关键词: C2C12细胞, 肌管细胞, 地塞米松, 骨疏康颗粒, 线粒体稳态, 含药血清, 肌肉减少症, 自噬小体

Abstract: BACKGROUND: Gushukang Granule is a Chinese herbal compound preparation, which is 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 3 weeks of normal feeding. The rats in the Gushukang group were given 0.48 g/kg Gushukang Granule suspension by intragastric administration after modeling, once a day, for 3 weeks. The rats in the blank group were given the same amount of distilled water by intragastric administration without modeling, once a day for 4 weeks. Two hours after the last administration, the left femur and gastrocnemius specimens of rats were taken and stained with hematoxylin-eosin to observe the morphological changes. At the same time, RNA was extracted from the right gastrocnemius and femur tissues, and the mRNA expression levels of mitochondrial function, autophagy and inflammatory genes were detected by real-time fluorescent quantitative PCR. (2) In vitro experiment: C2C12 cells were cultured and induced with 2% equine serum to differentiate into myotube cells. The differentiated myotube cells were divided into control group, model group and Gushukang Granule-containing serum group. The muscle atrophy model was constructed by incubation with 4 µmol/L dexamethasone for 48 hours, in which the Gushukang group was treated with dexamethasone for 24 hours followed by cultured with 10% Gushukang drug-containing serum for another 24 hours. Cell counting kit-8 was used to detect cell viability, flow cytometry was used to detect reactive oxygen species, and ultrastructural changes were observed by transmission electron microscopy. The expression levels of mitochondrial function related proteins, autophagy related proteins and antioxidant related proteins were detected by western blot.
RESULTS AND CONCLUSION: (1) Hematoxylin-eosin staining showed that compared with the model group, the muscle fiber structure recovered well in the Gushukang group. Real-time fluorescence quantitative PCR further verified the upregulatory effect of Gushukang Granule-containing serum on mitochondrial function and autophagy related genes, supporting its multi-target mechanism to alleviate muscle atrophy. (2) The results of flow cytometry and cell counting kit-8 showed that the reactive oxygen species level in C2C12 cells was significantly increased after dexamethasone treatment (P < 0.05), and the cell viability was significantly decreased (P < 0.05). Under the transmission electron microscopy, dexamethasone induced ultrastructure disorder, the number of organelles decreased significantly, and mitochondrial atrophy and fuzzy state appeared, accompanied by a large number of autophagosomes and cytoplasmic vacuoles. Western blot results showed that the expressions of mitochondrial function-related proteins (translocase of outer mitochondrial membrane 20 and heat shock protein 60) were significantly decreased after dexamethasone treatment, the autophagy related proteins (LC3 and Beclin-1) were abnormally expressed (P < 0.05), and the expression of silent information regulator 1 was significantly decreased (P < 0.05). It is suggested that dexamethasone can destroy mitochondrial homeostasis and inhibit autophagy. After treatment with drug-containing serum of Gushukang Granules, reactive oxygen species level was significantly decreased (P < 0.05), and cell viability was significantly increased (P < 0.05). Under the transmission electron microscopy, the ultrastructure of the cells was improved and the morphology of mitochondria was relatively restored, but not completely repaired. Western blot results further showed that the expressions of translocase of outer mitochondrial membrane 20, heat shock protein 60 and silent information regulator 1 were significantly up-regulated (P < 0.05), and the expressions of LC3 and Beclin-1 returned to normal levels (P < 0.05). These findings indicate that the drug-containing serum of Gushukang Granules plays a positive role in improving mitochondrial function, reducing oxidative stress and regulating autophagy.

Key words: C2C12 cells, myotube cells, dexamethasone, Gushukang Granules, mitochondrial homeostasis, drug-containing serum, sarcopenia, autophagosome