c2 cell injury was observed by measuring lactate dehydrogenase and creatine kinase myocardial band content, cell viability and nuclear morphology. The antioxidant effect was investigated by analyzing ROS accumulation and malondialdehyde, glutathione (GSH) peroxidase, GSH and glutathione reductase activity levels. The protein expression levels of Bax, Bcl‑2, caspase‑3 and cleaved caspase‑3 were analyzed using western blotting to determine cardiomyocyte apoptosis. Western blot analysis of the phosphorylation levels of Src and Erk1/2 were also performed to elucidate the molecular mechanism involved. The results showed that 3S, 3'S‑AST reduced the release of LDH and promoted cell viability, and attenuated ROS accumulation and cell apoptosis induced by H2O2. Furthermore, 3S, 3'S‑AST also restored apoptosis‑related Bax and Bcl‑2 protein expression levels in H2O2‑treated H9c2 cells. The phosphorylation levels of Src and Erk1/2 were significantly higher in the H2O2 treatment group, whereas 3S, 3'S‑AST pretreatment significantly decreased the levels of phosphorylated (p)‑Src and p‑ERK1/2. The results provided evidence that 3S, 3'S‑AST exhibited a cardioprotective effect against oxidative stress injury by attenuating NKA/Src/Erk1/2‑modulated ROS amplification.


Acute myocardial infarction (MI) is a common cause of death worldwide. The annual mortality and morbidity rates following MI are 7 and 22%, respectively (1). Reperfusion therapies, including primary percutaneous coronary intervention and thrombolytic therapy, are commonly used to attenuate MI damage (2). However, reperfusion therapies may cause myocardial ischemia reperfusion (I/R) injury when the blood flow returns to the myocardial tissue (2). The potential molecular mechanisms of I/R injury include oxidative stress, inflammation, calcium overload and cytokine release (3). A large number of reactive oxygen species (ROS) are produced within minutes of reperfusion, which could oxidize target proteins to trigger oxidative stress injury, as a physiological second messenger signaling molecule (4). Abundant natural biologically active substances from diverse sources (e.g. luteolin, coptisine, curcumin) can potentially decrease I/R injury through an antioxidant effect (57). Therefore, natural biologically active substances can be used to control the development of oxidative stress injury in order to improve the health of patients with I/R.

Astaxanthin (AST) belongs to a natural xanthophyll carotenoid that is well-known for its antioxidant, anti-inflammatory, anti-apoptosis and anticancer abilities (813). Haematococcus pluvialis (H. pluvialis) is the best natural AST resource as it primarily contains the 3S, 3′S-AST isomer. The 3S, 3′S-AST molecule is regarded as the most effective biological antioxidant to eliminate free radicals (14). AST has an α-hydroxyketone structure, which is responsible for its strong antioxidant activity by capturing singlet oxygen and reacting with free radicals (15). The antioxidant activity of AST is 500 times and 10 times higher compared with that in vitamin E and β-carotene, respectively (14,16). In recent years, AST has been reported to protect against cardiovascular disease. In rats with isoproterenol-induced MI, AST treatment decreased the weight of the heart, inflammatory cell infiltration and myocardial fibrosis by improving antioxidant enzyme activity (9). Furthermore, AST also attenuated cardiac dysfunction and fibrosis in a mouse MI model (17). However, the protective roles of 3S, 3′S-AST against ROS-induced damage of cardiomyocytes and the potential mechanisms involved have not been elucidated.

During I/R injury, cardiac Na+/K+-ATPase (NKA) function was found to be altered, and elucidation of the mechanism involved would be important to develop novel approaches for therapeutic intervention (18). NKA is a transmembrane enzyme responsible for transporting Na+ and K+ ions across the cytomembrane, which establishes and maintains the ion concentration gradient across the cell plasma membrane (19). In addition to its Na pump function, NKA was also discovered to function as a signal transduction protein. The NKA/Src/Erk1/2 signaling pathway was found to be used as a feed-forward amplifier for ROS signaling that aggravates atherosclerosis, dyslipidemia, obesity and diabetes (20,21). Wang et al (22) demonstrated that ROS could active the NKA/Src/Erk1/2 signaling pathway to further trigger ROS production. Emerging studies have demonstrated the NKA/Src/ROS signal pathway is an underlying target for protecting the heart from I/R injury (23). Therefore, the present study was designed to investigate whether 3S, 3′S-AST exerts protective effects against myocardial oxidative stress damage by attenuating NKA/Src/Erk1/2 signaling-modulated ROS amplification.

Materials and methods


The 3S, 3′S-AST compound was obtained from Sigma-Aldrich (cat. no. SML0982; Merck KGaA), with ≥97% purity. Antibodies against Bax (cat. no. 2772S), Bcl-2 (cat. no. 3498S), caspase-3 (cat. no. 9665S), cleaved-caspase-3 (cat. no. 9664S), Erk1/2 (cat. no. 4695S) and phosphorylated (p)-Erk1/2 (Thr202/Tyr204; cat. no. 9101S) were purchased from Cell Signaling Technology, Inc. NKA (cat. no. cs-58629) and anti-c-Src antibodies (cat. no. cs8056) were obtained from Santa Cruz Biotechnology, Inc., while anti-p-Src pY418 antibody (cat. no. 44-660G) was obtained from Thermo Fisher Scientific, Inc. Goat anti-rabbit IgG (cat. no. 925-68021) and goat anti-mouse IgG (cat. no. 925-68020) secondary antibodies were purchased from LI-COR Biosciences. Nitrocellulose (NC) membranes were purchased from EMD Millipore, while RIPA lysis buffer was purchased from Boster Biological Technology. FBS was obtained from Biological Industries and the MTT kit (cat. no. M2128) and DAPI fluorescent dye (cat. no. D9542) were purchased from Sigma-Aldrich (Merck KGaA).

Cell culture and treatment

H9c2 cells (rat embryonic cardiomyocytes) were obtained from the Cell Bank of Shanghai Academy of Sciences and cultured in DMEM (cat. no. C11995500BT; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO2. The H9c2 cells were passaged regularly and cultured to 70–80% confluence prior experimentation. Control group was treated with DMEM without FBS; DMSO group was treated with DMEM and DMSO without FBS. To generate a ROS-induced myocardial cell injury model, H9c2 cells were exposed to 100 µM H2O2 for 1 or 24 h and analyzed for further analysis. A total of 50 mg 3S, 3′S-AST powder was dissolved in 5 ml DMSO and prepared into a stock solution of 16.8 mM. Cells were pretreated with 3S, 3′S-AST (5, 10 and 20 µM) for 1 h prior to H2O2 administration.

Cell viability assay

Cell viability was detected using an MTT kit according to the manufacturer's instructions. H9c2 cells were plated into 96-well plates (1×103 cells/well), cultured with DMEM and treated with 3S, 3′S-AST (5, 10, 20 µM) and H2O2 (50, 100, 200, 400 µM) for 24 h. A total of 15 µl MTT solution was added into each well and co-incubated for 4 h, at 37°C. After removing the medium, 150 µl DMSO was added to each well. The absorbance was measured at a wavelength of 490 nm using a microplate absorbance reader (Multiskan; Thermo Fisher Scientific, Inc.) and analyzed to assess cell viability.

Cell apoptosis assay

To detect cell apoptosis, H9c2 cells were stained using a DAPI fluorescent dye to observe nuclear morphology. H9c2 cells were plated into six-well plates (1×105 cells/well), washed with PBS three times and fixed in 4% paraformaldehyde (Beyotime Institute of Biotechnology) at room temperature for 10 min. The cells were then washed three times with PBS and stained using 3 ml DAPI staining solution (1 µg/ml) for 10 min at 37°C. The nuclear morphology was observed using fluorescence microscopy (Olympus Corporation; magnification, ×200). Healthy living cells presented with uniform staining of the nuclei. Apoptotic cells presented with pyknosis and fragmentation of the nuclei.

Lactate dehydrogenase (LDH) release analysis

Cell damage was determined using an LDH assay kit (cat. no. A020; Nanjing Jiancheng Bioengineering Institute) by detecting LDH release of myocardial cells. H9c2 cells were cultured at a density of 1×105 cells/well in six-well plates. A total of 20 µl cell culture medium was added into another 96-well plate, with 300 µl reaction mixture from the kit and the mixture was incubated for 30 min at 37°C. LDH activity was measured using a microplate reader at a wavelength of 450 nm.

Creatine kinase-myocardial band (CK-MB) activity analysis

CK-MB activity levels were measured using a CK-MB assay kit (cat. no. E006; Nanjing Jiancheng Bioengineering Institute) according to manufacturer's protocol. Briefly, H9c2 cells were lysed with PBS buffer, and freeze/thawed three times on ice for 30 min, followed by centrifugation at 12,000 × g for 15 min at 4°C to isolate the cell debris. The protein content of samples was examined using a bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology). CK-MB activity levels were detected at a wavelength of 340 nm using a microplate absorbance reader and expressed as U/g protein.

Malondialdehyde (MDA) and glutathione peroxidase (GSH-px) level detection

The levels of MDA and GSH-px in H9c2 cells were detected using MDA (cat. no. A003) and GSH-px assay kits (cat. no. A005) (both from Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions. Briefly, H9c2 cells were lysed with PBS buffer and freeze/thawed 3 times on ice for 30 min. The supernatant was isolated by centrifugation at 12,000 × g for 15 min at 4°C. The MDA content was measured at a wavelength of 532 nm and expressed as nmol/mg protein. The GSH-px activities of the cell lysate was detected at a wavelength of 412 nm according to the produced enzyme-catalyzed reaction product.

GSH and glutathione reductase (GR) level detection

The levels of GSH and GR in H9c2 cells were detected using GSH (cat. no. A006) and GR assay kits (cat. no. A062) (both from Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions. Briefly, the H9c2 cells were lysed with PBS buffer and freeze/thawed three times on ice for 30 min then. The mixture was then centrifuged at 12,000 × g for 15 min at 4°C to isolate the cell debris. GSH activity levels were detected at a wavelength of 405 nm and expressed as µmol/g protein, while GR activity levels were detected at a wavelength of 340 nm using a spectrophotometer and expressed as U/g protein.

Intracellular ROS measurements

A ROS assay kit (cat. no. S0033; Beyotime Institute of Biotechnology) was used to detect intracellular ROS levels according to the manufacturer's instructions. 2′-7′-dichlorofluorescin diacetate (DCFH-DA) was diluted with serum-free medium (1:1,000). Cells were seeded into a six-well plate (1×105 cells/well) and treated with H2O2 and 3S, 3′S-AST for 24 h. The culture medium was removed and the cells were incubated with DCFH-DA (10 µM) for 20 min at 37°C. Subsequently, the cells were washed with serum-free medium (three times for 5 min each time) and images were obtained using a fluorescence microscope (Olympus Corporation; magnification ×100). ROS fluorescence intensity was analyzed using Image-Pro Plus (version 5.0; MediaCybernetics, Inc.).

Western blot analysis

Following treatment, H9c2 cells were scraped using a scraper and the protein lysates were extracted using a solubilization buffer (containing RIPA and 1% protease inhibitor). The debris was removed by centrifugation at 12,000 × g for 15 min at 4°C. The protein concentration was determined using a BCA assay kit. The protein samples (80 µg) were added to 5X loading buffer (Beyotime Institute of Biotechnology) and heated at 95°C for 5 min using metal heating block. Protein samples were separated using 10% SDS-PAGE and transferred onto a NC membrane, at a constant current of 300 mA for 1 h under wet transfer conditions. The NC membranes were blocked with 5% skimmed milk at room temperature, and the proteins were incubated with the following antibodies overnight at 4°C: NKA (1:200), c-Src (1:200), p-Src (1:1,000), Erk1/2 (1:1,000), p-Erk1/2 (1:1,000), Bax (1:500), Bcl-2 (1:500), caspase-3 (1:500), cleaved-caspase-3 (1:500) and GAPDH (1:1,000). The membranes were then incubated with the following fluorescent secondary antibodies: Goat anti-rabbit (1:10,000) and goat anti-mouse (1:10,000). The bands were captured and quantified using an Odyssey Imaging System (LI-COR Biosciences).

Statistical analysis

Statistical analysis was performed using SPSS software (version 22.0; IBM Corp.). The results are represented as the mean ± SEM from at least three independent experiments. Analysis between two groups were performed using Student's t-test. Analysis between multiple groups were performed using one-way ANOVA analysis followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.


3S, 3′S-AST attenuates ROS-induced cell injury in rat cardiomyocytes

To investigate the protective effects of 3S, 3′S-AST in cardiomyocytes under oxidative stress damage in vitro, the rat H9c2 embryonic heart-derived cell line was treated with H2O2 to simulate oxidative stress injury. The cell viability was evaluated using an MTT assay. As shown in Fig. 1A and B, H2O2 significantly decreased cell viability in H9c2 cells in dose-dependent manner, and pretreatment with 3S, 3′S-AST significantly inhibited the decrease of cell viability caused by H2O2. Furthermore, with the increase in the dose of 3S, 3′S-AST, the effect of inhibition was more significant. Because the 100 µM H2O2 decreased cell viability to ~60%, this concentration was used for subsequent experiments. LDH and CK-MB are well-known indicators of cell injury (24); Therefore, they were measured in the present study. The LDH content in the culture medium of H2O2-treated cells was significantly higher compared with the control group, and treatment with H2O2 significantly increased CK-MB content in H9c2 cells. However, pretreatment with 3S, 3′S-AST significantly reduced this increase (Fig. 1C and D). No significant difference was observed in the 5 µM 3S, 3′S-AST-treated group compared with the H2O2-treated group.

3S, 3′S-AST inhibits ROS-induced cardiomyocyte apoptosis

DAPI DNA fluorescent staining was utilized to investigate the effect of 3S, 3′S-AST on cell apoptosis. The morphology of the nucleus in the H9c2 cells showed uniform staining in the control and DMSO groups. By contrast, H2O2-treated cells showed nuclear pyknosis and fragmentation. Compared with the H2O2-treated group, the 3S, 3′S-AST-treated group showed markedly attenuated cell apoptosis, but no significant difference was observed in the 5 µM 3S, 3′S-AST-treated group (P>0.05) (Fig. 2A). Western blot analysis was performed to evaluate the effect of 3S, 3′S-AST on the expression levels of pro-apoptosis proteins (Bax, caspase-3 and cleaved-caspase-3) and anti-apoptosis protein (Bcl-2) in each group. The protein expression levels of caspase-3, cleaved-caspase-3 and Bax were significantly downregulated, while the expression level of Bcl-2 was significantly upregulated in the 3S, 3′S-AST-treated groups compared with the H2O2-treated group. These effects were attenuated treatment with 10 and 20 µM 3S, 3′S-AST (Fig. 2B-F).

Effects of 3S, 3′S-AST on ROS accumulation and antioxidant activity

DCFH-DA staining was used to determine ROS accumulation. Cells treated with 3S, 3′S-AST significantly attenuated intracellular ROS level induced by H2O2 (Fig. 3A and B). It was reported that the NKA/Src signaling pathway, which regulates ROS amplification, plays a key role in oxidative stress damage (23). In the present study, the results indicated that 3S, 3′S-AST may act as an antioxidant to reduce the ROS level and inhibit the NKA/Src/ROS amplification loop. Furthermore, the effects of 3S, 3′S-AST on MDA content and GSH-px, GSH and GR activity levels were also measured. The results revealed that treatment with H2O2 significantly increased MDA content and significantly decreased GSH-px, GSH and GR activity levels in H9c2 cells. The effects of 10 and 20 µM 3S, 3′S-AST were more effective compared with cells treated with 5 µM 3S, 3′S-AST (Fig. 3C-F).

Effects of 3S, 3′S-AST on the NKA/Src signaling pathway

ROS is a specific ligand of NKA and could induce activation of Src and Erk1/2 and activated NKA/Src/Erk1/2 signaling further promoted ROS production (22). Therefore, the effects of 3S, 3′S-AST on the phosphorylation of Src and its downstream regulator Erk1/2 in H2O2-treated H9c2 cells was determined. As shown in Figs. 4 and 5, the phosphorylation of Src and Erk1/2 was significantly upregulated in H2O2-treated cells compared with controls. However, 3S, 3′S-AST inhibited the activation of Src and Erk1/2 and did not alter the protein expression levels of NKA in cells with ROS-induced damage. These results indicated that 3S, 3′S-AST attenuated the activities of the NKA/Src/Erk1/2 signaling pathway, which acted as one upstream pathway of ROS production and apoptosis.


Restoring blood flow has been confirmed to be an efficient approach to recover ischemic myocardium (25). However, myocardial reperfusion will elicit an increase in ROS production in cardiomyocytes (26). Some therapeutic drugs, such as rosuvastatin (27), have been available to prevent oxidative stress injury from I/R; however, the therapeutic effects for I/R injury remain unsatisfactory (28). Therefore, it is essential to discover safe and effective therapeutic agents. The present study demonstrated the function of 3S, 3′S-AST in protecting cardiomyocytes from ROS-induced injury in vitro. The results indicated that 3S, 3′S-AST treatment inhibited cardiomyocyte apoptosis and improved antioxidant activity. It was further indicated that 3S, 3′S-AST treatment prevented intracellular ROS generation by reducing the activity of the NKA/Src/Erk1/2/ROS amplification signal transduction pathway.

AST exists in three stereoisomeric forms (3S, 3′S; 3R, 3′R and 3R, 3′S)(29). H. pluvialis-derived AST primarily contains the 3S, 3′S-form, and synthetic AST is a mixture of the three stereoisomers [(3S, 3′S):(3R, 3′S):(3R, 3′R), 1:2:1 ratio] (29). It was reported that synthetic AST could reduce MI size in I/R rabbits (30); however, humans are unable to use synthetic AST (31), while H. pluvialis-derived AST is the only AST permitted for human consumption (31). Synthetic AST has 20 times lower antioxidant capacity than natural AST and to date has not been approved for human consumption. The United States Food and Drug Administration and the European Food Safety Authority has approved the use of AST from H. pluvialis, as a food ingredient in the food production process and it can be used as an additive at dosages below 12–24 mg/day within 30 days (32). Initial preclinical studies also support the antioxidant ability of AST at a particular dose. For example, Iwamoto et al (33) found that the resistance of volunteers to low-density lipoprotein oxidation was enhanced when AST was administered at doses of 1.8–21.6 mg/day for 14 days. Yoshida et al (34) showed that triglyceride and high-density lipoprotein-cholesterol levels were ameliorated in patients administered with AST at doses of 12–18 mg/day for 12 weeks. Furthermore, in a human clinical study, the beneficial action of dietary AST was identified at doses of 2 and 8 mg/day for 8 days and was found to regulate the immune response, oxidative damage and inflammation (35). However, a previous study investigating the anti-fibrotic effects of different concentrations of AST (10, 20 and 40 µM) on LX-2 cells revealed that AST could modulate proliferation and apoptosis of LX-2 cells at the cellular level (36). H9c2 cells pre-treated with 0.5–8 µM AST for 6 h and co-incubated with homocysteine for 72 h revealed that AST alleviated homocysteine induced cytotoxicity (37). In the present study, different concentrations of 3S, 3′S-AST (5, 10 and 20 µM) was used to treat H9c2 cells to investigate the effects and mechanism of 3S, 3′S-AST on oxidative stress. It was found that 10 and 20 µM 3S, 3′S-AST was more effective compared with that for 5 µM, and almost no significant difference was observed in cells treated with 5 µM 3S, 3′S-AST compared with H2O2-treated cells.

The bioavailability of AST is affected a number of factors, including host-related conditions, such as sex, age, obesity, smoking and alcohol consumption, as well as the structure of AST or its dispersion medium (14). Some trials have investigated the bioavailability of AST from H. pluvialis under different factors. Okada et al (38) showed that the metabolism of AST in a group of smokers was faster compared with the non-smokers group, which may be attributed to the antioxidant effect of AST. A previous study found that dietary oils may enhance the absorption of AST bioavailability in humans (39). In addition, Mercke et al (40) determined the bioavailability of AST from H. pluvialis dispersed in synthetic hydrophilic surfactant polysorbate 80 (B), which is a lipid-based formulation, was 1.7–3.7 times higher compared with the commercial formulation. Furthermore, studies have shown that the antioxidant ability of AST from H. pluvialis was improved in rat plasma and liver tissues after being dissolved in olive oil (41,42). Satoh et al (43) administered participants with an oil-based natural AST-rich product for 4 weeks at 20 mg daily, and there were no safety concerns reported by evaluating toxicity and efficacy levels. As aforementioned, further research should focus on improving host-related conditions and identifying new biomaterials to act as astaxanthin vectors to improve its bioavailability.

Baburina et al (44) revealed that AST could inhibit opening of a large-conductance channel (mitochondrial permeability transition pore) in the rat heart following mitochondrial calcium overload or oxidative stress by reducing the serine/threonine protein kinase B/cAMP-responsive element-binding protein signaling pathways in the mitochondria. Nakao et al (45) found that 0.08% AST had a significantly positive effect on cardiac function of mice. AST treatment reversed enhanced lipid peroxidation and reduced antioxidant enzyme activities in heart tissues caused by isoproterenol (ISO) administration in rats, which suggested that AST may protect cardiac tissue in ISO-administered rats by suppressing oxidative stress and enhancing antioxidant enzyme functions (9). However, the effects and mechanism of 3S, 3′S-AST on oxidative stress injury in myocardial cells caused by I/R was unknown. In the current study, oxidative stress injury in the H9c2 cells induced by H2O2 was used to mimic I/R injury. It was found that 3S, 3′S-AST significantly increased cell viability in H2O2-treated H9c2 cells. LDH and CK-MB are known to be markers of cell injury, and release of LDH and content of CK-MB were significantly reduced by 3S, 3′S-AST.

AST shows effective anti-apoptosis, anti-inflammatory, anti-cancer, and cardioprotective capacities, which were associated with its antioxidant activity (14,29). ROS is a universal inducer of myocardial apoptosis in reperfusion injury (46). Excessive ROS production has been considered to be important to induce I/R injury (46). MDA is the end product of the lipid peroxidation reaction; therefore, the content of MDA could reflect the degree of lipid peroxidation (47). To protect molecules from ROS, cells stimulate antioxidant defense systems, including GSH-px, GSH and GR, which play important roles in cellular antioxidant activity (48,49). The present study found that 3S, 3′S-AST could decrease the content of MDA and increase GSH-px, GSH and GR activity levels in the H9c2 cells. The results indicated that 10 and 20 µM 3S, 3′S-AST could improve the antioxidant ability of cardiomyocytes, inhibit excessive ROS accumulation and the occurrence of lipid peroxidation induced by oxidative damage. Apoptosis-related proteins, such as Bax, Bcl-2, caspase-3 and p53 play vital roles in cardiomyocyte damage (50,51). The present study showed that myocardial apoptosis was significantly inhibited by 3S, 3′S-AST. In addition, the pro-apoptotic proteins Bax and caspase-3 were significantly upregulated, while the anti-apoptotic protein Bcl-2 was significantly decreased in H2O2-treated H9c2 cells. Treatment with 3S, 3′S-AST inhibited the increase of Bax, caspase-3 and cleaved-caspase-3 and the decrease of Bcl-2 protein expression.

The NKA was discovered as an ion pump for moving Na+ and K+ across the cell membrane ~60 years ago (52). It is a key enzyme in maintaining the cellular Na+ and K+ ion gradient in human cardiac myocytes (53). In addition to its ion pumping function, NKA also serves as a scaffold protein, interacting with neighboring proteins and facilitating multiple cell signaling pathways. Wang et al (22) reported that endogenous cardiotonic steroids could activate the NKA/Src/Erk1/2 receptor complex to increase ROS levels, and ROS act as endogenous cardiotonic steroids to stimulate NKA/Src/Erk1/2 activation. Inhibition of the NKA/Src/Erk1/2/ROS amplification loop exhibited a beneficial effect on I/R injury (23). The present study found that 3S, 3′S-AST could reduce ROS generation induced by H2O2, and exerted antioxidant effects by restoring the activation of antioxidant enzymes.

The NKA receptor not only acts as an ion pump, maintains cell membrane potential and stabilizes cell volume, but also participates in a variety of protein-protein interactions, which activates a number of protein cascades and participate in a variety of signal transductions, including Raf/MEK/ERK, phospholipase C/protein kinase C, PI3K/Akt, Ca2+ signal transduction and ROS production (54). However, a number of studies have shown that these signal transduction pathways can often cross-react and generate specific cell regulation according to different signal stimulation. Wu et al (55) PI3K signaling could be stimulated by NKA with or without the presence of Src. Src inhibitors can downregulate PI3K signaling in cells treated with ouabain. Haas et al (56) showed that Src regulate interactions between NKA and EGFR, resulting in activated Ras/MAPK kinases cascade reaction. Further study will be performed to investigate the effects of AST on other signaling pathways, such as Raf/MEK/ERK or PI3K/Akt.

Decrease of Src and Erk1/2 phosphorylation levels could inhibit ROS generation, and p-Src and p-Erk1/2 were reduced by pNaKtide, which showed a cardioprotective effect in I/R injury (23). In the present study, treatment with H2O2, for 1 and 24 h significantly upregulated the levels of p-Src and p-Erk1/2. Pretreatment with 3S, 3′S-AST attenuated the activation of Src and Erk1/2 induced by H2O2; however, NKA protein expression levels were not changed. The present study demonstrated that 3S, 3′S-AST could inhibit oxidative stress injury of myocardial cells by reducing the NKA/Src/Erk1/2/ROS amplification signaling pathway.

In conclusion, 3S, 3′S-AST could inhibit ROS production and apoptosis by inhibiting the activation of Src and Erk1/2, and associated oxidant amplification signaling in myocardial cells following ROS treatment. The current study showed the therapeutic effects of 3S, 3′S-AST and its potential to be developed as a clinical approach for oxidative stress-related myocardial cell injury.


Not applicable.


This study was supported in part by the Nature Science Foundation of Zhejiang Province (grant no. LQ18H260003), the Technology Program of Zhejiang Province (grant no. 2017F30001), Key Subjects of Nutrition of Zhejiang Province (grant no. 16-zc03), Key Research and Development Program of Zhejiang Province (grant no. 2019C02028) and Zhejiang Provincial Bureau of Traditional Chinese Medicine (grant no. 2019ZZ005).

Authors' contributions

XQ and YW designed the project and supervised all research. XQ wrote the manuscript. ZZ, WH, ML, BZ, LZ, SM, ZH, DL, ZL and JC performed all experiments and analyzed data. All authors read and approved the final manuscript.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.



Ong SB, Hernández-Reséndiz S, Crespo-Avilan GE, Mukhametshina RT, Kwek XY, Cabrera-Fuentes HA and Hausenloy DJ: Inflammation following acute myocardial infarction: Multiple players, dynamic roles, and novel therapeutic opportunities. Pharmacol Ther. 186:73–87. 2018. View Article : Google Scholar : PubMed/NCBI


Hausenloy DJ and Yellon DM: Myocardial ischemia-reperfusion injury: A neglected therapeutic target. J Clin Invest. 123:92–100. 2013. View Article : Google Scholar : PubMed/NCBI


Xie B, Liu X, Yang J, Cheng J, Gu J and Xue S: PIAS1 protects against myocardial ischemia-reperfusion injury by stimulating PPARγ SUMOylation. BMC Cell Biol. 19:242018. View Article : Google Scholar : PubMed/NCBI


Sies H: Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress. Redox Biol. 11:613–619. 2017. View Article : Google Scholar : PubMed/NCBI


Yu D, Li M, Tian Y, Liu J and Shang J: Luteolin inhibits ROS-activated MAPK pathway in myocardial ischemia/reperfusion injury. Life Sci. 122:15–25. 2015. View Article : Google Scholar : PubMed/NCBI


Guo J, Wang SB, Yuan TY, Wu YJ, Yan Y, Li L, Xu XN, Gong LL, Qin HL, Fang LH and Du GH: Coptisine protects rat heart against myocardial ischemia/reperfusion injury by suppressing myocardial apoptosis and inflammation. Atherosclerosis. 231:384–391. 2013. View Article : Google Scholar : PubMed/NCBI


Mokhtari-Zaer A, Marefati N, Atkin SL, Butler AE and Sahebkar A: The protective role of curcumin in myocardial ischemia-reperfusion injury. J Cell Physiol. 234:214–222. 2018. View Article : Google Scholar : PubMed/NCBI


Faraone I, Sinisgalli C, Ostuni A, Armentano MF, Carmosino M, Milella L, Russo D, Labanca F and Khan H: Astaxanthin anticancer effects are mediated through multiple molecular mechanisms: A systematic review. Pharmacol Res. 155:1046892020. View Article : Google Scholar : PubMed/NCBI


Alam MN, Hossain MM, Rahman MM, Subhan N, Mamun MAA, Ulla A, Reza HM and Alam MA: Astaxanthin prevented oxidative stress in heart and kidneys of isoproterenol-administered aged rats. J Diet Suppl. 15:42–54. 2018. View Article : Google Scholar : PubMed/NCBI


Fang Q, Guo S, Zhou H, Han R, Wu P and Han C: Astaxanthin protects against early burn-wound progression in rats by attenuating oxidative stress-induced inflammation and mitochondria-related apoptosis. Sci Rep. 7:414402017. View Article : Google Scholar : PubMed/NCBI


Coombes JS, Sharman JE and Fassett RG: Astaxanthin has no effect on arterial stiffness, oxidative stress, or inflammation in renal transplant recipients: A randomized controlled trial (the XANTHIN trial). Am J Clin Nutr. 103:283–289. 2016. View Article : Google Scholar : PubMed/NCBI


Xu L, Zhu J, Yin W and Ding X: Astaxanthin improves cognitive deficits from oxidative stress, nitric oxide synthase and inflammation through upregulation of PI3K/Akt in diabetes rat. Int J Clin Exp Pathol. 8:6083–6094. 2015.PubMed/NCBI


Kim YJ, Kim YA and Yokozawa T: Protection against oxidative stress, inflammation, and apoptosis of high-glucose-exposed proximal tubular epithelial cells by astaxanthin. J Agric Food Chem. 57:8793–8797. 2009. View Article : Google Scholar : PubMed/NCBI


Ambati RR, Phang SM, Ravi S and Aswathanarayana RG: Astaxanthin: Sources, extraction, stability, biological activities and its commercial applications-a review. Mar Drugs. 12:128–152. 2014. View Article : Google Scholar : PubMed/NCBI


Kishimoto Y, Yoshida H and Kondo K: Potential anti-atherosclerotic properties of astaxanthin. Mar Drugs. 14:352016. View Article : Google Scholar


Zhang ZW, Xu XC, Liu T and Yuan S: Mitochondrion-permeable antioxidants to treat ROS-burst-mediated acute diseases. Oxid Med Cell Longev. 2016:68595232016.PubMed/NCBI


Shi Y, Lin P, Wang X, Zou G and Li K: Sphingomyelin phosphodiesterase 1 (SMPD1) mediates the attenuation of myocardial infarction-induced cardiac fibrosis by astaxanthin. Biochem Biophys Res Commun. 503:637–643. 2018. View Article : Google Scholar : PubMed/NCBI


Belliard A, Sottejeau Y, Duan Q, Karabin JL and Pierre SV: Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning. Am J Physiol Heart Circ Physiol. 304:H94–H103. 2013. View Article : Google Scholar : PubMed/NCBI


Li Z and Xie Z: The Na/K-ATPase/Src complex and cardiotonic steroid-activated protein kinase cascades. Pflugers Arch. 457:635–644. 2009. View Article : Google Scholar : PubMed/NCBI


Yan Y, Shapiro AP, Haller S, Katragadda V, Liu L, Tian J, Basrus V, Malhotra D, Xie Z, Abraham NG, et al: Involvement of reactive oxygen species in a feed-forward mechanism of Na/K-ATPase-mediated signaling transduction. J Biol Chem. 288:34249–34258. 2013. View Article : Google Scholar : PubMed/NCBI


Liu J, Tian J, Haas M, Shapiro JI, Askari A and Xie Z: Ouabain interaction with cardiac Na+/K+-ATPase initiates signal cascades independent of changes in intracellular Na+ and Ca2+ concentrations. J Biol Chem. 275:27838–27844. 2000.PubMed/NCBI


Wang Y, Ye Q, Liu C, Xie JX, Yan Y, Lai F, Duan Q, Li X, Tian J and Xie Z: Involvement of Na/K-ATPase in hydrogen peroxide-induced activation of the Src/ERK pathway in LLC-PK1 cells. Free Radic Biol Med. 71:415–426. 2014. View Article : Google Scholar : PubMed/NCBI


Li H, Yin A, Cheng Z, Feng M, Zhang H, Xu J, Wang F and Qian L: Attenuation of Na/K-ATPase/Src/ROS amplification signal pathway with pNaktide ameliorates myocardial ischemia-reperfusion injury. Int J Biol Macromol. 118:1142–1148. 2018. View Article : Google Scholar : PubMed/NCBI


Ding S, Liu D, Wang L, Wang G and Zhu Y: Inhibiting MicroRNA-29a protects myocardial ischemia-reperfusion injury by targeting SIRT1 and suppressing oxidative stress and NLRP3-mediated pyroptosis pathway. J Pharmacol Exp Ther. 372:128–135. 2020. View Article : Google Scholar : PubMed/NCBI


Karu I, Tähepõld P, Ruusalepp A and Starkopf J: Pretreatment by hyperoxia-a tool to reduce ischaemia-reperfusion injury in the myocardium. Curr Clin Pharmacol. 5:125–132. 2010. View Article : Google Scholar : PubMed/NCBI


Cadenas S: ROS and redox signaling in myocardial ischemia-reperfusion injury and cardioprotection. Free Radic Biol Med. 117:76–89. 2018. View Article : Google Scholar : PubMed/NCBI


Wang L, Lin R, Guo L and Hong M: Rosuvastatin relieves myocardial ischemia/reperfusion injury by upregulating PPAR-γ and UCP2. Mol Med Rep. 18:789–798. 2018.PubMed/NCBI


Hoffman JJ Jr, Gilbert TB, Poston RS and Silldorff EP: Myocardial reperfusion injury: Etiology, mechanisms, and therapies. J Extra Corpor Technol. 36:391–411. 2004.PubMed/NCBI


Visioli F and Artaria C: Astaxanthin in cardiovascular health and disease: Mechanisms of action, therapeutic merits, and knowledge gaps. Food Funct. 8:39–63. 2017. View Article : Google Scholar : PubMed/NCBI


Lauver DA, Lockwood SF and Lucchesi BR: Disodium Disuccinate Astaxanthin (Cardax) attenuates complement activation and reduces myocardial injury following ischemia/reperfusion. J Pharmacol Exp Ther. 314:686–692. 2005. View Article : Google Scholar : PubMed/NCBI


Shah MM, Liang Y, Cheng JJ and Daroch M: Astaxanthin-producing green microalga Haematococcus pluvialis: From single cell to high value commercial products. Front Plant Sci. 7:5312016. View Article : Google Scholar : PubMed/NCBI


EFSA Panel on Dietetic Products Nutrition and Allergies (NDA), . Scientific opinion on the safety of astaxanthin-rich ingredients (AstaREAL A1010 and AstaREAL L10) as novel food ingredients. EFSA J. 15–July;2014.(Epub ahead of print). doi: org/10.2903/j.efsa.2014.3757. PubMed/NCBI


Iwamoto T, Hosoda K, Hirano R, Kurata H, Matsumoto A, Miki W, Kamiyama M, Itakura H, Yamamoto S and Kondo K: Inhibition of low-density lipoprotein oxidation by astaxanthin. J Atheroscler Thromb. 7:216–222. 2000. View Article : Google Scholar : PubMed/NCBI


Yoshida H, Yanai H, Ito K, Tomono Y, Koikeda T, Tsukahara H and Tada N: Administration of natural astaxanthin increases serum HDL-cholesterol and adiponectin in subjects with mild hyperlipidemia. Atherosclerosis. 209:520–523. 2010. View Article : Google Scholar : PubMed/NCBI


Ruen-Ngam D, Shotipruk A and Pavasant P: Comparison of extraction methods for recovery of astaxanthin from Haematococcus pluvialis. Sep Sci Technol. 46:64–70. 2010. View Article : Google Scholar


Zhu S, Wang T, Luo F, Li H, Jia Q, He T, Wu H and Zou T: Astaxanthin inhibits proliferation and induces apoptosis of LX2 cells by regulating the miR29b/Bcl2 pathway. Mol Med Rep. 19:3537–3547. 2019.PubMed/NCBI


Fan CD, Sun JY, Fu XT, Hou YJ, Li Y, Yang MF, Fu XY and Sun BL: Astaxanthin attenuates homocysteine-induced cardiotoxicity in vitro and in vivo by inhibiting mitochondrial dysfunction and oxidative damage. Front Physiol. 8:10412017. View Article : Google Scholar : PubMed/NCBI


Okada Y, Ishikura M and Maoka T: Bioavailability of astaxanthin in Haematococcus algal extract: The effects of timing of diet and smoking habits. Biosci Biotechnol Biochem. 73:1928–1932. 2009. View Article : Google Scholar : PubMed/NCBI


Qiao X, Yang L, Zhang T, Zhou Q, Wang Y, Xu J and Xue C: Synthesis, stability and bioavailability of astaxanthin succinate diester. J Sci Food Agric. 98:3182–3189. 2018.PubMed/NCBI


Mercke OJ, Lignell A, Pettersson A and Hoglund P: Oral bioavailability of the antioxidant astaxanthin in humans is enhanced by incorporation of lipid based formulations. Eur J Pharm Sci. 19:299–304. 2003. View Article : Google Scholar : PubMed/NCBI


Barros MP, Marin DP, Bolin AP, de Cássia Santos Macedo R, Campoio TR, Fineto C Jr, Guerra BA, Polotow TG, Vardaris C, Mattei R and Otton R: Combined astaxanthin and fish oil supplementation improves glutathione-based redox balance in rat plasma and neutrophils. Chem-Biol Interact. 197:58–67. 2012. View Article : Google Scholar : PubMed/NCBI


Rao AR, Reddy RLR, Baskaran V, Sarada R and Ravishankar GA: Characterization of microalgal carotenoids by mass spectrometry and their bioavailability and antioxidant properties elucidated in rat model. J Agr Food Chem. 58:8553–8559. 2010. View Article : Google Scholar


Satoh A, Tsuji S, Okada Y, Murakami N, Urami M, Nakagawa K, Ishikura M, Katagiri M, Koga Y and Shirasawa T: Preliminary clinical evaluation of toxicity and efficacy of a new astaxanthin-rich Haematococcus pluvialis extract. J Clin Biochem Nutr. 44:280–284. 2009. View Article : Google Scholar : PubMed/NCBI


Baburina Y, Krestinin R, Odinokova I, Sotnikova L, Kruglov A and Krestinina O: Astaxanthin inhibits mitochondrial permeability transition pore opening in rat heart mitochondria. Antioxidants (Basel). 8:5762019. View Article : Google Scholar


Nakao R, Nelson OL, Park JS, Mathison BD, Thompson PA and Chew BP: Effect of astaxanthin supplementation on inflammation and cardiac function in BALB/c mice. Anticancer Res. 30:2721–2725. 2010.PubMed/NCBI


Kalogeris T, Bao Y and Korthuis RJ: Mitochondrial reactive oxygen species: A double edged sword in ischemia/reperfusion vs preconditioning. Redox Biol. 2:702–714. 2014. View Article : Google Scholar : PubMed/NCBI


Ma JJ, Yu YG, Yin SW, Tang CH and Yang XQ: Cellular uptake and intracellular antioxidant activity of Zein/Chitosan nanoparticles incorporated with quercetin. J Agric Food Chem. 66:12783–12793. 2018. View Article : Google Scholar : PubMed/NCBI


Goc Z, Szaroma W, Kapusta E and Dziubek K: Protective effects of melatonin on the activity of SOD, CAT, GSH-Px and GSH content in organs of mice after administration of SNP. Chin J Physiol. 60:1–10. 2017. View Article : Google Scholar : PubMed/NCBI


Couto N, Wood J and Barber J: The role of glutathione reductase and related enzymes on cellular redox homoeostasis network. Free Radic Biol Med. 95:27–42. 2016. View Article : Google Scholar : PubMed/NCBI


Lopez-Neblina F, Toledo AH and Toledo-Pereyra LH: Molecular biology of apoptosis in ischemia and reperfusion. J Invest Surg. 18:335–350. 2005. View Article : Google Scholar : PubMed/NCBI


Eefting F, Rensing B, Wigman J, Pannekoek WJ, Liu WM, Cramer MJ, Lips SJ and Doevendans PA: Role of apoptosis in reperfusion injury. Cardiovasc Res. 61:414–426. 2004. View Article : Google Scholar : PubMed/NCBI


Skou JC: The influence of some cations on an adenosine triphosphatase from peripheral nerves. Biochim Biophys Acta. 23:394–401. 1957. View Article : Google Scholar : PubMed/NCBI


Pierre SV and Xie Z: The Na,K-ATPase receptor complex: Its organization and membership. Cell Biochem Biophys. 46:303–316. 2006. View Article : Google Scholar : PubMed/NCBI


Cui X and Xie Z: Protein interaction and Na/K-ATPase-mediated signal transduction. Molecules. 22:9902017. View Article : Google Scholar


Wu J, Akkuratov EE, Bai Y, Gaskill CM, Askari A and Liu L: Cell signaling associated with Na(+)/K(+)-ATPase: Activation of phosphatidylinositide 3-kinase IA/Akt by ouabain is independent of Src. Biochemistry. 52:9059–9067. 2013. View Article : Google Scholar : PubMed/NCBI


Haas M, Wang H, Tian J and Xie Z: Src-mediated inter-receptor cross-talk between the Na+/K+-ATPase and the epidermal growth factor receptor relays the signal from ouabain to mitogen-activated protein kinases. J Biol Chem. 277:18694–18702. 2002. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

Volume 22 Issue 6

Print ISSN: 1791-2997
Online ISSN:1791-3004

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
Spandidos Publications style
Qu X, Zhang Z, Hu W, Lou M, Zhai B, Mei S, Hu Z, Zhang L, Liu D, Liu Z, Liu Z, et al: Attenuation of the Na/K‑ATPase/Src/ROS amplification signaling pathway by astaxanthin ameliorates myocardial cell oxidative stress injury. Mol Med Rep 22: 5125-5134, 2020
Qu, X., Zhang, Z., Hu, W., Lou, M., Zhai, B., Mei, S. ... Wang, Y. (2020). Attenuation of the Na/K‑ATPase/Src/ROS amplification signaling pathway by astaxanthin ameliorates myocardial cell oxidative stress injury. Molecular Medicine Reports, 22, 5125-5134.
Qu, X., Zhang, Z., Hu, W., Lou, M., Zhai, B., Mei, S., Hu, Z., Zhang, L., Liu, D., Liu, Z., Chen, J., Wang, Y."Attenuation of the Na/K‑ATPase/Src/ROS amplification signaling pathway by astaxanthin ameliorates myocardial cell oxidative stress injury". Molecular Medicine Reports 22.6 (2020): 5125-5134.
Qu, X., Zhang, Z., Hu, W., Lou, M., Zhai, B., Mei, S., Hu, Z., Zhang, L., Liu, D., Liu, Z., Chen, J., Wang, Y."Attenuation of the Na/K‑ATPase/Src/ROS amplification signaling pathway by astaxanthin ameliorates myocardial cell oxidative stress injury". Molecular Medicine Reports 22, no. 6 (2020): 5125-5134.