Key Takeaways & Executive Findings
- •• Gushukang Granule-containing serum alleviates dexamethasone-induced C2C12 myotube atrophy by restoring mitochondrial homeostasis and modulating autophagy. • In vivo, Gushukang Granule improves muscle fiber structure and upregulates mitochondrial function and autophagy-related genes in a rat model of dexamethasone-induced muscle atrophy. • In vitro, Gushukang-containing serum reduces reactive oxygen species levels, increases cell viability, and restores mitochondrial morphology and function in dexamethasone-treated C2C12 myotubes. • The therapeutic mechanism involves upregulation of TOMM20, HSP60, and SIRT1, and normalization of LC3 and Beclin-1 expression, suggesting a multi-target approach.
Abstract
BACKGROUND: Gushukang Granule is a Chinese herbal compound preparation, commonly used clinically for the treatment of osteoporosis and other bone-related diseases. However, its role in the regulation of muscle metabolism is not clear. OBJECTIVE: To investigate the inhibitory effect of Gushukang Granule-containing serum on dexamethasone-induced C2C12 muscle atrophy. METHODS: (1) In vivo experiment: Thirty-six 3-month-old female Sprague-Dawley rats were randomly divided into model group, blank group and Gushukang group, with 12 rats in each group. The rats in the model group were given 2.5 mg/kg dexamethasone by intragastric administration, once a day, for 1 week, followed by normal feeding for 3 weeks; the Gushukang group was given 0.48 g/kg Gushukang Granule suspension by gavage after modeling, once a day, for 3 weeks; the blank group was not modeled and given equal volume of distilled water by gavage, once a day, for 4 weeks. Two hours after the last administration, left femur and gastrocnemius muscle specimens were taken for hematoxylin-eosin staining, and RNA was extracted from right gastrocnemius and femur tissues; real-time fluorescence quantitative PCR was used to detect mRNA expression levels of mitochondrial function, autophagy and inflammation-related genes. (2) In vitro experiment: C2C12 cells were cultured and induced to differentiate into myotubes with 2% horse serum. Differentiated myotubes were divided into control group, model group and Gushukang-containing serum group. Muscle atrophy model was constructed by incubating with 4 µmol/L dexamethasone for 48 h, and the Gushukang group was treated with 10% Gushukang-containing serum for 24 h after dexamethasone treatment for 24 h. CCK-8 was used to detect cell viability, flow cytometry to detect reactive oxygen species levels, transmission electron microscopy to observe ultrastructural changes, and western blot to detect expression levels of mitochondrial function-related proteins, autophagy-related proteins and antioxidant-related proteins. RESULTS AND CONCLUSION: (1) Hematoxylin-eosin staining showed that compared with the model group, the muscle fiber structure of rats in the Gushukang group recovered better; real-time fluorescence quantitative PCR further verified that Gushukang Granule up-regulated mitochondrial function and autophagy-related genes, supporting its multi-target mechanism to alleviate muscle atrophy. (2) Flow cytometry and CCK-8 results showed that after dexamethasone treatment, reactive oxygen species levels in C2C12 cells were significantly increased (P < 0.05), and cell viability was significantly decreased (P < 0.05); transmission electron microscopy revealed that dexamethasone induced ultrastructural disorder, reduced number of organelles, atrophic and blurred mitochondria, accompanied by a large number of autophagosomes and cytoplasmic vacuoles; western blot results showed that after dexamethasone treatment, the expression of mitochondrial function-related proteins translocase of outer mitochondrial membrane 20 and heat shock protein 60 was significantly decreased, autophagy-related proteins LC3 and Beclin-1 were abnormally expressed (P < 0.05), and silent information regulator 1 expression was significantly decreased (P < 0.05), suggesting that dexamethasone disrupted mitochondrial homeostasis and inhibited autophagy. After treatment with Gushukang Granule-containing serum, reactive oxygen species levels were significantly decreased (P < 0.05), cell viability was significantly increased (P < 0.05); transmission electron microscopy showed that ultrastructure was improved and mitochondrial morphology was relatively restored; western blot results showed that the expression of translocase of outer mitochondrial membrane 20, heat shock protein 60, and silent information regulator 1 was significantly up-regulated (P < 0.05), and LC3 and Beclin-1 expression returned to normal levels (P < 0.05), indicating that Gushukang Granule-containing serum played a positive role in improving mitochondrial function, reducing oxidative stress, and regulating autophagy.
1. Introduction
Sarcopenia is a syndrome characterized by progressive loss of skeletal muscle mass, strength, and function, often associated with aging or certain disease states such as prolonged bed rest, chronic illness, or medication use [1]. With the global aging population, sarcopenia has become a major public health concern [2]. Epidemiological data indicate that approximately 30% of adults over 65 years and more than 50% of those over 80 years suffer from varying degrees of sarcopenia [3]. Sarcopenia not only impairs quality of life but also significantly increases the risk of falls, fractures, hospitalization, and mortality [4]. The pathogenesis of sarcopenia is complex, involving age-related physiological muscle loss and pathological muscle atrophy, with underlying mechanisms including muscle fiber reduction, decreased satellite cell activity, reduced protein synthesis, increased protein degradation, and chronic inflammation [5]. In this process, inflammatory cytokines and oxidative stress play critical roles in disrupting muscle homeostasis.
Glucocorticoids, such as dexamethasone, are widely used clinically for their anti-inflammatory and immunosuppressive effects, but they are also known to induce muscle atrophy as a side effect. Dexamethasone has been shown to impair mitochondrial function, increase oxidative stress, and dysregulate autophagy in muscle cells, leading to protein degradation and muscle wasting. Therefore, dexamethasone-induced C2C12 myotube atrophy serves as a valuable in vitro model for studying muscle atrophy and evaluating potential therapeutic agents.
Traditional Chinese medicine (TCM) has gained attention for its potential in treating muscle wasting diseases. Gushukang Granule, a Chinese herbal compound, is commonly used for osteoporosis and other bone-related disorders. However, its effects on muscle metabolism remain unclear. Given the close relationship between bone and muscle, and the shared regulatory pathways, it is plausible that Gushukang Granule may also benefit muscle health. This study aims to investigate the effects of Gushukang Granule-containing serum on dexamethasone-induced C2C12 myotube atrophy, focusing on mitochondrial homeostasis and autophagy, to provide experimental evidence for its potential application in sarcopenia treatment.
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WEI Wei, LIU Hongfei, QI Xiaonan, LIU Yantong, WANG Deyu, YU Zhitong, QIAO Chunlin, WANG Shixuan, TENG Hai (2026). Gushukang Granule-containing drug serum improves dexamethasone-induced atrophy of C2C12 myotubes via regulating mitochondrial homeostasis. Chinese Journal of Tissue Engineering Research. https://doi.org/10.12307/2026.21233
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Frequently Asked Questions
What is the main finding of this study?
The study demonstrates that Gushukang Granule-containing serum effectively alleviates dexamethasone-induced atrophy of C2C12 myotubes by restoring mitochondrial homeostasis and modulating autophagy, as evidenced by improved cell viability, reduced reactive oxygen species, and normalized expression of key proteins such as TOMM20, HSP60, SIRT1, LC3, and Beclin-1.
How was the muscle atrophy model established in this study?
In vitro, C2C12 myotubes were treated with 4 µmol/L dexamethasone for 48 hours to induce atrophy. In vivo, female SD rats were injected with dexamethasone (2.5 mg/kg) for one week to establish a muscle atrophy model.
What are the potential clinical implications of this research?
The findings suggest that Gushukang Granule, a traditional Chinese medicine, may be a promising therapeutic agent for sarcopenia and other muscle wasting conditions, particularly those associated with glucocorticoid use, by targeting mitochondrial function and autophagy.
What methods were used to assess mitochondrial function and autophagy?
The study employed real-time quantitative PCR to measure mRNA expression of mitochondrial and autophagy-related genes, western blot to analyze protein levels (TOMM20, HSP60, SIRT1, LC3, Beclin-1), flow cytometry for reactive oxygen species, transmission electron microscopy for ultrastructural observation, and CCK-8 for cell viability.
What is the significance of mitochondrial homeostasis in muscle atrophy?
Mitochondrial homeostasis is crucial for maintaining muscle cell function. Disruption of mitochondrial dynamics, increased oxidative stress, and impaired autophagy contribute to muscle protein degradation and atrophy. Restoring mitochondrial homeostasis and autophagy is a key therapeutic strategy for muscle wasting diseases.
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