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Biol. Pharm. Bull. 34(4) 575—579 (2011)
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Sauchinone Attenuates Oxidative Stress-Induced Skeletal Muscle Myoblast Damage through the Down-Regulation of Ceramide Min-Ho JUNG,a Min-Cheol SONG,a Kiho BAE,a Han Sung KIM,b Seung Hyun KIM,c Sang Hyun SUNG,d Sang Kyu YE,e Kwang Ho LEE,f Yeo-Pyo YUN,g and Tack-Joong KIM*,a a Division of Biological Science and Technology, College of Science and Technology, Yonsei University; b Department of Biomedical Engineering, College of Health Science, Yonsei University; Wonju 220–710, Korea: c Institute for Life Science, Elcomscience Co., Ltd.; Seoul 152–742, Korea: d College of Pharmacy, Seoul National University; Seoul 151–742, Korea: e Department of Pharmacology, Seoul National University College of Medicine; Seoul 110–799, Korea: f Department of Biotechnology, College of Biomedical & Health Science, Konkuk University; Chungju 380–701, Korea: and g College of Pharmacy, Chungbuk National University; Cheongju 361–763, Korea. Received October 19, 2010; accepted January 24, 2011; published online January 28, 2011
We investigated the effects of sauchinone, isolated from the root of Saururus chinensis, on muscle disorders and the underlying mechanism of oxidative stress-induced C2C12 skeletal muscle myoblast damage. To assess the protective effects of sauchinone on oxidative stress-induced C2C12 skeletal muscle myoblasts, we measured the viability of the cells, showing that sauchinone pre-treatment significantly reduced the decreased cell viability after H2O2 treatment. We also investigated the mechanism of this protective effect of sauchinone. In Western blot analysis, the heat shock protein (HSP)-70 level increased significantly in the sauchinone-pretreated myoblasts. We used high performance liquid chromatography (HPLC) to examine the level of endogenous ceramide after pre-treatment with sauchinone followed by exposure to H2O2. While hydrogen peroxide increased the ceramide 38.93% of the control level, pre-treatment with sauchinone inhibited this content to approximately 166.60 increase, maintaining the ceramide content at the control level. We demonstrated that sauchinone regulates intracellular HSP70 expression as well as ceramide levels to protect against oxidative stress-induced C2C12 muscle myoblast damage. We suggest the potential benefits of herbal medicines in the treatment of oxidative stressrelated muscle disorders. Key words
sauchinone; muscle atrophy; ceramide
Skeletal muscle atrophy typically results from reduced muscle use, as in unloading, bed rest, denervation, and space flight.1,2) Skeletal muscle cells are susceptible to oxidative stress induced through electron transport and oxygen flux during normal contraction, and this stress may increase with exercise intensity.1—6) Muscle redox status, especially during atrophy, is more oxidative than the redox status in other organs. Augmentation of cellular antioxidant defense presents a basic strategy for controlling oxidative muscle injury and related disease conditions. Various cellular stresses have been shown to induce apoptosis, including heat shock (HS), H2O2 treatment, tumor necrosis factor (TNF)-a , ultraviolet (UV) radiation, ionizing radiation, osmotic stress, and anti-cancer agents.7—9) Simultaneously, subsets of some stresses (heat, oxidative stress, TNF-a , Fas ligand, and UV irradiation) activate sphingomyelinase (SMase), resulting in ceramide production.9) Recently, Ferreira et al. suggested that SMase stimulated a ceramide-oxidant signaling pathway that results in muscle weakness and fatigue.10) Ceramide has been recognized as a lipid mediator in the induction of apoptosis,11) since a diverse array of stresses that lead to apoptosis were reported to increase ceramide levels in many cell types.12) Recently, Kondo et al. demonstrated that the ceramide induction of heat shock-induced apoptosis occurs through the suppression of anti-apoptotic heat shock protein (HSP)-70 in HL-60 cells.13) HSPs consist of a family that includes HSP90, HSP70, HSP27, and other small HSPs.14) In terms of the role of HSP70 on apoptosis, it has been reported that the induction of thermotolerance correlates with an increase of HSP70 pro∗ To whom correspondence should be addressed.
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teins in fibroblasts15) and that this induction is blocked by the inhibition of HSP70 expression in K-562 leukemia cells.16) HS-induced thermotolerance showed a resistance to apoptosis induction due to pro-apoptotic stresses including TNF-a , UV, oxidative stress, and ceramide.17,18) Moreover, the overexpression of HSP70 induced resistance to TNF-a -induced cytotoxity19) and to ischemic heart injury.20) Sauchinone, a lignan isolated from Saururus chinensis, has been used not only as a anti-pyretic, a diuretic, and an anti-inflammatory herbal agent in Korea, but also as a medicine to treat edema, jaundice, and gonorrhea.10) In a previous study, sauchinone was reported to be hepatoprotective in rat hepatocytes.21) Sauchinone has also shown anti-inflammatory activity to reduce inducible nitric oxide synthase (iNOS) expression and viral inflammation on RAW265.7 cells.22,23) It also suppresses inflammation in Th2 cells24) and osteoclasts.25) However, only a few studies have been performed on the protective effects of sauchinone on muscle atrophy. The aim of the present study is to evaluate the effects of sauchinone on signal transduction mechanisms involving skeletal muscle myoblast damage induced by oxidative stress. MATERIALS AND METHODS Materials Cell culture materials were purchased from Gibco-BRL (Gaithersburg, MD, U.S.A.), while the HSP27, HSP70, HSP90 and b -actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.). C17- and C2-ceramide were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, U.S.A.) The sauchinone was prepared as described previously.26) All other chemicals used were of the © 2011 Pharmaceutical Society of Japan
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highest analytical grade commercially available. Cell Culture C2C12 skeletal muscle myoblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM: Sigma-Aldrich, St. Louis, MO, U.S.A.) supplemented with 10% fetal bovine serum (FBS), penicillin 100 U/ml, streptomycin 100 m g/ml, N-(2-hydroxyethyl)piperazine-N-2ethanesulfonic acid (HEPES) 8 mM, and L-glutamine 2 mM. Cells were maintained at 37 °C in a humidified 5% CO2 incubator. Cell Viability Cell viability was determined using the EZ-Cytox cell viability kit (Daeil Lab., Seoul, Korea) following the manufacturer’s instructions. Initially, the cells were seeded into 96-well culture plates at 1104 cells/ml and were cultured in DMEM containing 10% FBS at 37 °C. When cells reached 70% confluence, the medium was replaced with serum-free DMEM containing various concentrations of sauchinone for 24 h. These culture media were then replaced with H2O2. Control cells were cultured in DMEM in the absence of H2O2. EZ-Cytox cell viability kit reagents were added to the medium, and the cells were incubated for 1 h. To evaluate the strength of the sauchinone effect on the cellular response to stress, N-acetyl cysteine (NAC) was included as a reference (positive control). The optical density was determined at 450 nm using a microplate reader (BioTek Instruments Inc., Winooski, VT, U.S.A.). Western Blot Analysis C2C12 skeletal muscle myoblasts were cultured for 24 h with or without sauchinone. The myoblasts were then treated with H2O2 1 mM (by addition to the medium) for 1 h. The cell lysates were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 7.5—10% polyacrylamide gels according to the method described by Ahn et al.27) The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, U.S.A.), blocked overnight at 4 °C in Tris-buffered saline containing 0.1% Tween 20 (TBS/T) and 5% skim milk powder, and then incubated with 1 : 2000 dilutions of HSP27, HSP70, HSP90 and b -actin antibodies. Blots were washed with TBS/T and incubated with a 1 : 5000 dilution of horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) antibody (Cell Signaling Technology, Danvers, MA, U.S.A.). Proteins were detected using an enhanced chemiluminescence (ECL) detection reagent for Western blots (GE Healthcare, Buckinghamshire, U.K.). Measurement of Ceramide Content The analyses of ceramide levels were performed as previously described.28) In brief, total lipids were extracted using chloroform/methanolic KOH (1 : 2, v/v) with an addition of C17 sphingosine-based ceramide as an internal standard. Ceramide was converted simultaneously to sphingosine by ceramidase in a reaction buffer containing 25 mM Tris–HCl buffer, pH 7.5, containing 1% sodium chlorate, and 15% fatty acid-free bovine serum albumin. The released sphingosine from ceramide was analyzed using HPLC following o-phthalaldehyde derivatization. Detection of Apoptosis C2C12 skeletal muscle myoblasts were cultured on a cover glass, fixed in 4% paraformaldehyde, and then membrane-permeabilized through a 30-min exposure to 0.1% Triton X-100 in phosphate-buffered saline at room temperature. Cells were then exposed to 4,6-diamino-2-phenylindole (DAPI) solution. Apoptotic cells were identified by the morphological changes observed in the stained cells under fluorescence microscopy.
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Statistical Analysis Experimental results are expressed as meansS.D. One-way analysis of variance (ANOVA) was followed by Dunnett’s test for multiple comparisons. p values 0.05 and 0.01 were considered statistically significant, as indicated. RESULTS AND DISCUSSION Sauchinone Protected C2C12 Skeletal Muscle Myoblasts against H2O2-Induced Oxidative Stress Damage The chemical structure of sauchinone, isolated from the root of Saururus chinensis, is shown in Fig. 1A. To determine the cytotoxicity of sauchinone, we treated C2C12 skeletal muscle myoblasts with sauchinone at concentrations from 10 to 50 m M for 24 h and measured the cell viability using the EZCytox cell viability kit. Within this dose range, sauchinone showed no evidence of cytotoxicity in C2C12 skeletal muscle myoblasts (Fig. 1B). Skeletal muscle is susceptible to injury by reactive oxygen species (ROS) even under physiological conditions because of the rapid bursts of electron transport and oxygen flux during contraction.3) Intense exercise may lead to muscle injury through an increase in ROS production.4—6) Cell injury associated with ROS may contribute to a variety of muscle diseases and pathologic conditions.29) To clarify the effect of sauchinone on the oxidative stress response in muscle cells, we exposed C2C12 skeletal muscle myoblasts to H2O2. In control groups the cell viability decreased in a dose-dependent manner following H2O2 exposure (Fig. 2A). In contrast, cell viability recovered significantly as the sauchinone concentration increased from 10 to 25 m M. As compared with the antioxidant N-acetyl cysteine (NAC) (positive control), sauchinone showed a greater reduction in the H2O2-induced decrease in cell viability in C2C12 skeletal muscle myoblasts (Fig. 2B). Therefore, the sauchi-
Fig. 1. Cytotoxicity of Sauchinone in C2C12 Skeletal Muscle Myoblasts (A) Chemical structure of sauchinone, isolated from the root of Saururus chinensis. (B) Cytotoxicity of sauchinone in C2C12 skeletal muscle myoblasts. C2C12 skeletal muscle myoblasts were cultured in 96-well plates until confluent, after which the medium was replaced with serum-free medium with or without sauchinone (0—50 m M) for 24 h. The EZ-Cytox reagent was added to the medium, and cells were incubated for 1 h. The optical density was determined at 450 nm using a microplate reader. Shown are the mean values (S.D.) from four experiments. Sau: Sauchinone.
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Fig. 2. Effect of Sauchinone on H2O2-Treated C2C12 Skeletal Muscle Myoblast Cell Viability
Fig. 3. Effect of Sauchinone on HSP27, HSP70 and HSP90 Expression in H2O2-Treated C2C12 Skeletal Muscle Myoblasts
(A) Dose-dependent effect of H2O2 on C2C12 skeletal muscle myoblast cell viability. C2C12 skeletal muscle myoblasts were cultured in 96-well plates until confluent, after which the medium was replaced with serum-free medium with or without sauchinone at 10 or 25 m M. After pre-incubation for 24 h, graded concentrations of H2O2 (0—4 mM, as indicated) were added for 1 h. The EZ-Cytox reagent was then added to the medium, and the cells were incubated further for 1 h. (B) Comparative effects of sauchinone and N-acetylcysteine (NAC) on viability in H2O2-treated C2C12 skeletal muscle myoblasts. C2C12 skeletal muscle myoblasts were cultured in 96-well plates until confluent and the medium was then replaced with serum-free medium with or without sauchinone (10 m M), NAC (10 m M and 5 mM) for 24 h. After this pre-incubation, cells were treated with H2O2 (1 mM) for 1 h. Optical density was determined at 450 nm using a microplate reader. The cell viability was calculated according to the following equation: cell viability (%): [(absorbance of H2O2-treated sample/absorbance of H2O2-untreated control)100]. Shown are the mean values (S.D.) from three experiments, each performed in triplicate. (∗ p0.05, ∗∗ p0.01). Sau, Sauchinone. NAC, N-acetylcysteine. UN, chemically-untreated control.
C2C12 skeletal muscle myoblasts were cultured in six-well plates until confluent, and the medium was then replaced with serum-free medium with or without sauchinone (10, 25 m M) for 24 h. C2C12 skeletal muscle myoblasts were treated with 1 mM H2O2 for 1 h and lysed, and proteins in the lysates were analyzed using SDS-PAGE. Band intensities were quantified using densitometry. Shown are the mean values (S.D.) of HSPs/b -actin from three independent experiments, each performed in triplicate. (∗∗ p0.01). Sau, Sauchinone.
none pre-treatment was clearly shown to modulate the antioxidant defenses of C2C12 skeletal muscle myoblasts. This compound may potentiate cell therapy regimens now in development for muscular dystrophies. Our findings suggest that sauchinone enhances the inherent oxidative stress defenses. Sauchinone Increased HSP70 Expression in H2O2Treated C2C12 Skeletal Muscle Myoblasts HSPs consist of a family that includes HSP90, HSP70, HSP27, and other small HSPs.14) HSP70 is significantly down-regulated in various models of skeletal muscle atrophy.17,30—33) The cell-protective effects of HSP70 are closely linked to apoptosis inhibition,19,34—40) and evidence suggests that HSP70 prevents apoptosis by inhibiting the stress-activated protein kinase/cjun N-terminal kinase (SAPK/JNK) signaling cascade.18,41) Since HSP70 is an inhibitor of caspase-mediated apoptosis, we investigated whether its expression was affected by sauchinone treatment, which is believed to exert its protective effects through the modulation of HSP70. Following culture for 24 h in the presence or absence of sauchinone, C2C12 skeletal muscle myoblasts were treated for 1 h with 1 mM H2O2. As shown in Fig. 3, the HSP70 protein level increased significantly in the sauchinone-pretreated cells, whereas HSP27 and HSP90 have no effects. This result suggests that sauchinone may protect against cell damage through the in-
duction of HSP70 in the C2C12 skeletal muscle myoblasts. HSP70 protects cells from a number of apoptotic stimuli, including HS, TNF-a , growth factor withdrawal, oxidative stress, chemotherapeutic agents, ceramide, and radiation.19,35—40) A decrease in HSP70 may, therefore, positively contribute to pathogenic stress responses, such as those that occur during skeletal muscle disuse. Sauchinone Down-Regulated Ceramide Levels in H2O2Treated C2C12 Skeletal Muscle Myoblasts Ceramide is involved in the regulation of cell death and acts as a lipid mediator of cellular stress responses.11,12) The ceramide level in cells is up-regulated by various types of stress conditions including ionizing radiation, serum deprivation, and anti-cancer drugs.8) To determine the apoptotic effects of ceramide, we treated C2C12 skeletal muscle myoblasts with C2-ceramide at concentrations of 10 or 20 m M for 4 h and measured the apoptosis using the DAPI staining (Fig. 4A). The results indicated that the C2-ceramide induced the apoptosis on C2C12 skeletal muscle myoblasts. Next, we examined to evaluate the effects of sauchinone on endogenous ceramide levels involving skeletal muscle myoblast damage induced by oxidative stress. When we examined the content of endogenous ceramide using an HPLC assay after treatment with H2O2, there was an increase in ceramide content to approximately 166.6038.93% of the control level. Conversely, the sauchinone pre-treatment maintained the ceramide content at the control level (Fig. 4B). Therefore, the sauchinone pretreatment clearly modulated the antioxidant defenses of C2C12 skeletal muscle myoblasts. To test the effect of sauchinone on apoptotic cell death, we evaluated the changes in the chromatin morphology of H2O2-
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through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (2010-00757). REFERENCES
Fig. 4. Effects of Sauchinone on Ceramide Content and Apoptosis in H2O2-Treated C2C12 Skeletal Muscle Myoblasts (A) Effect of ceramide on apoptosis of C2C12 skeletal muscle myoblasts. C2C12 skeletal muscle myoblasts (2.5105 cells/cm2) were cultured on a cover glass for 4 h with or without ceramide (10 or 20 m M). Cells grown on cover glass were stained with DAPI and examined under fluorescence microscopy. (B) Effects of sauchinone on ceramide content in H2O2-treated C2C12 skeletal muscle myoblasts. C2C12 skeletal muscle myoblasts were cultured in six-well plates until confluent, and the medium was then replaced with serum-free medium with or without sauchinone (25 m M) for 24 h. C2C12 skeletal muscle myoblasts were treated with 1 mM H2O2 for 1 h and lysed. The ceramide content in the lysates were analyzed using HPLC. Shown are the mean values (S.D.) from three experiments. (∗∗ p0.01). (C) Effects of sauchinone on apoptosis in H2O2treated C2C12 skeletal muscle myoblasts. C2C12 skeletal muscle myoblasts (2.5105 cells/cm2) were cultured on a cover glass for 24 h with or without sauchinone (25 m M), then treated with 1 mM H2O2 for 1 h. Cells grown on cover glass were stained with DAPI and examined under fluorescence microscopy. Cer, ceramide. Sau, Sauchinone. UN, chemically-untreated control.
treated C2C12 skeletal muscle myoblasts using DAPI staining. Treatment with H2O2 for 1 h caused apoptosis characterized by chromatin condensation, small membrane-bound bodies (apoptotic bodies), cytoplasmic condensation, and cell shrinkage. Pre-treatment for 24 h with sauchinone reduced apoptosis in H2O2-treated C2C12 skeletal muscle myoblasts (Fig. 4C), suggesting that sauchinone protects against cell death through down-regulation of the ceramide level in C2C12 skeletal muscle myoblasts. However, the direct effect of ceramide on HSP70 remains to be clarified. Although we focused on the ceramide level in muscle cells and linked its protective function to antioxidant defense, the full role of sauchinone in this function remains to be elucidated. Sauchinone may participate in other pathways involving the antioxidative enzymes and cellular components that generate free radicals as an antioxidant molecule that engages in complex signal transduction reactions in a stress response. In conclusion, we have shown that sauchinone increases intracellular HSP70 and decreases the ceramide level caused by H2O2-induced oxidative stress in C2C12 skeletal muscle myoblasts. Through its ability to down-regulate ceramide, sauchinone treatment may be used to delay or prevent skeletal muscle atrophy. Acknowledgements This research was supported by Leading Foreign Research Institute Recruitment Program
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