• Journal of Ginseng Research
• /
• v.47 no.6
• /
• pp.726-734
• /
• 2023

Background: Skeletal muscles play a key role in physical activity and energy metabolism. The loss of skeletal muscle mass can cause problems related to metabolism and physical activity. Studies are being conducted to prevent such diseases by increasing the mass and regeneration capacity of muscles. Ginsenoside Rg5 has been reported to exhibit a broad range of pharmacological activities. However, studies on the effects of Rg5 on muscle differentiation and growth are scarce. Methods: To investigate the effects of Rg5 on myogenesis, C2C12 myoblasts were induced to differentiate with Rg5, followed by immunoblotting, immunostaining, and qRT-PCR for myogenic markers and promyogenic signaling (p38MAPK). Immunoprecipitation confirmed that Rg5 increased the interaction between MyoD and E2A via p38MAPK. To investigate the effects of Rg5 on prevention of muscle mass loss, C2C12 myotubes were treated with dexamethasone to induce muscle atrophy. Immunoblotting, immunostaining, and qRT-PCR were performed for myogenic markers, Akt/mTOR signaling for protein synthesis, and atrophy-related genes (Atrogin-1 and MuRF1). Results: Rg5 promoted C2C12 myoblast differentiation through phosphorylation of p38MAPK and MyoD/E2A heterodimerization. Furthermore, Rg5 stimulated C2C12 myotube hypertrophy via phosphorylation of Akt/mTOR. Phosphorylation of Akt induces FoxO3a phosphorylation, which reduces the expression of Atrogin-1 and MuRF1. Conclusion: This study provides an understanding of how Rg5 promotes myogenesis and hypertrophy and prevents dexamethasone-induced muscle atrophy. The study is the first, to the best of our knowledge, to show that Rg5 promotes muscle regeneration and to suggest that Rg5 can be used for therapeutic intervention of muscle weakness and atrophy, including cancer cachexia.

• Journal of Korean Medicine Rehabilitation
• /
• v.34 no.2
• /
• pp.15-28
• /
• 2024

Objectives We used the D-galactose (D-gal) induced C2C12 myoblast senescence model to investigate whether ethanol extract of Perilla. fructescens leaves (EEPF) could delay cellular senescence and regulate related mechanisms. Methods C2C12 myogenic cells were cultured in an incubator under 37 ℃ and 5% CO₂ conditions. EEPF, dried perilla leaves were pulverized and extracted at 1:10 (v/v) at 50 ℃ for 4 hours. Cell counting kit-8 and western blot analysis was performed. Annexin V-FITC apoptosis detection kit and DAPI staining was applied. Catalase (CAT), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and malondialdehyde analysis kits were used. To measure the level of reactive oxygen species generation, staining and flow cytometry was used. To analyze the mitochondrial activity, membrane potential changes were measured using JC-1. 𝛽-gal activity was analyzed using SA-𝛽-gal staining solution, and DNA damage was analyzed by using 𝛾-H2AX. Quantikine ELISA kit was used to analyze inflammatory cytokine production. Results According to the results of this study, EEPF significantly alleviated the decrease in cell viability in C2C12 cells treated with D-gal and suppressed the decrease in the expression of proliferating cell nuclear antigen. EEPF also markedly blocked D-gal-induced C2C12 cell apoptosis and restored reduced activity of CAT, GSH-Px, T-AOC, SOD. In addition, EEPF suppressed the decrease in 𝛽-galactosidase activity, the induction of DNA damage and the increase in expression of senescence-associated secretory phenotype proteins such as p16, p53 and p21 in D-gal-treated C2C12 cells. Furthermore, EEPF significantly attenuated D-gal-induced production and expression of inflammatory cytokines such as interleukin (IL)-6 and IL-18. Conclusions The results of this study indicate that EEPF can be used as a potential candidate for the prevention and treatment of muscle aging.

• Journal of the korean academy of Pediatric Dentistry
• /
• v.28 no.2
• /
• pp.238-246
• /
• 2001

Bone morphogenetic proteins(BMPs), members of the transforming growth factor $\beta$(TGF-$\beta$) superfamily were first identified as the factors that induce ectopic bone formation in vivo, when implanted into muscular tissue. Especially BMP-2 inhibits terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells. In the molecular mechanism of the signal transduction of TGF-$\beta$ and related factors, intracellular signaling proteins were identified as Smad. In previous study, it has been reported that Smad 1 and Smad 5, which belong to the R-Smad family mediate BMP signaling, were involved in the induction of osteoblast differentiation in C2C12 cells. To understnad the role of Smads involved in osteogenic transdifferentiation in C2C12 cell, in present study, after we stably transfected C2C12 cells with each. Smad(Smad 1,Smad 5) expression vector, cultured for 3 days and stained for alkaline phophatase activity. ALP activity positive cells appeared in the Smad 1, Smad 5 stably transfected cell even in the abscence of BMP. After transiently co-transfected C2C12 cells with each Smad expression vector and ALP promoter, it was examined that Smad 1 and Smad 5 expression vector had increased about 2 fold ALP promoter activity in the abscence of BMP. These result suggested that both Smad 1 and Smad 5 were involved in the intracellular BMP signals which induce osteoblast differentiation in C2C12 cells. The effect of BMP on C2C12 cells with Smad 1, Smad 5 transfected were studied by using northern blot analysis. the treatment of BMP upregulated ALP mRNA level in three groups, especially upregulation of ALP was larger in Smad 1, Smad 5 transfected cell than control group. Pretreatment with cycloheximide($10{\mu}g/ml$), a protein synthesis inhibitor resulted in blocking the ALP gene expression even in BMP(100ng/ml) treated cell. These results suggested that Smad increased the level of ALP mRNA via the synthesis of a certain transcriptional regulatory protein.

• Journal of Life Science
• /
• v.29 no.2
• /
• pp.215-222
• /
• 2019

Muscle dysfunction may arise from skeletal muscle atrophy caused by aging, injury, oxidative stress, and hereditary disease. Powdered heat-killed Enterococcus faecalis (EF-2001) has anti-allergy, anti-inflammatory, and anti-tumor effects. However, its antioxidant and anti-atrophy effects are poorly characterized. In this study, we examined the effects of EF-2001 on muscle atrophy. To determine the protective effect of EF-2001 on oxidative stress, C2C12 myoblasts were treated with $H_2O_2$ to induce oxidative stress. This induced cell damage, which was reduced by treatment with EF-2001. The mechanism of EF-2001's effect was examined in response to oxidative stress. Treatment with EF-2001 reversed the expression of HSP70 and SOD1 proteins. Also, mRNA levels of Atrogin-1/MAFbx and MuRF1 increased under oxidative stress conditions but decreased following EF-2001 treatment. To evaluate muscle volume, two and three dimensional models of the muscles were analyzed using micro-CT. As expected, muscle volume decreased after sciatic denervation and recovered after oral administration of EF-2001. Therefore, EF-2001 is a candidate for the treatment of muscular atrophy, and future discovery of the additional effects of EF-2001 may yield further applications as a functional food with useful activities in various fields.

• Journal of Nutrition and Health
• /
• v.56 no.6
• /
• pp.602-614
• /
• 2023

Purpose: The balance between synthesis and degradation of proteins plays a critical role in the maintenance of skeletal muscle mass. Mitochondrial dysfunction has been closely associated with skeletal muscle atrophy caused by aging, cancer, and chemotherapy. Polygalacin D is a saponin derivative isolated from Platycodon grandiflorum (Jacq.) A. DC. This study aimed to investigate the effects of polygalacin D on myoblast differentiation and muscle atrophy in association with mitochondrial function in in vitro and in zebrafish models in vivo. Methods: C2C12 myoblasts were cultured in differentiation media containing different concentrations of polygalacin D, followed by the immunostaining of the myotubes with myosin heavy chain (MHC). The mRNA expression of markers related to myogenesis, muscle atrophy, and mitochondrial function was determined by real-time quantitative reverse transcription polymerase chain reaction. Wild type AB^* zebrafish (Danio rerio) embryos were treated with 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI) with or without polygalacin D, and immunostained to detect slow and fast types of muscle fibers. The Tg(Xla.Eef1a1:mitoEGFP) zebrafish expressing mitochondria-targeted green fluorescent protein was used to monitor mitochondrial morphology. Results: The exposure of C2C12 myotubes to 0.1 ng/mL of polygalacin D increased the formation of MHC-positive multinucleated myotubes (≥ 8 nuclei) compared with the control. Polygalacin D significantly increased the expression of MHC isoforms (Myh1, Myh2, Myh4, and Myh7) involved in myoblast differentiation while it decreased the expression of atrophic markers including muscle RING-finger protein-1 (MuRF1), mothers against decapentaplegic homolog (Smad)2, and Smad3. In addition, polygalacin D promoted peroxisome proliferator-activated receptor-gamma coactivator (Pgc1α) expression and reduced the level of mitochondrial fission regulators such as dynamin-1-like protein (Drp1) and mitochondrial fission 1 (Fis1). In a zebrafish model of FOLFIRI-induced muscle atrophy, polygalacin D improved not only mitochondrial dysfunction but also slow and fast muscle fiber atrophy. Conclusion: These results demonstrated that polygalacin D promotes myogenesis and alleviates chemotherapy-induced muscle atrophy by improving mitochondrial function. Thus, polygalacin D could be useful as nutrition support to prevent and ameliorate muscle wasting and weakness.

• Journal of Nutrition and Health
• /
• v.57 no.1
• /
• pp.53-64
• /
• 2024

Purpose: Mitochondria play a crucial role in preserving skeletal muscle mass, and damage to mitochondria leads to muscle mass loss. This study investigated the effects of oxypeucedanin hydrate, a furanocoumarin isolated from Angelica dahurica radix, on myogenesis and mitochondrial function in vitro and in zebrafish models. Methods: C2C12 myotubes cultured in media containing 0.1, 1, 10, or 100 ng/mL oxypeucedanin hydrate were immunostained with myosin heavy chain (MHC), and then multinucleated MHC-positive cells were counted. The expressions of markers related to muscle differentiation, muscle protein degradation, and mitochondrial function were determined by quantitative reverse transcription polymerase chain reaction. To investigate the effects of oxypeucedanin hydrate on mitochondrial dysfunction, Tg(Xla.Eef1a1:mito-EGFP) zebrafish embryos were treated with 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI) with or without oxypeucedanin hydrate and analyzed for mito-EGFP intensity and mitochondrial length. Results: Oxypeucedanin hydrate significantly increased MHC-positive multinucleated myotubes (≥ 3 nuclei) and increased the expression of the myogenic marker myosin heavy chain 4. However, it decreased the expressions of muscle-specific RING finger protein 1 and muscle atrophy f-box (markers of muscle protein degradation). Furthermore, oxypeucedanin hydrate enhanced the expressions of markers of mitochondrial biogenesis (peroxisome proliferator-activated receptor-gamma coactivator 1 alpha, transcription factor a mitochondrial, succinate dehydrogenase complex flavoprotein subunit A, and cytochrome c oxidase subunit 1) and mitochondrial fusion (optic atrophy 1). However, it reduced the expression of dynamin-related protein 1 (a mitochondrial fission regulator). Consistently, oxypeucedanin hydrate reduced FOLFIRI-induced mitochondrial dysfunction in the skeletal muscles of zebrafish embryos. Conclusion: The study indicates that oxypeucedanin hydrate promotes myogenesis by improving mitochondrial function, and thus, suggests oxypeucedanin hydrate has potential use as a nutritional supplement that improves muscle mass and function.