Yoo-hwan Lee ¹, Jung-hee Kim ¹, Choon-ho Song ¹, Kyung-jeon Jang ¹, Cheol-hong kim ¹, Ji- Sook Kang ², Yung-hyun Choi ^2,3, Hyun-Min Yoon ^{1 *}

The mushroom Ganoderma lucidum has been widely used as a traditional herbal medicine for many years. Although several studies have focused on the anti-oxidative activity of this mushroom, the molecular mechanisms underlying its activity have not yet been clearly established. The present study investigated the cytoprotective effect of ethanol extract of Ganoderma lucidum (EGL) against oxidative stress (hydrogen peroxide, H₂O₂) and elucidated the underlying mechanisms in a C2C12 myoblast cell line.

Oxidative stress markers were determined by using the comet assay to measure reactive oxygen species (ROS) generation and deoxyribonucleic acid (DNA) damage. Cell viability and Western blotting analyses were employed to evaluate the cellular response to EGL and H₂O₂ in C2C12 cells. Transfection with nuclear factor erythroid 2-related factor 2 (Nrf2)-specific small interfering ribonucleic acid (siRNA) was conducted to understand the relationship between Nrf2 expression and H₂O₂-induced growth inhibition.

The results showed that EGL effectively inhibited H₂O₂-induced growth and the generation of ROS. EGL markedly suppressed H₂O₂-induced comet-like DNA formation and phosphorylation of histone H2AX at serine 139 (p-γH2AX), a widely used marker of DNA damage, suggesting that EGL prevented H₂O₂-induced DNA damage. Furthermore, the EGL treatment effectively induced the expression of Nrf2, as well as heme oxygenase-1 (HO-1), with parallel phosphorylation and nuclear translocation of Nrf2 in the C2C12 myoblasts. However, zinc protoporphyrin IX, a HO-1 inhibitor, significantly abolished the protective effects of EGL against H₂O₂-induced accumulation of ROS and reduced cell growth. Notably, transient transfection with Nrf2-specific siRNA attenuated the cytoprotective effects and HO-1 induction by EGL, indicating that EGL induced the expression of HO-1 in an Nrf2-dependent manner.

Collectively, these results demonstrate that EGL augments the cellular anti-oxidant defense capacity through activation of Nrf2/HO-1, thereby protecting C2C12 myoblasts from H₂O₂-induced oxidative cytotoxicity.

anti-oxidant, Ganoderma lucidum, heme oxygenase-1, Nrf2, reactive oxygen species

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

The decrease in anti-oxidant mechanisms, together with excessive accumulation of reactive oxygen species (ROS), including superoxide radical anions, hydroxyl radicals, singlet oxygen, and hydrogen peroxide (H₂O₂), are causally linked to various health problems [1, 2]. Within healthy muscle cells, ROS are generated both as by-products of metabolism and as critical effectors of signaling cascades [3]. However, ROS also have genotoxicological effects, causing damage to major cellular components, including proteins, lipids, and nucleic acids [4, 5]. Excessive generation of ROS in skeletal muscles can influence the contractile function of skeletal muscles by causing fatigue and increasing cell oxidative damages, which is implicated in the development of multiple diseases, including muscular dystrophy and sarcopenia [6, 7]. Therefore, increasing the anti-oxidant capacity of skeletal muscles can play a role in preventing disease, improving muscle performance, and enhancing quality of life.

The fruiting body of Ganoderma lucidum (G. lucidum; Polyporaceae) has been used as a traditional Oriental medicine in tonics for promoting longevity and health for more than 2000 years in China, Korea, Japan and other Asian countries [8, 9]. Accumulating evidence indicates that this mushroom possesses diverse and potentially significant pharmacological effects, including anti-cancer, anti-inflammatory, anti-diabetic, and anti-bacterial effects, in addition to hepatoprotective, neuroprotective, and immunoenhancing activities [8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19]. Studies also suggest that G. lucidum possesses strong anti-oxidant properties, which protect cells against oxidative-induced damage through the induction of phase II enzymes, such as heme oxygenase-1 (HO-1), quinone reductase, glutathione S-transferase (GST), and glutathione reductase [20-25]. G. lucidum was reported to protect macrophages against inflammatory stress via the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2), mediated by the expression of HO-1 [22]. However, to date, the mechanisms underlying the inhibition of oxidative stress by G. lucidum have not been well characterized.

Nrf2, a basic leucine zipper transcription factor ubiquitously expressed in most tissues, is a master cellular sensor of oxidative stress and represents the primary response to changes in the cellular redox state [26, 27]. Under normal conditions, Nrf2 is bound to Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm. Phase II enzyme inducers can disrupt the Nrf2/Keap1 complex, resulting in the release of Nrf2 and its subsequent translocation to the nucleus [28, 29]. In the nucleus, Nrf2 binds with and activates the anti-oxidant response element (ARE), a cis-acting enhancer present in the promoter region of a large and distinct set of target genes, with the aim of restoring redox homeostasis [27, 30]. HO-1 was reported to be active in protecting cells exposed to oxidizing agents by catalyzing the oxidation of heme to biologically active products, including carbon monoxide, biliverdin, and ferrous iron [31]. The final products of heme catabolism exert antioxidant effects by neutralizing intracellular ROS [32, 33]. Moreover, enhanced HO-1 expression, which is mainly regulated by the Nrf2-ARE pathway, is associated with protection against stress, such as oxidative stress [26, 34, 35]. Recent studies reported that the stabilization or phosphorylation of the Nrf2 protein increased the activation of the Nrf2/HO-1 pathway. Several signaling molecules have been shown to participate in this process in response to a variety of phase II gene inducers [36, 37]. Therefore, pharmacological activation of Nrf2/HO-1 signaling is critical for the protection of cells exposed to oxidative stress. The present study investigated the ability of an ethanol extract of G. lucidum (EGL) to protect cells from H₂O₂-induced cell damage and to elucidate the mechanism underlying those protective effects by using a mouse myoblast C2C12 cell line.

EGL was supplied by the Department of Acupuncture and Moxibustion, Dongeui University College of Korean Medicine (Busan, Korea). For the preparation of EGL, freezedried and milled G. lucidum fruiting bodies (200 g) were extracted with 25% ethanol (4 L) at room temperature for 10 hours by using a blender. The extracts were filtered through a Whatman no. 2 filter (pore size: 0.22 μm, Whatman, Maidstone, UK), concentrated to 500 mL under vacuum conditions and then stored at ─ 20°C [38]. The EGL solution was directly diluted in culture medium to the desired concentration prior to use.

The C2C12 mouse myoblast cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were grown in Dulbecco’s modified Eagle’s medium (GibcoBRL, Gaithersburg, MD, USA) and supplemented with 10% heat-inactivated fetal bovine serum (GibcoBRL) and 100 μg/mL of penicillin/streptomycin antibiotics in a humidified 5% CO₂ atmosphere at 37°C. Nrf2 small interfering ribonucleic acid (siRNA) and control siRNA were purchased from Santa Cruz Biotechnology, Inc. Lipofectamine^® RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA, USA) was used according to the manufacturer’s instructions to transfect the siRNA into the cells. For transfection, the cells were seeded in 6-well culture plates and incubated with the control siRNA or Nrf2 siRNA at 50 nM for 6 hours in serum-free OPTI-MEM media (Invitrogen). After incubation, the transfected cells were subjected to the treatment described in the Fig. 6.

For the cell viability assay, C2C12 cells were seeded in 6-well plates at a density of 1 × 10⁵ cells per well. After incubation for 24 hours, the cells were treated with EGL at different concentrations in the presence or absence of H₂O₂ (Sigma-Aldrich Chemical Co., St. Louis, MO, USA), zinc protoporphyrin IX (ZnPP; Sigma-Aldrich Chemical Co.) or N-acetyl-L-cysteine (NAC, Sigma-Aldrich Chemical Co.). After incubation for 24 hours, the medium was discarded, and the cells were incubated with 0.5 mg/mL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Chemical Co.) solution for 3 hours at 37°C. The supernatant was discarded, and the formazan blue that formed in the cells was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Chemical Co.). The optical density was then measured at 540 nm by using an enzyme-linked immunosorbent assay (ELISA) plate reader (Dynatech MR-7000; Dynatech Laboratories, Chantilly, VA, USA).

The measurement of the ROS was performed using ROS-sensitive 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA, Molecular Probes, Eugene, OR, USA) reagent, a fluorescent dye that visualizes ROS. Briefly, the cells were incubated with 10-μM DCF-DA for 30 minutes at room temperature in the dark. Spectrofluorimetry analysis using an ELISA plate reader was performed to quantify the intracellular ROS at excitation and emission wavelengths of 488 and 525 nm, respectively.

To assess deoxyribonucleic acid (DNA) damage, we mixed the cell suspension with 0.5% low-melting agarose (LMA) at 37°C, and we spread the mixture on a fully-frosted microscopic slide, which had been pre-coated with 1% normal-melting agarose. After the solidification of the agarose, the slide was covered with 0.5% LMA and was subsequently immersed in a lysis solution [2.5-M sodium chloride (NaCl), 100-mM Na-ethylenediaminetetraacetic acid (EDTA), 10-mM Tris, 1% Triton X100, and 10% DMSO (pH 10)] for 1 hour at 4°C. The slides were placed in a gel electrophoresis apparatus containing 300-mM sodium hydroxide (NaOH) and 10-mM Na-EDTA (pH 13) for 40 minutes to allow for the unwinding of the DNA and for the expression of alkali-labile damage. An electrical field was then applied (300 mA, 25 V) for 20 minutes at 4°C to draw the negatively-charged DNA toward the anode. After electrophoresis, the slides were washed three times for 5 minutes each time at 4°C in a neutralizing buffer (0.4-M Tris, pH 7.5), followed by staining with 20 μg/mL of propidium iodide (Sigma-Aldrich Chemical Co.). The slides were washed twice with phosphate buffer saline (PBS), and images were then captured using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany). The images were also analyzed using an image analysis system (Komet 5.5, Kinetic Imaging, Liverpool, UK) to evaluate the degree of DNA damage. The tail length and moment were used as measures of the extent of the DNA damage. One hundred cells were randomly selected and measured (two slides made for one sample, 50 randomly-selected cells per slide) from one sample. The values of the mean tail length per sample and the percentages of cells with different tail lengths (i.e., undamaged cells without a tail, cells with a tiny tail, cells with a dim tail, cells with a clear tail, and cells with only a tail) were calculated [39].

To prepare the whole-cell proteins, we collected and lysed cells with lysis buffer (25 mM of Tris-Cl (pH 7.5), 250 mM of NaCl, 5 mM of EDTA, 1% Nonidet P-40, 0.1 mM of sodium orthovanadate, 2 μg/mL of leupeptin, and 100 μg/ mL of phenylmethylsulfonyl fluoride) containing protease inhibitor cocktail tablets for 30 minutes at 4°C. In a parallel experiment, nuclear and cytosolic proteins were prepared using nuclear extraction reagents (Pierce Biotechnology, Rockford, IL, USA) according to the manufacturer’s protocol. The cell debris was discarded following centrifugation at 13,000 g for 15 minutes, and the supernatants were collected. The protein content was determined using a BioRad protein assay reagent (BioRad, Hercules, CA, USA) according to the manufacturer’s instructions. For Western blot analyses, equal amounts of protein extracts were subjected to electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels and subsequently transferred onto nitrocellulose membranes (Schleicher & Schuell Bioscience, Inc., Keene, NH, USA) by electroblotting. The membranes were blocked with 5% skimmed milk for 1 hour at room temperature and incubated overnight with primary anti-bodies (Cell Signaling Technology, Inc., Boston, MA, USA; Santa Cruz Biotechnology, Santa Cruz, CA, USA; Abcam, Inc., Cambridge, UK), followed by incubation for 1 hour with horseradish peroxidase-conjugated donkey anti-rabbit and sheep anti-mouse immunoglobulin (Amersham Biosciences, Arlington Heights, IL, USA). The immunoreactive bands were revealed by using enhanced chemiluminescence (ECL) with a commercially-available ECL kit (Amersham Biosciences) according to the recommended procedure. Actin and poly [adenosine diphosphate (ADP)- ribose] polymerase (PARP) were used as internal controls for total cellular and nuclear proteins, respectively.

Data from at least three independent experiments were expressed as the mean ± standard deviation (SD). Statistical comparisons between the different groups were performed using a one-way analysis of variance (ANOVA), followed by Student’s t-tests, after comparing each treated group to the negative control. Values of P < 0.01 were considered statistically significant.

To evaluate the protective effect of EGL on H₂O₂-induced cytotoxicity, we treated the C2C12 cells with various concentrations of EGL for 24 hours, and the optimal dose (i.e., the dose that had the least effect on cell viability) was selected by determining the percentage of MTT reduction. As shown in Fig. 1A, treatment of the cell cultures with 5 ─ 50 μg/mL of EGL for 24 hours had no effect on the cell viability. However, 100 μg/mL EGL induced a partial reduction in the cell viability. Thus, 50 μg/mL of EGL was chosen as the highest optimal dose for studying the cytoprotective effect of EGL against H₂O₂-induced cell damage. To determine the protective effects of EGL against H₂O₂-induced cytotoxicity in the C2C12 cells, we pretreated the cells with EGL for 1 hour and exposed them to H₂O₂ for an additional 24 hours. The viability of the C2C12 cells when exposed to H₂O₂ at a concentration of 1 mM for 6 hours decreased significantly as compared with that of the control group, and the survival rate was approximately 58.70% that of the control. However, when the C2C12 cells were pretreated with 25 and 50 μg/mL of EGL, the cell viability significantly increased as compared with that of the H₂O₂ group, and the survival rates were 71.20% and 81.70% that of the control, respectively (Fig. 1B).

To examine the inhibitory effect of EGL on H₂O₂-induced ROS production, we stimulated the C2C12 cells with 1-mM H₂O₂ for 30 minutes in the presence or absence of 50 μg/mL of EGL; then, we determined the intracellular levels of ROS. Compared to the non-treated control cells, the C2C12 cells treated with 1-mM H₂O₂ for 30 minutes showed levels of intracellular ROS that were markedly increased, as indicated by an increase in the DCF-liberated fluorescent signal (Fig. 1C). However, pretreatment with EGL significantly reduced H₂O₂-induced ROS production as compared with that of the control. As a positive control, 10-mM NAC, a specific ROS scavenger, also markedly attenuated H₂O₂-induced generation of ROS. The results indicate that EGL possesses scavenging activity, preventing the accumulation of ROS generated by H₂O₂ in C2C12 myoblasts.

Oxidative stress-induced DNA damage produces lesions that lead to a loss of cell viability. H₂O₂-mediated DNA damage in the C2C12 cells was detected using an alkaline comet assay (single-cell gel electrophoresis) and a Western blot analysis. The comet assay revealed that the treatment with H₂O₂ alone significantly increased the number of DNA breaks, enhancing the fluorescence intensity in the tails of the comet-like structures associated with an increase in the tail length and moment (Fig. 2A, Table 1). In contrast, the untreated control cells showed only typical, representative nuclei. The pretreatment with EGL markedly prevented the adverse effects. An immunoblot image revealed that the levels of phosphorylation of nuclear histone H2AX (p-γH2AX) at serine 139, a sensitive marker of DNA double-strand break formation [40], were markedly increased in the H₂O₂-treated C2C12 cells (Fig. 2B). In contrast, the expression of p-γH2AX decreased significantly in the EGL-pretreated cells, indicating that EGL helped to protect against DNA damage induced by oxidative stress.

As regulation by Nrf2 signaling of the cellular anti-oxidant response has been well documented [27, 30], we next examined whether EGL protected cells from oxidative stress by activating the Nrf2 signaling pathway. The immunoblotting results indicated that the EGL treatment effectively induced the expression of the Nrf2 protein in a concentration- and time-dependent manner in the C2C12 cells (Fig. 3) The results also showed that the EGL treatment induced the expression of the HO-1 protein, an Nrf2 target gene, in the C2C12 cells (Fig. 3). However, the EGL treatment had no effect on other anti-oxidant enzymes, such as nicotinamide adenine dinucleotide phosphate (NADPH)-quinone oxidoreductase 1 (NQO-1) and thioredoxin reductase 1 (TrxR1) (Fig. 3).

To investigate the role of HO-1 induction in EGL-mediated protective effects against oxidative stress in the C2C12 cells, we applied ZnPP, a selective inhibitor of HO-1, in the present study. As indicated in Fig. 4A, ZnPP significantly reversed the inhibition of ROS generation by EGL in the H₂O₂-stimulated C2C12 cells. In addition, the results of the MTT assay showed that the addition of ZnPP significantly attenuated the protective effect of EGL against H₂O₂-induced cytotoxicity (Fig. 4B), suggesting that the cytoprotective effect of EGL was partly mediated by HO-1 induction.

As the phosphorylation of Nrf2 at Ser40 by several kinases is a critical process in its stabilization and nuclear translocation [30, 36, 37], the phosphorylation of Nrf2 in the EGL-treated cells was examined to confirm whether Nrf2 was induced by EGL. As shown in Fig. 5A and Fig. 5B, the EGL treatment caused concentration- and time-dependent increases in the levels of phosphorylated Nrf2 expression. Additionally, a Western blot analysis was carried out using the nuclear and the cytosolic fractions of the C2C12 cells to determine whether EGL promoted the nuclear translocation of Nrf2. As shown in Fig. 5C and Fig. 5D, the amounts of total and phosphorylated Nrf2 proteins in the nucleus were markedly increased following exposure to EGL, indicating that EGL induced the translocation of Nrf2 from the cytosol to the nucleus.

We next developed an Nrf2 gene knockdown model by using siRNA transfection to demonstrate the contribution of Nrf2 signaling to the negative effects of EGL on H₂O₂-induced cytotoxicity. The results of a Western blot analysis indicated that the silencing of Nrf2 by using specific siRNA greatly reduced the EGL-induced expression of Nrf2 compared with that of the untransfected control cells and the control siRNA-transfected cells (Fig. 6A). Therefore, we investigated whether Nrf2 siRNA transfection blocked EGL-induced up-regulation of HO-1 and found that Nrf2 siRNA attenuated these effects, providing evidence that Nrf2 mediated EGL-induced augmentation of HO-1. Furthermore, Nrf2 siRNA significantly cancelled the protective effect of EGL against the H₂O₂-induced reduction of cell viability (Fig. 6B).

Although few studies have investigated how oxidative stress quantitatively affects the load-carrying capacity of muscle cells [6, 7], oxidative stress is known to accompany muscle dysfunction [3, 7]. Oxidative stress occurs when the production of ROS exceeds their catabolism. ROS belong to a class of endogenous signaling molecules, the functions of which depend on their subcellular localization, local concentration, and duration of production. Moderate levels of ROS transiently oxidize cysteine sulphydryl, which contributes to the active sites of most proteins [4, 5]. However, when the accumulation of ROS exceeds a certain threshold, ROS have adverse effects on cellular functions [1, 4], leading to cellular dysfunction and cell death in skeletal muscles [6, 7]. The mitochondrial electron transport system is a major source of intracellular ROS generation [3-5], with mitochondria playing a pivotal role in the ROS-mediated cell-death process. H₂O₂ directly induces mitochondrial dysfunction, followed by a rapid efflux of intracellular ROS, which increases the permeabilization and depolarization of the mitochondrial membrane [1, 4]. Therefore, the discovery of a reagent that could reduce the levels of ROS would be valuable in the treatment of muscle- related diseases.

This study, which is part of a research program to derive new anti-oxidant agents from medicinal mushrooms, used a C2C12 myoblast cell model to discover whether EGL conferred protection against oxidative stress-induced cytotoxicity. The results indicated that H₂O₂ treatment of C2C12 cells caused marked intracellular accumulations of ROS and inhibited cell survival. However, pretreatment of the C2C12 cells with EGL significantly attenuated the H₂O₂-induced generation of ROS and inhibition of cell viability. Thus, we conjectured that EGL might improve the mitochondrial function by eliminating the H₂O₂-induced overproduction of ROS. In addition, the present study showed that H₂O₂ treatment increased the DNA tail moment and length on a comet assay and the expression of p-γH2AX, both of which are widely-used markers for the detection of DNA damage [40]. The EGL treatment markedly reduced these increases, indicating that EGL provided protection against H₂O₂-induced apoptosis by reducing DNA damage caused by oxidative stress in C2C12 myoblasts.

Recent studies have suggested that Nrf2 plays an indispensable role in protecting a variety of tissues from a wide array of toxic insults, including oxidative stress [27, 29]. Although the role of Nrf2 signaling has not been previously characterized in skeletal muscles, coordinated regulation of Nrf2 redox signaling is believed to preserve the redox state and protect the structure and the function of skeletal muscles [2, 27]. Previous studies demonstrated increased Nrf2 levels in active skeletal muscles and subsequent activation of its major target anti-oxidant enzymes, including HO-1 [1, 2]. They also showed that the loss of Nrf2 was strongly coupled with dysregulation of anti-oxidant pathways and progression of muscle dysfunction [2, 3, 6]. Therefore, Nrf2/HO-1 signaling may represent a potential therapeutic target in the management of oxidative stress-related diseases. Studies have proposed that the induction of phosphorylation plays a key role in the regulation of the transcriptional activity of Nrf2 [36, 37]. A number of protein kinases have been implicated in the regulation of Nrf2 activity, with upstream signals facilitating the translocation of Nrf2 into the nucleus, whereupon it binds to ARE in promoter regions [2-30]. In the present study, we found that EGL increased the expression of the Nrf2 protein in C2C12 myoblasts in a concentration- and duration-dependent manner. Subsequently, we found that EGL triggered the induction of HO-1 expression and led to Nrf2 phosphorylation, along with concomitant translocation of Nrf2 into the nuclei. However, ZnPP-induced inhibition of the expression of HO-1 significantly weakened the inhibitory effects of EGL on H₂O₂-induced inhibition of cell growth by blocking the generation of ROS. In addition, the knockdown of Nrf2 by Nrf2-targeted siRNA completely abrogated EGL-induced HO-1 expression. This finding suggests that Nrf2 is a critical upstream regulator of EGL-mediated induction of HO-1 in C2C12 myoblasts. Furthermore, the knockdown of Nrf2 halted the ability of EGL to restore the growth of C2C12 cells inhibited by H₂O₂. These results suggest that the Nrf2-dependent induction of HO-1 by EGL may participate, at least partly, in the protection against oxidative stress in C2C12 myoblasts.

In summary, the results of this study clearly demonstrate that EGL provided protection against H₂O₂-induced cytotoxicity and DNA damage in C2C12 myoblasts through suppression of intracellular ROS generation. Overall, the results imply that EGL may activate Nrf2 signaling and contribute to the induction of the phase II anti-oxidant HO-1 in C2C12 cells, thereby contributing, at least partly, to a cellular defense mechanism against oxidative stress-induced genotoxic events. These findings suggest that EGL may be a potential anti-oxidant agent in C2C12 myoblasts.

This research was supported by the Basic Science Research Program through a National Research Foundation of Korea (NRF) grant funded by the Korea government (2015R1A2A2A01004633).

The authors declare that there are no conflict of interest.

Hyun-Min Yoon. http://orcid.org/0000-0003-3645-6109.

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