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Updated: Apr 4, 2026
Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans
Published on: February 16, 2017
[Myogenic induction activity and mechanism of rhein in aged mouse myoblasts C2C12 cell line]
Cancan Pan1, Lei Qi, Jiawen Si
1Department of Oral and Craniomaxillofacial Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology. Shanghai 200011, China.
Purpose:
To establish an in vitro model of aged murine myogenic cell line C2C12, clarify rhein's anti-inflammatory function, and explore its effects on myogenic induction activity and related immunomodulatory mechanisms in aged C2C12 cells.
Methods:
CCK-8 assay was used to assess cell proliferation rates of C2C12 cells pretreated with different concentrations of hydrogen peroxide(H2O2) for varying duration. Real-time quantitative reverse transcription PCR (RT-qPCR) was employed to observe myogenic and aged-related gene expression changes. The expression of reactive oxygen species(ROS) and senescence-associated β-galactosidase (SA-β-gal) was measured. Immunofluorescence was used to compare the diameter of the formed myotubes. The effects of different concentrations of rhein(0, 1, 2, 4, 8, 16, 32 μmol/L) on the proliferation of murine macrophage cell line RAW264.7 over 3 days were measured using CCK-8 assay. The expression of polarization-related factors in RAW264.7 cells was detected by RT-qPCR and immunofluorescence. The effects of macrophage-conditioned medium(CM) induced by rhein on myogenic function and age-related phenotypes in aged C2C12 cells were explored through experiments including RT-qPCR to detect relevant gene expression, ROS and SA-β-gal staining, and immunofluorescence staining.
Results:
Compared with the control group, treatment of C2C12 cells with 200 and 400 μmol/L H2O2 for 24 hours significantly reduced the proliferation ability of C2C12 without causing apoptosis, increased the expression of age-related genes, ROS, and SA-β-gal, decreased the expression of myogenic genes, and inhibited myotube formation, with more pronounced effects at 400 μmol/L(P＜0.05). Rhein at a concentration of 1 μmol/L was non-cytotoxic to RAW264.7 cells, reduced the expression of M1-related phenotypes (TNF-α, IL-1β, iNOS, IL-6), and promoted the expression of M2-related phenotypes (IL-1Ra, CD206)(P＜0.05). Macrophage-conditioned medium (CM) induced by rhein enhanced the expression of myogenic genes in aged C2C12 cells, increased the diameter of myotube, and reduced the expression of age-related genes, ROS, and SA-β-gal(P＜0.05).
Conclusions:
An in vitro model of aged muscle stem cells can be established using H2O2. Rhein can enhance the myogenic capacity of aged muscle, likely through the regulation of macrophage M2 polarization.
