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Introduction

Cardiovascular disease (CVD) is a prime cause of death worldwide, accounting for 17.3 million deaths per year (1). Acute myocardial infarction is the most serious type of CVD. There are >3 million patients with acute ST-segment elevation myocardial infarction each year. The most effective treatment for these patients is timely and effective reperfusion therapy (2). Reperfusion therapy improves the myocardial blood supply and is accompanied by a series of pathophysiological reactions, including peroxidation, inflammation, intracellular calcium overload, and finally irreversible apoptosis and necrosis. Myocardial injury resulting from reperfusion is termed reperfusion injury (3), and it presents a clinical problem that urgently needs to be solved.

During myocardial ischemia-reperfusion (I/R), myocardial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme 4 (NOX4) expression is upregulated, and myocardial metabolic activity is enhanced, producing a large amount of reactive oxygen species (ROS) which contributes to myocardial injury (4). ROS can trigger a variety of signal transduction pathways, including enzyme-coupled receptor signaling pathways and G protein-coupled receptor signaling pathways, among which the MAPK signaling pathways play a key role in numerous cell activities (such as proliferation, differentiation, survival and death) (5). The inhibition of overactivated p38 MAPK can significantly reduce experimental myocardial I/R injury (6).

Plumbagin (PLB; 5-hydroxy-2-methyl-naphthalene-1,4-dione) is a major bioactive compound extracted from the roots of Plumbago zeylanica that acts as an inhibitor of NOX4(7). PLB not only inhibits adenosine diphosphate (ADP)-induced platelet aggregation in the cardiovascular system but also suppresses NOX4 expression, which can significantly improve the redox state imbalance of myocardial I/R (8-10). The aforementioned studies suggest that PLB can alleviate myocardial I/R injury and has robust potential for application in the treatment of CVD. As an organic hydroperoxide, tertiary butyl hydrogen peroxide (TBHP) is more stable than hydrogenperoxide (H2O2), and it selectively inhibits mitochondrial function, induces membrane lipid peroxidation, and promotes nucleotide degradation and adenosine formation (11). Based on the relatively well-defined mechanisms of TBHP action, TBHP-treated H9c2 cells were selected as a biological model likely to result in peroxidation injury in response to oxidative stress. In the present study, the protective effects of PLB in the prevention of TBHP-induced oxidative stress and apoptosis were verified in H9c2 cardiomyocytes.

Materials and methods

Materials

PLB (cat. no. S4777) was obtained from Selleck Chemicals. Anti-microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I (cat. no. PAB34124), anti-NOX4 (PAB30655), anti-phosphorylated (p)-p38 MAPK (PAB43139-P) and anti-p38 MAPK (PAB40560) were purchased from Bioswamp; Wuhan Bienle Biotechnology Co., Ltd. Anti-cleaved caspase-3 (ab214430) and anti-GAPDH (ab181602) were obtained from Abcam.

Cell culture and treatments

H9c2 cardiomyocytes were obtained from the American Type Culture Collection (ATCC: CRL-1446). Cells were grown in DMEM (product no. SH30022.01B; HyClone; Cytiva) supplemented with 10% FBS (Gibco), 100 µg/ml penicillin (Sigma) and 100 µg/ml streptomycin (Sigma) and maintained at 37˚C in a humidified 5% CO2 incubator. Confluent cardiomyocytes were cultured in DMEM supplemented with 2% FBS for an additional 12 h prior to experimentation. For experiments, cells were preincubated with PLB (5, 10 or 20 µM) for 24 h and TBHP (75 µM) for another 4 h. PLB was dissolved in DMSO and then diluted with DMEM to a final concentration of <0.1% DMSO. TBHP was dissolved in DMEM.

Cell viability assays

Cardiomyocytes were seeded in 96-well plates at a density of 5x103 cells/well. Cells were pretreated with PLB (5, 10, and 20 µM) for 24 h and then treated with TBHP for another 4 h. The number of viable cells was determined using a Cell Counting Kit-8 (CCK-8) assay. Briefly, the DMEM culture medium was discarded, and 100 µl CCK-8 reagent (Beyotime Institute of Biotechnology) was added to fresh DMEM. The 96-well plate was placed in a CO2 incubator for 2 h. The optical density (OD) values were determined at a wavelength of 450 nm. The cell proliferation rate (%) was calculated as follows: (OD value of experimental well - OD value of control well)/OD value of control well x100%. The CCK-8 assay was repeated 3 times for consistency.

Lactate dehydrogenase (LDH) and creatine kinase (CK) leakage

Cytotoxicity was evaluated by detecting plasma membrane damage using commercially available LDH-estimation (cat. no. A020-1) and CK-estimation (cat. no. A032-1-1) kits (both from Beyotime Institute of Biotechnology). For LDH and CK leakage assays, H9c2 cells were grown in 24-well plates at a density of 3x105 cells/well, and cells were subjected to further experiments after 24 h. The LDH and CK activities were measured after 24 h of treatment per the manufacturer's protocols.

Estimation of intracellular ROS production

ROS production was determined by detecting the fluorescence intensity of dichlorofluorescin (DCF) via flow cytometry. Cells were treated with 2'-7'dichlorofluorescin diacetate (DCFH-DA) (10 µM) at 37˚C in the dark for 20 min. Cells were then collected and suspended in PBS. The DCF fluorescence intensity was analyzed using flow cytometry (ACEA NovoCyte, ACEA Biosciences) at an excitation wavelength of 488 nm and an emission wavelength of 519 nm. Each assay was performed 3 times. Data were analyzed with NovoExpress 1.5 developed by ACEA Biosciences Inc.

Measurement of apoptosis

Following treatment, cardiomyocytes (1.5x105-1x106) were collected and immobilized in 75% cold ethanol for 12 h at 4˚C. Immobilized cells were double-stained with Annexin V-FITC (10 µl) and PI (10 µl; cat. no. 556547, BD Biosciences) in the dark at room temperature for 30 min. The apoptotic rate of cardiomyocytes was analyzed using flow cytometry (ACEA NovoCyte, ACEA Biosciences). Each test was repeated 3 times. Data were analyzed with NovoExpress 1.5 (ACEA Biosciences Inc.).

Western blot analysis

Protein levels were analyzed in whole cardiomyocyte lysates. H9c2 cell lysates were prepared with RIPA buffer containing protease inhibitor cocktail (cat. no. PAB180006; Bioswamp), and protein concentration were determined by BCA Protein Assay Kit. A total of 30 µg of protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore). Membranes were blocked in 5% bovine serum albumin (Beyotime) for 2 h at room temperature and then incubated with the following primary antibodies overnight at 4˚C: NOX4, cleaved caspase-3, LC3-II/LC3-I, p-p38 MAPK, p38 MAPK, and GAPDH (all 1:1,000). The following day, the membranes were incubated with HRP-conjugated secondary antibody (cat. no. SAB43714, Bioswamp, 1:10,000) at room temperature for 1.5 h. The secondary antibody was detected by ECL (Beyotime Institute of Biotechnology). The bands were scanned and quantified by densitometry analysis using Tanon GIS software (GIS 1D Ver.4.00, Tanon, Shanghai, China).

Statistical analysis

Data are presented as the mean ± SD. The significant differences between groups were assessed with SPSS version 13.0. Comparisons of results were performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Results

PLB protects H9c2 cells from cell death

Cell viability was assessed using the CCK-8 assay, as shown in Fig. 1A. TBHP significantly reduced cell viability compared with the control group (P<0.01). PLB (5, 10 or 20 µM) pretreatment attenuated the TBHP-induced reduction in H9c2 cell viability (P<0.01). Additionally, TBHP increased LDH and CK activities in H9c2 cardiomyocytes, and these increases were reduced by PLB pretreatment (P<0.01; Fig. 1B and C). The chemical structure of PLB is presented in Fig. 1D.

Figure 1 PLB protects H9c2 cells from cell death. (A) Cell viability. (B) LDH activity. (C) CK activity. (D) Chemical structure of PLB. Data are expressed as the mean ± SD; n=6. **P<0.01 vs. control; ##P<0.01 vs. TBHP; determined using one-way ANOVA with Tukey's post hoc analysis. PLB, plumbagin; LDH, lactate dehydrogenase; CK, creatine kinase; TBHP, tertiary butyl hydrogen peroxide; OD, optical density.

PLB alleviates TBHP-induced increase of ROS

The ROS levels in H9c2 cells were quantified by DCF-DA staining. TBHP treatment significantly increased intracellular ROS levels in H9c2 cells, and pretreatment with PLB (5, 10 and 20 µM) decreased ROS generation (P<0.01; Fig. 2).

Figure 2 PLB reduces ROS production. ROS levels were determined using DCFH-DA. (A) Control, (B) TBHP, (C) TBHP + PLB (5 µM), (D) TBHP + PLB (10 µM), and (E) TBHP + PLB (20 µM). (F) Bar graph represents DCF fluorescence intensity. Data are expressed as the mean ± SD; n=3. **P<0.01 vs. the control; ##P<0.01 vs. TBHP; determined using one-way ANOVA with Tukey's post hoc analysis. PLB, plumbagin; ROS, reactive oxygen species; DCFH-DA, 2'-7'dichlorofluorescin diacetate; TBHP, tertiary butyl hydrogen peroxide; DCF, dichlorofluorescin.

PLB prevents TBHP-induced apoptosis

As revealed in Fig. 3, the apoptotic rate was significantly increased in the TBHP group compared with the control group (P<0.01). Pretreatment with PLB (5, 10 or 20 µM) significantly reduced the apoptotic rate compared with TBHP treatment alone (P<0.01).

Figure 3 PLB prevents TBHP-induced apoptosis. Apoptotic rates were detected by Annexin V-FITC/PI assay. (A) Control, (B) TBHP, (C) TBHP + PLB (5 µM), (D) TBHP + PLB (10 µM), and (E) TBHP + PLB (20 µM). (F) Bar graph represents the apoptotic rates. Data are expressed as the mean ± SD; n=3. **P<0.01 vs. the control; ##P<0.01 vs. TBHP; determined using one-way ANOVA with Tukey's post hoc analysis. PLB, plumbagin; TBHP, tertiary butyl hydrogen peroxide.

PLB promotes autophagy in H9c2 cells

LC3-II/LC3-I levels, a marker of active autophagosomes, were analyzed by western blot analysis (Fig. 4). The ratio of LC3-II/LC3-I was decreased in TBHP-treated H2c9 cells, and this decrease was attenuated by PLB pretreatment compared with TBHP treatment alone (P<0.05). These data indicated that PLB induces autophagy in TBHP-treated H9c2 cells.

Figure 4 PLB promotes the autophagy of H9c2 cells. (A) Representative western blots for LC3-II/LC3-I. (B) Densitometric analysis for the ratio of LC3-II/LC3-I. Data are expressed as the mean ± SD; n=3. *P<0.05 vs. control; #P<0.05 vs. TBHP; determined using one-way ANOVA with Tukey's post hoc analysis. PLB, plumbagin; LC3, microtubule-associated protein 1 light chain 3; TBHP, tertiary butyl hydrogen peroxide.

PLB suppresses the NOX4/p38 MAPK pathway

NOX4, cleaved caspase-3, and p-p38/p38 MAPK protein expression was significantly increased in the TBHP group compared with the control group (P<0.05 and P<0.01). Pretreatment with PLB (5, 10 or 20 µM) suppressed TBHP-induced NOX4, cleaved caspase-3, and p-p38/p38 MAPK protein expression when compared with the TBHP only group (P<0.01 and P<0.05; Fig. 5).

Figure 5 PLB suppresses the NOX4/p38 MAPK pathway. (A) Representative western blots for NOX4, cleaved caspase-3, p-p38/p38 MAPK protein. (B) Densitometric analysis for expression of NOX4, cleaved caspase-3, p-p38/p38 MAPK protein. Data are expressed as the mean ± SD; n=3. *P<0.05 and **P<0.01 vs. the control; #P<0.05 and ##P<0.01 vs. TBHP; determined using one-way ANOVA with Tukey's post hoc analysis. PLB, plumbagin; NOX4, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme 4; p-, phosphorylated; TBHP, tertiary butyl hydrogen peroxide.

Discussion

Myocardial I/R injury is closely related to oxidative stress. Under physiological conditions, small amounts of oxygen free radicals can be quickly eliminated in the body. However, when cells are ischemic and hypoxic, intracellular metabolism becomes disordered, and oxygen free radical scavenging capacity is insufficient. When blood supply is suddenly restored in previously ischemic tissue, oxygen free radicals are produced at a level that is unable to be quickly eliminated, causing damage to myocardial tissues and surrounding cells (12-14). PLB is a natural naphthoquinone compound that protects against myocardial damage by modulating cardiac biomarkers, antioxidants, and apoptotic signaling in doxorubicin-induced cardiotoxicity in rats (15). PLB can also inhibit NOX4 and regulate redox signals (16). Thus, in the present study, the effect of PLB on oxidative stress-induced H9c2 cardiomyocyte injury was investigated. Experimental doses of PLB were based on a previous in vitro study (17).

TBHP is a pro-oxidant that increases membrane permeability, lipid peroxidation, ATP consumption, protein thiol group modification, and cytoplasmic calcium ion concentration imbalance by generating tert-butoxy groups (18). TBHP also causes cytotoxicity, mitochondrial-mediated apoptosis, and necrosis through the loss of membrane integrity, characterized by the release of cytochrome c, increased p53 expression, and mitochondrial membrane transformation (19). In the present study, the classic oxidant TBHP was selected to induce oxidative stress damage in the H9c2 cell line and to explore the cytoprotective effects of PLB on TBHP-induced cardiomyocyte injury. The electron carrier 1-methoxy PMS mediates CCK-8 reduction by dehydrogenase in cell mitochondria to form a highly water-soluble yellow formazan. The amount of formazan produced is directly proportional to the number of living cells. Therefore, CCK-8 measurement can indirectly reflect the number of living cells. The results of the present study demonstrated that TBHP reduced H9c2 proliferation rate, suggesting that TBHP-induced oxidative stress decreased cell viability. Pretreatment with PLB reduced the negative impact of TBHP on cellular proliferation and improved cell viability in a dose-dependent manner. LDH and CK are important diagnostic indicators of acute myocardial infarction, angina pectoris, myocarditis, and other myocardial injuries. Following ischemia and reperfusion, the myocardial cell membrane is damaged and its permeability increases, causing leakage of intracellular LDH and other enzymes. As a result, plasma LDH levels increase. CK is an important enzyme in energy metabolism. When various tissues and organs of the body are injured, intracellular CK leaks and cell vitality is reduced, which can particularly be observed with myocardial tissue damage that occurs during ischemia (20). PLB pretreatment reduces LDH and CK activity induced by TBHP, suggesting that PLB may improve TBHP-induced cardiomyocyte damage.

Excessive ROS production during the I/R period leads to oxidative stress, an important pathogenic factor of myocardial I/R injury (21). Under physiological conditions, there is a certain amount of ROS in the myocardium. When myocardial ischemia and hypoxia occur, the function of the ROS scavenging system decreases and the function of the generating system increases. Once the blood oxygen supply is restored, ROS are produced in large quantities and accumulate quickly, causing acute or chronic cardiomyocyte injury (22). During myocardial I/R, ROS is produced in large quantities by myocardial cell mitochondria, vascular endothelial cell purine oxidase and other oxidases, neutrophil respiratory bursts, catecholamines, and other pathways that allow the myocardial cell membrane and subcellular organelle membrane to communicate. Increased permeability and loss of membrane integrity result in membrane dysfunction, allowing a large amount of Ca2+ to flood into cells and the direct attack of cell structural proteins and nucleic acids by ROS. Consistent with this, TBHP significantly increased intracellular ROS levels in H9c2 cells in the present study, whereas TBHP-induced ROS production was reduced by PLB pretreatment.

Apoptosis is a main pathogenic mechanism of I/R injury (23,24). Oxidative stress leads to changes in the metabolic and functional properties of mitochondria in ischemic myocardium, thus activating the mitochondrial apoptotic pathway. ROS cause oxidative damage to membrane proteins and lipids, leading to mitochondrial dysfunction and cytochrome c release and ultimately activating caspases, particularly caspase-3, that induce apoptosis (25). In the present study, it was determined that PLB reduced the TBHP-induced apoptosis of H9c2 cells by reducing lysed caspase-3, indicating that PLB prevents apoptosis by inhibiting the intrinsic apoptotic pathway mediated by mitochondria.

Autophagy is a ubiquitous protein degradation process that removes abnormal proteins and organelles to promote energy recycling (26). The specific process is divided into induction of macroautophagy, formation of autophagosomes, autophagosome docking and fusion, and autophagic body breakdown (27). Autophagy occurs at a basic level to allow sustained metabolic recycling of intracellular components in most tissues. Under pathological conditions, autophagy can act as a cytoprotective mechanism to degrade and recycle defective cytoplasmic proteins (28). Autophagy inhibition in cardiomyocytes has adverse effects (29). Additionally, the dual effect of autophagy in CVD has been investigated. An increasing number of investigations have confirmed that autophagy stimulates the inflammatory response and is responsible for ceroid formation in atherosclerosis (30-33). LC3 plays a key role in autophagosome formation during autophagy. Activated LC3-I conjugates with the target lipid phosphatidylethanolamine (PE) on the outer membrane, forming LC3-II. LC3-II is cleaved from LC3-I and released back to the cytosol or degraded upon autophagosome maturation (34). Thus, expression of LC3-II/LC3-I has been regarded as a classic autophagy marker. In the present study, PLB induced autophagy in H9c2 cells, as evidenced by the increased LC3-II/LC3-I ratio determined by western blot analysis. Crosstalk between apoptosis (type 1 cell death) and autophagy (type 2 cell death) occurs in myocardial injury. When autophagy is promoted, cell death via apoptosis is inhibited (35). Likewise, results of the present study demonstrated that TBHP enhanced apoptosis in H9c2 cells, and PLB abolished this apoptosis when autophagy was induced.

MAPK activation constitutes a pattern of intracellular signaling and participates in myocardial I/R injury (36). The p38 MAPK pathway regulates the cellular response to growth, apoptosis, and stress signals in different cell models. Phosphorylation of threonine and tyrosine residues in p38 leads to conformational changes, thereby increasing accessibility of the p38 active site and enhancing catalysis (37). Some researchers have suggested that autophagy may be induced by activating p38 MAPK and that upregulating autophagy via the p38 MAPK pathway may protect H9c2 cells from oxidative stress (38,39). In addition, the downstream effects of p38 MAPK pathway activation depends on the assembly of Nox subunits into the NAPDH oxidase complex responsible for ROS production (40). Considering that ROS leads to p38 MAPK phosphorylation and cytotoxicity, the effect of PLB treatment on p38 MAPK phosphorylation was evaluated in the present study. Compared with the use of TBHP alone, PLB treatment significantly reduced TBHP-induced phosphorylation of p38 MAPK in H9c2 cells.

Scavenging ROS fails to effectively prevent CVD progression (41). Large-scale multicenter clinical studies (HOPE, SECURE, GISSI and HPS) (42-44) have not confirmed the therapeutic value of vitamin E (a free radical scavenger) in slowing atherosclerosis or in reducing major cardiovascular events (45). This may be due to the fact that simply administering antioxidant vitamins fails to eliminate the root cause of large amounts of ROS-oxidase, especially NOX (46). The NOX family is a tissue-specific homolog of NADPH oxidase, which exists on the plasma membrane of various nonphagocytic cells. Among the NOX family members, NOX1 and NOX5 are mainly expressed in smooth muscle and endothelial cells, respectively. NOX2 and NOX4 are found in endothelial cells, adventitia fibroblasts, and vascular smooth muscle cells, and NOX4 is the main source of intracellular ROS (47). The expression and activity of NOX4 are increased in myocardial I/R, leading to increased ROS and representing an important mechanism of myocardial injury (48). The results of the present study demonstrated that PLB suppressed NOX4 expression induced by TBHP in H9c2 cells, indicating that PLB may help reduce ROS production. Even though H9c2 cardiomyocytes have been widely used to investigate the molecular mechanisms underlying the cellular response to oxidative stress, it may not be fully consistent with other models. Thus, further studies based on in vivo models are still ongoing.

In summary, the present study suggested that PLB alleviated TBHP-induced cytotoxicity by reducing ROS-induced apoptosis and modulating autophagy in cardiomyocytes.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by the Science and Technology Project of the Changjiang River Administration of Navigational Affairs (grant no. 201810010) and Science Foundations of Hubei Provincial Health Commission (grant no. WJ2021M040).

Availability of data and materials

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

Authors' contributions

QZ and TW designed the study and wrote the manuscript. HF, WG and FC performed some of the experiments and collected the main data. FH conducted the statistical analysis. TW conceived the study. QZ and TW confirm the authenticity of all the raw data. All authors have read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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.

References