Excessive production of vascular reactive oxygen species (ROS) plays a central role in the development and progression of cardiovascular diseases (CVD), including hypertension, atherosclerosis, and myocardial infarction (Kockx and Knaapen, 2000). In normal arteries, there is a low turnover of endothelial cells because endothelial cells produce nitric oxide (NO) to protect against endothelial apoptosis (Kang-Decker et al., 2007; Nematbakhsh et al., 2008). ROS act as a cellular mediator of endothelial dysfunction and apoptosis (Hsu et al., 2010; Niwa et al., 2003). Previous research has shown that excessive production of H2O2 impairs cell proliferation, reduces cell viability, and decreases the bioavailability of nitric oxide (Antunes and Cadenas, 2001; Price et al., 2000; Stone and Collins, 2002).
There are three mitogen-activated protein kinases (MAPKs): c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38-MAPK. All three are serine/threonine kinases. MAPK signaling is a response to stress. ERK is usually associated with cell survival, whereas JNK and p38 are usually associated with cell apoptosis (Carvalho et al., 2004; Colavitti et al., 2002; Peshavariya et al., 2009). It has been shown that H2O2 induces apoptosis through MAPK (Ramachandran et al., 2002).
Mangosteen (Garcinia mangostana L.) is a tropical fruit found extensively in Southeast Asia, including in Thailand. Many parts of the mangosteen tree have been used in traditional medicine, for example, the leaves and bark are used to treat eczema, and the pericarp is used to treat dysentery. The peel of the mangosteen fruit has been found to contain more than 50 xanthones (such as α-mangostin, γ-mangostin, β-mangostin, and garcinone), and it also contains flavonoids and proanthocyanidins (Fu et al., 2007). The xanthones are nonpolar, while the proanthocyanidins are polar. Xanthones have been shown to possess anti-inflammatory (Gutierrez-Orozco et al., 2013), anti-tumor (Hung et al., 2009; Kaomongkolgit et al., 2011; Krajarng et al., 2011; Kosem et al., 2013; Kritsanawong et al., 2016; Kwak et al., 2016; Lee et al., 2016; Li et al., 2014), and antioxidant properties (Jung et al., 2006; Kosem et al., 2007; Kondo et al., 2009). The polar fraction of mangosteen extract (ME) is also antioxidant. Moongkarndi et al. (2014) showed specifically that polar compounds extracted from mangosteen peel are more antioxidant than the nonpolar compounds. Previous studies have also shown that ME has neuroprotective effects during oxidative stress in SK-N-SH neuronal cells (Moongkarndi et al., 2010; Sattayasai et al., 2013). In addition, ME suppresses amyloid peptide-induced neural injury and prevents memory impairment in mice. However, very little research exists on the effects of ME on H2O2-induced endothelial cell injury. The current study examines the antioxidant effects of ME on H2O2-induced endothelial cell death.
MATERIALS AND METHODS
Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin-EDTA, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT), penicillin-streptomycin, and fetal bovine serum (FBS) were purchased from Gibco-Invitrogen (Grand Island, NY, USA). 2ʹ, 7ʹ dichlorodihydrofluorescein diacetate (DCF-DA) was obtained from Sigma-Aldrich (St. Louis, MO) and the apoptosis assay kit was purchased from Merck (Mendota Heights, MN).
Plant material and extraction
Mangosteen extraction followed the procedure previously described by Moongkarndi et al. (Moongkarndi et al., 2010; 2014). Briefly, the peel of the mangosteens was chopped, dried, crushed, and then macerated with ethanol. This crude ethanolic extract was then partitioned with ethyl acetate and water to fractionate the low polar and high polar fractions, respectively. That step separated xanthone and α-mangostin away from the water-soluble constituents. The water-soluble fraction was then spray dried to yield a powder extract. This powder extract is the mangosteen extract referred to as ME in this study. A preliminary analysis of the chemical constituents and biological activities of the ME were performed by thin-layer chromatography and high-performance liquid chromatography.
Human endothelial cell lines (EA.hy926; ATCC number CRL-2922) were cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin, and incubated with 5% CO2 at 37oC. This same incubation atmosphere and temperature were used throughout the study. This medium is hereafter referred to as “complete medium” (CM). At 70% confluence, the cells were prepared for subsequent experiments.
Cell cytotoxicity assay
Cytotoxicity of the ME and H2O2 were separately determined by MTT assay. 1 × 103 cells were incubated with CM in each well of a 96-well plate for 24 hours. After that, the original CM medium was removed and replaced with a low serum medium (LSM) consisting of DMEM supplemented with 1% FBS and 0.5% penicillin-streptomycin for 8 hours. The cells were then treated by adding various concentrations of ME (0, 1, 5, 10, 20, or 40 μg/ml) or H2O2 (0, 50, 100, 200, or 300 μM) to the existing medium and incubating them for another 24 hours. After that incubation, the medium was suctioned off and into each well was added fresh LSM and 5 mg/ml of MTT. The cells were then incubated at 37oC in a humidified atmosphere containing 5% CO2 for 4 hours. The cells were then again drained of their medium and DMSO was added to dissolve the formazan crystals. Finally, the optical density of each well’s contents was measured at 540 nm using a microplate reader.
Intracellular ROS assay
In order to first find the most effective concentration of H2O2 to produce ROS, cells were incubated with CM in 24 well plates. At 70% confluence, the CM was replaced with LSM and incubated for another 8 hours. Then to each well was added 20 μM DCF-DA. The cells were incubated for another 30 minutes and then washed with PBS and drained. Fresh LSM was added, along with H2O2 at concentrations of 0, 50, 100, 200, or 300 μM. The fluorescence intensity was immediately determined by a microplate reader with excitation 504 nm and emission 529 nm. Next, in order to determine the effect of ME on H2O2-induced ROS production, cells were prepared as per the above procedure, except just before adding DCF-DA, three different concentrations (1, 5, and 10 μg/ml) of ME were added as a pretreatment, followed by an additional 30 minutes of incubation and another PBS wash and replenishment of fresh LSM. Then DCF-DA was added just as before and the original procedure continued as above, except that only the optimal concentration of H2O2 was used (200 μM). Finally, fluorescence intensity was determined as before.
Cell viability assay
In order to determine the effect of ME on H2O2-induced cell death, cells were cultured and then pretreated with three non-toxic concentrations of ME (1, 5, and 10 μg/ml) for 30 minutes. After that, the cells were treated with 200 μM H2O2 and then incubated for 24 hours. MTT was added, followed by 4 hours of additional incubation. Finally, DMSO was added. The optical density was measured using a microplate reader at 540 nm.
In brief, cells were seeded onto an 18-mm coverslip in a 12-well plate and incubated until 70% confluence. The cells were then pretreated with ME for 30 minutes and then treated with H2O2 for 24 hours. Apoptotic cells were detected using the Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay according to the manufacturer’s protocol.
Western blot analysis
Cells were pretreated with ME for 30 minutes and then treated with H2O2 for 15 minutes. Cells were washed with PBS and then extracted with RIPA buffer. 20 μg of protein was separated using 8%–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane. After washing and blocking, the membrane was incubated with the primary antibodies (p-p38, t-p38, p-JNK, t-JNK, p-ERK, t-ERK, cleaved caspase3, cleaved poly(ADP-ribose) polymerase-1 (PARP-1), and β-actin) overnight at 4oC. The membranes were then incubated again with horseradish peroxidase-conjugated secondary antibodies against the primary antibodies (1:2,000) for 1 hour more at room temperature. Blots were visualized by enhanced chemiluminescence and detected using the Gel Doc XR+ system. Band densities were analyzed with Image J software.
All data are expressed as the mean ± standard error of the mean (SEM). Statistical comparisons were analyzed using one-way ANOVA with the Tukey Post hoc test. A p-value of less than 0.05 was considered as statistically significant.
Cell cytotoxicity and intracellular ROS production
Results of the cell cytotoxicity assay on ME (Fig. 1) showed that ME concentrations of 20 and 40 μg/ml significantly reduced the percentage of viable cells, by 10% and 60%, respectively, whereas ME concentrations of 1, 5, and 10 μg/ml did not significantly reduce cell viability. These three concentrations are, therefore, considered non-toxic and were used in the subsequent experiments. Similarly, results of H2O2 cytotoxicity are shown in Figure 2a. Only H2O2 concentrations of 200 and 300 μM significantly increased cell death. The results of the intracellular ROS assay for selecting the optimal H2O2 concentrations are seen in Figure 2b. H2O2 enhanced ROS production in a concentration-dependent manner. Specifically, H2O2 concentrations of 200 and 300 μM significantly increased ROS production and cell death. 200 μM H2O2 was used in the subsequent experiments. Since 200 μM was the minimum concentration that caused both maximum ROS production and significant cell death.
Effect of ME on H2O2-induced intracellular ROS production
Next, the results of the intracellular ROS assay for determining the effect of ME on H2O2-induced ROS production are displayed in Figure 3. ME at 5 and 10 μg/ml significantly reduced H2O2-induced ROS. Both of these ME concentrations yielded a 30% reduction.
Effect of ME on H2O2 induced cell death
Figure 4 shows that ME at 5 and 10 μg/ml significantly inhibited H2O2-induced cell death. Both of these ME concentrations enhanced cell viability by 10%.
|Figure 1. Effect of ME on cell viability. Cells were treated with various concentrations of ME. Cell viability was assessed by MTT assay (n = 3, ** p < 0.01, *** p < 0.001 vs. control).|
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|Figure 2. Effects of H2O2 on cell death and ROS formation. Cells were treated with different concentrations of H2O2 for 24 hours. (A) Cell viability was determined by MTT assay and (B) ROS production was detected by DCF-DA (n = 3, * p < 0.05, ** p < 0.01 vs. control).|
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Effect of ME on H2O2-induced apoptosis
Mangosteen extract’s inhibition of H2O2-induced apoptosis can be seen in Figure 5. ME concentrations of 5 and 10 μg/ml significantly inhibited cell apoptosis. The inhibition by either concentration was 33%.
ME suppresses H2O2-induced apoptosis through p38 MAPK
The western blot results are displayed in Figure. 6. H2O2 significantly activated p-p38 MAPK, cleaved caspase 3, and cleaved PARP-1. ME significantly inhibited these three processes.
Endothelial cells are the innermost layer of blood vessels and they regulate vascular tone. Endothelial cells produce and release not only vasorelaxant substances such as NO and endothelium-derived hyperpolarizing factors but also vasoconstrictors, including endothelin, thromboxane A2, and prostaglandin H2. In normal physiology, there is a low turnover of endothelial cells in arteries because of the action of NO against apoptosis. However, excessive vascular ROS can induce endothelial dysfunction and apoptosis, leading to the development and progression of CVD. Therefore, ROS can greatly affect the molecular signaling pathway of endothelial apoptosis. Previous studies have established that H2O2 induces apoptosis in endothelial cells (Niwa et al., 2003; Hsu et al., 2010), epithelial cells (Carvalho et al., 2004), and neuronal cells (Sattayasai et al., 2013).
Polyphenols from mangosteen have been shown to possess antioxidant properties. For example, α-mangostin prevented cardiac reperfusion injury by reducing oxidative stress in the Lagendorff-reperfused heart model (Buelna-Chontal et al., 2011). In other studies, α-mangostin also attenuated ROS production induced by hypoxia in retinal endothelial cells (Jittiporn et al., 2014) by H2O2 in human retinal pigment epithelial cells (Fang et al., 2016) and by high glucose in endothelial cells (Jittiporn et al., 2017). Also, α-mangostin reduced total ROS and mitochondrial ROS in high fat-induced hepatic steatosis (Tsai et al., 2016). The mangosteen extracts used in the above-mentioned studies were all semi-polar solvent extracts with purified α-mangostin (>98%), while the studies mentioned below all used a powdered ME. The finished ME powder contained less than 2% α-mangostin but included a broad spectrum of other polyphenols from the fruit. A previous study by Moongkarndi et al. (2010) showed that ME suppresses beta-amyloid-induced oxidative stress in SK-N-SH neuroblastoma cells (Moongkarndi et al., 2010). In addition, Sattayasai et al. (2013) found that ME demonstrates antioxidant action in H2O2-induced or polychlorinated-biphenyl induced oxidative stress in human neuroblastoma cells. In the same study, ME also protected against memory deficit in mice. Suthammarak et al. (2016) studied the antioxidant action of ME in healthy volunteers aged 20–60. That study showed that ME enhances the antioxidant ability of red blood cells (RBC) and decreases oxidative damage of proteins within RBCs. Jaisupa et al. (2018) showed that ME attenuated H2O2-induced intracellular ROS production through enhancement of catalase and heme oxygenase-1 in human neuroblastoma cells and human embryonic kidney cells. Dovetailing with the findings of the previous ME studies mentioned here, the results of the present study demonstrated that ME attenuated intracellular ROS in endothelial cells.
|Figure 3. Impact of ME on H2O2-induced ROS formation. Human endothelial cells were treated with or without H2O2 in the presence or absence of ME. ROS production was detected by DCF-DA (n = 3, ###p < 0.001 vs. vehicle, * p < 0.05 vs. vehicle + H2O2).|
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|Figure 4. Impact of ME on H2O2-induced cell damage. Cells were treated with or without H2O2 in the presence or absence of ME. Cell viability was determined by MTT assay (n = 3, #p < 0.05, ###p < 0.001 vs. vehicle, *p < 0.05 vs. vehicle + H2O2).|
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Specifically, this study showed that ME significantly suppressed H2O2-induced endothelial apoptosis in a dose-dependent manner by inhibiting ROS production. The results indicate that ME significantly suppressed TUNEL positive cell death and MTT cell death induced by H2O2. The signaling pathway of ROS induced apoptosis relies on MAPKs (Carvalho et al., 2004; Matsuzawa and Ichijo, 2008; Son et al., 2011). This study showed that ME inhibits H2O2-induced phosphorylation of p38 but does not inhibit ERK, JNK, or Akt (data not shown). ME also significantly suppressed apoptotic downstream signaling including cysteine-dependent aspartate-specific proteases 3 (caspase 3) and PARP-1. Caspase 3, which belongs to the cysteine proteases group, is considered the main terminal cleavage enzyme in the apoptotic cascade. Activation of caspase 3 results in the cleaving of PARP-1. Cleavage of PARP-1 is another hallmark of apoptosis (Mallat and Tedgui, 2000; Nicholson et al., 1995). Previous studies have shown that α-mangostin attenuated hepatic cell apoptosis in high fat-induced hepatic steatosis in rats (Tsai et al., 2016) as well as high glucose-induced apoptosis in endothelial cells through inhibition of phosphorylation of JNK and p38 MAPK (Jittiporn et al., 2017) and inhibition of acid sphingomyelinase (Luo and Lei, 2017). ME also suppressed amyloid and H2O2-induced neuronal cell apoptosis (Moongkarndi et al., 2010; Sattayasai et al., 2013) and decreased scopolamine-induced memory impairment in mice (Sattayasai et al., 2013). The current study indicates that by downregulating the p38 MAPK, caspase3, and PARP-1 signaling pathway, ME is able to protect against H2O2-induced apoptosis in endothelial cells.
|Figure 5. Impact of ME on H2O2-induced cell apoptosis. (A) Cells were treated with or without H2O2 in the presence or absence of ME. Apoptotic cells detected by TUNEL assay. (B) Quantification of apoptotic cells was determined manually (n = 3, ##p < 0.01 vs. vehicle, *p < 0.05 vs. vehicle + H2O2).|
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|Figure 6. Determination of the apoptosis signaling in response to treatment of ME. Cells were treated with or without H2O2 in the presence or absence of ME for 15 minutes. (A) p38, (B) caspase3 and PARP-1 were detected by Western blot (n = 3, # p < 0.05, ## p < 0.01 vs. vehicle, *p < 0.05, **p < 0.01 vs. vehicle + H2O2).|
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This study has demonstrated that water extracts of mangosteen exert antioxidant and anti-apoptotic actions on endothelial cells through p38 MAPK, and these abilities make ME a good candidate for use as a natural substance in the prevention of CVD.
This study was made possible in part by a generous grant from the National Research Council of Thailand in cooperation with Naresuan University (grant R2561B037). The authors would like to thank the Naresuan University Writing Clinic (DIALD) for their editing assistance.
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
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