Original papers
Protective Effects of Tyrosol Against Oxidative Damage in L6 Muscle Cells
2018 年 24 巻 5 号 p. 943-947
詳細
2018 年 24 巻 5 号 p. 943-947
Tyrosol (2-(4-hydroxyphenyl) ethanol) is a phenylethanoid present in olive oil, with anti-oxidative, anti-inflammatory, and cerebroneural protection effects. In this study, the protective effect of tyrosol against oxidative damage was measured in L6 muscle cells. Tyrosol effectively inhibited H2O2-induced L6 cell death in part through regulation of ERK, JNK, and p38 MAP kinase and increased ATP production. In addition, it increased HO-1 expression in the cell. Based on results, tyrosol is effective in inhibiting oxidative damage of muscle cells.
Regular and proper exercise prevents and reduces hypertension, stroke, cardiovascular disease, diabetes, hyperlipidemia, and cancer (Bassuk and Manson, 2005; Roberts and Barnard, 2005). However, strenuous exercise produces excessive reactive oxygen species, causing oxidative damage to muscle tissues (Hyun, 2009; Ji, 1996). Reactive oxygen species is a term for chemically reactive molecules containing oxygen such as super-oxide radical, hydroxyl radical, hydrogen peroxide (H2O2), and singlet oxygen, produced during normal metabolic processes and play key physiological roles in the body. However, excessively produced reactive oxygen species induce peroxidation of lipids, the main components of cell membranes triggering tissue damage and disrupting DNA sequences of nucleic acids in the cell (Djordjevi, 2004). The human body is protected by producing antioxidant enzymes as a defense system against such oxidative damage, but protective capacity varies significantly depending on age and health status (Irshad and Chaudhuri, 2002; Urso and Clarkson, 2003). Antioxidants may enable the body's anti-oxidative defense system.
As a phenylethanoid present in olive oil, tyrosol (Fig. 1) has anti-oxidative, anti-inflammatory, and neural protection effects (Owen et al., 2000; Giovannini et al., 2002; Bu et al., 2007). However, studies on protective effect against oxidative damage in muscle cells have not been conducted. In this study, protective effect of tyrosol against reactive oxygen species-induced damage was examined in L6 muscle cells by measuring effect on cell viability after H2O2 treatment. Effects on mitogen-activated protein kinase (MAPK) signaling pathway and intracellular HO-1 production were also measured.
Chemical structure of tyrosol
Materials Tyrosol was purchased from Chromadex (Irvine, USA). Dulbecco's modification of Eagle medium (DMEM), fetal bovine serum (FBS), and Antibiotic Antibiotics (AA) were purchased from Gibco-BRL (Gaithersburg, USA). HO-1 antibody was purchased from Enzo Life Science (Farmingdale, USA). Cleaved caspase-3, p44/42 MAPK (Erk1/2), phospho-p44/42 MAPK (Erk1/2), p38 MAPK, anti-phospho-p38 MAPK, anti-JNK, anti-phospho-JNK, and tubulin antibodies were purchased from Cell Signaling Technology (Danvers, USA)
Cell culture L6 muscle cell line was obtained from the Korean Cell Line Bank (Seoul, Korea). Cells were cultured at 37°C, 5% CO2 using DMEM containing 10% FBS and 1% AA.
L6 cells were seeded at 1×105 cells/mL in a 96-well plate and incubated. Once the cells reached confluence, the medium was changed to DMEM containing 2% horse serum and 1% AA for differentiation into myotubes. Experiments were undertaken after differentiation.
Cell viability Cell viability was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay. To measure protective effect of tyrosol in muscle cells, cultured cells were treated with each concentration of tyrosol and 0.5 mM H2O2 at the same time and incubated again for 24 h. Cells were stained with MTT (0.5 mg/mL in PBS) solution, and absorbance was measured at 540 nm. Protective effect of tyrosol was expressed as percentage (%) by calculating recovery rate with tyrosol treatment of cell death rate with H2O2 treatment.
Western immunoblotting Cells were subsequently collected and lysis buffer (50 mM Tris-HCl, pH 8.0, 0.1% SDS, 150 mM NaCl, 1% NP-40, 0.02% sodium azide, 10 µg/mL PMSF, 1 µg/mL aprotinin) was added and the mixture sonicated to extract proteins inside cells. After cell lysis, proteins were separated using 10% SDS-poly-acrylamide gel electrophoresis and transferred to Hybond ECL nitrocellulose membrane. Membranes were blocked with 5% skim milk, and incubated with various primary antibodies: anti-cleaved caspase-3 (1: 2,000 dilution), anti-p44/42 MAPK (Erk1/2) (1: 2,000 dilution), anti-phospho-p44/42 MAPK (Erk1/2) (1: 2,000 dilution), anti-p38 MAPK(1: 1,000 dilution), anti-phospho-p38 MAPK (1: 2,000 dilution), anti-JNK (1: 2,000 dilution), anti-phospho-JNK (1: 1,000 dilution), or anti-HO-1 (1: 2,000 dilution) followed by horseradish peroxidase (HRP) conjugated anti-rabbit or mouse secondary antibody. Membranes were developed using enhanced chemiluminescence detection (Bio-Rad, Hercules, USA). Band intensities were analyzed using Bio-Rad Image Lab Software.
Adenosine triphosphate (ATP) production ATP production in L6 cells was measured using ATP bioluminescence assay kit HS II (Roche, Germany). Cultured cells were treated with each concentration of tyrosol and 0.5 mM H2O2 at the same time and incubated again for 24 h. Cells were lysised and ATP bioluminescence assay was conducted. Absorbance was measured using luminometer (Tecan, Switzerland).
Statistical analysis Results were expressed as mean ± standard deviation, and statistical significance was analyzed using one-way ANOVA followed by Tukey's post hoc test. P-values less than 0.05 were statistically significant.
Protective effects in muscle cells Strenuous exercise produces excessive reactive oxygen species, causing oxidative damage to muscle tissues (Morales-Alamo and Calbet, 2014). Protective effect of tyrosol against H2O2-induced oxidative damage was measured in L6 muscle cells to determine muscle cell-protecting effect. After confirming L6 cell death was not induced by 100 µM of tyrosol, L6 cells were treated with 1, 30, and 100 µM tyrosol samples with 0.5 mM H2O2 for 24 h. As a result, that measured cell viability, L6 cells treated with H2O2 revealed 43.2% of cell viability, but tyrosol treatment decreased cell death in a concentration-dependent manner (Fig. 2). The inhibitory effect of tyrosol on L6 cell death was 22.3, 33.1, and 58.3% at 1, 30, and 100 µM, respectively. The images of L6 cells are shown in Fig. 3.
Protective effects of tyrosol against H2O2-induced oxidative damage in L6 muscle cells. L6 cells were treated with 0.5 mM H2O2 and tyrosol for 24 h. Data represent the mean ± S.E. of three experiments. *p<0.05, significantly different from the H2O2-treated group.
Microscopic images of L6 cells. (A) Normal. (B) Differentiation. (C) H2O2. (D) H2O2 + 100 µM of tyrosol. L6 cells were treated with 0.5 mM H2O2 and tyrosol for 24 h. Scale bar = 100 µm.
Effect of tyrosol on H2O2-induced caspase-3 activation in muscle cells Caspase-3, a cysteine-dependent aspartate protease, is a key regulator of cell death (Nicholson, 1999). In order to verify the protective effect of tyrosol against H2O2-induced oxidative damage, we tested appearance of cleaved caspase-3 with Western immunoblotting analysis. Cleaved caspase-3 was significantly increased in cells treated with H2O2, and was markedly reduced after tyrosol treatment in a concentration-dependent manner (Fig. 4).
Effects of tyrosol on cleaved caspase-3 level in L6 cells.
(A) Western immunoblotting analysis of cleaved caspase-3 in L6 cell lysate. Tubulin was used as the loading control. (B) Quantification of Western immunoblotting. L6 cells were treated with 0.5 mM H2O2 and tyrosol for 24 h. Data represent the mean ± S.E. of three experiments. ***p<0.005, significantly different from the H2O2-treated group. ###p<0.005, significantly different from the unstimulated control group.
Effect of tyrosol on H2O2-induced ERK1/2, p38 MAPK, and JNK 1/2 activation in muscle cells We investigated if the MAPK signaling pathway, including extra cellular signal-regulated kinase (ERK), p38 MAP kinase (p38 MAPK), and c-Jun N-terminal kinase (JNK), was involved in protective effects of tyrosol against H2O2-induced cell death since numerous studies suggested ROS may activate MAPK signaling pathway (McCubrey et al., 2006; Ruffels et al., 2004). Activation of MAPK signaling pathway was determined with Western immunoblotting. As shown Figure 4, incubation of L6 muscle cells in presence of H2O2 induced ERK, p38, and JNK activation. Interestingly, we found that phosphorylation of those proteins was decreased after tyrosol treatment in a dose-dependent manner (Fig. 5). Especially, the level of phospho-JNK (p-JNK) was significantly reduced in presence of tyrosol under against H2O2-induced cell death. Many studies have reported that oxidative stress lead to increased level of p-JNK and subsequent cell death (Qian et al., 2015; Zheng et al., 2017).
Effects of tyrosol on MAPK phosphorylation in H2O2-treated L6 cells.
(A) Western immunoblotting analysis with indicated antibodies in L6 cell lysate. Tubulin was used as the loading control. (B–D) Quantification of Western immunoblotting. L6 cells were treated with 0.5 mM H2O2 and tyrosol for 24 h. Data represents the mean ± S.E. of four experiments. *p<0.05, **p<0.01, ***p<0.005, significantly different from the H2O2-treated group. ###p<0.005, significantly different from the unstimulated control group.
Effects on ATP production in muscle cells ATP is essentially required for survival of cells (Fei et al., 2015). Inhibitory effect of tyrosol on H2O2 induced ATP reduction was measured in L6 cells. As a result, ATP production was effectively increased after tyrosol treatment in L6 cells (Fig. 6).
Effects of tyrosol on ATP production.
After L6 cells were exposed to H2O2 in the presence or absence of tyrosol for 24 h, ATP contents were determined. Data represent the mean ± S.E. of three experiments. *p<0.05
Effects on HO-1 expression in muscle cells HO-1 plays a role of antioxidant, a transcription factor in the nucleus (Ogborne et al., 2004). Increased HO-1 expression is associated with decreased ROS and maintenance of the intracellular conditions. In this study, expression of HO-1 was measured using Western immunoblotting to assess the effect of tyrosol on expression of HO-1 as the representing factor of the anti-oxidation mechanism. As a result, expression of intracellular HO-1 protein was increased compared to the control when treated with tyrosol (Fig. 7).
Effects of tyrosol on HO-1 protein expression in L6 cells.
(A) Western immunoblotting analysis. (B) Quantification of Western immunoblotting. Data represent the mean ± S.E. of three experiments. ***p<0.005
Oxidative stress has a significant role in the etiology of variety of human diseases. Numerous studies have demonstrated that oxidative stress induced by chemical oxidants, such as H2O2, leads to cell loss (Cai et al., 2008). H2O2 has also been reported to induce apoptotic changes, which subsequently lead to death in a variety of cell systems including L6 cells (Xie et al., 2017). In this study, we investigated the antioxidant properties of tyrosol using an oxidative stress in L6 muscle cells. Numerous studies have used H2O2 (one of the major agents generated by oxidative stress) to induce cellular damage, accordingly, we also used H2O2 as an oxidative stress inducer.
From our data, it was shown that cell viability against H2O2-induced oxidative damage was dramatically increased by tyrosol treatment (Fig. 2). This observation was further confirmed by Western immunoblotting analysis for cleaved caspase-3, which is appeared by H2O2 as an effector of apoptotic cell death (Fig. 4). These results show that tyrosol has protective effects to H2O2-induced apoptosis through the inhibition of caspase-3 activation.
We also considered the possible involvement of three subfamilies of MAPK in the protective effects of tyrosol treatment: ERK1/2, JNK and p38 MAP kinase. These have been shown to be activated in response to the generation of ROS (Ferrer et al., 2001). Moreover, H2O2 can rapidly activate ERK, JNK, and p38, which are all involved in the cell death induced by ROS. We observed that H2O2 activates MAPKs, which is consistent with previous reports (Pan et al., 2013); however, tyrosol could effectively inhibit H2O2-induced phosphorylation of the three MAPKs, especially JNK (Fig. 5). The findings could be interpreted that JNK inactivation may be susceptible to tyrosol treatment for the protection.
It has been recently reported that tyrosol increased HO-1 expression against LPS-induced lung injury model (Wang et al., 2017). However, there is no study in muscle cells. In this study, we also demonstrated that tyrosol significantly increased HO-1 protein expression and ATP production in muscle cells (Fig. 6 and 7). Further study of HO-1/Nrf2 pathways is remains, because the activation of the HO-1/Nrf2 pathway is related to oxidative stress.
In this study, tyrosol significantly inhibited oxidative stress-induced cell death via at tenuation of JNK phosphorylation and increased HO-1 protein expression in L6 muscle cells. Therefore, tyrosol has considerable potential as a substance that inhibits muscle damage from oxidative stress triggered by strenuous exercise.
Acknowledgement This study was supported by the Korea Food Research Institute (E0186701).
