Plumbagin (PLB) has been previously reported to alleviate myocardial ischemia/reperfusion injury
Cardiovascular disease (CVD) is a prime cause of death worldwide, accounting for 17.3 million deaths per year (
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 (
Plumbagin (PLB; 5-hydroxy-2-methyl-naphthalene-1,4-dione) is a major bioactive compound extracted from the roots of
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.
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.
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.
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.
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.
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.).
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).
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.
Cell viability was assessed using the CCK-8 assay, as shown in
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;
As revealed in
LC3-II/LC3-I levels, a marker of active autophagosomes, were analyzed by western blot analysis (
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;
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 (
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 (
Excessive ROS production during the I/R period leads to oxidative stress, an important pathogenic factor of myocardial I/R injury (
Apoptosis is a main pathogenic mechanism of I/R injury (
Autophagy is a ubiquitous protein degradation process that removes abnormal proteins and organelles to promote energy recycling (
MAPK activation constitutes a pattern of intracellular signaling and participates in myocardial I/R injury (
Scavenging ROS fails to effectively prevent CVD progression (
In summary, the present study suggested that PLB alleviated TBHP-induced cytotoxicity by reducing ROS-induced apoptosis and modulating autophagy in cardiomyocytes.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
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.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
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 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. 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 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. (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.