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Abstract
Schisandrae fructus (SF) has recently been reported to increase skeletal muscle mass and inhibit atrophy in mice. We investigated the effect of SF extract on human myotube differentiation and its acting pathway. Various concentrations (0.1–10 μg/mL) of SF extract were applied on human skeletal muscle cells in vitro. Myotube area and fusion index were measured to quantify myotube differentiation. The maximum effect was observed at 0.5 μg/mL of SF extract, enhancing differentiation up to 1.4-fold in fusion index and 1.6-fold in myotube area at 8 days after induction of differentiation compared to control. Phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 and 70 kDa ribosomal protein S6 kinase, which initiate translation as downstream of mammalian target of rapamycin pathway, was upregulated in early phases of differentiation after SF treatment. SF also attenuated dexamethasone-induced atrophy. In conclusion, we show that SF augments myogenic differentiation and attenuates atrophy by increasing protein synthesis through mammalian target of rapamycin/70 kDa ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1 signaling pathway in human myotubes. SF can be a useful natural dietary supplement in increasing skeletal muscle mass, especially in the aged with sarcopenia and the patients with disuse atrophy.
Supplementary materials
Immunofluorescent staining
Primary cultured human skeletal muscle cells (2×104 cells/well) were seeded on a cover glass in a 12-well plate, which was coated with 1% gelatin. After 2 days, cells were washed with phosphate buffered saline (PBS) and fixed by 4% paraformaldehyde. Then, cells were permeabilized with PBS containing 0.25% Triton X-100 (PBST) for 25 minutes. These cells were incubated in 2% BSA for 30 minutes and then with PAX3 (ab180754; Abcam) or PAX7 (ab34360; Abcam) antibodies overnight at 4°C. After washing with PBS, cells were incubated with fluorescent antirabbit IgG antibodies (Alexa 594 or Alexa 488; Thermo Fisher Scientific, Waltham, MA, USA) for 1 hour. Nuclei were stained by Hoechst 33342 (DAPI, 1 μg/mL) for 3 minutes. All antibodies were diluted in PBST containing 1% BSA. These cells were mounted on a glass slide. These results were recorded using fluorescence microscopy (Eclipse 80i; Nikon Corporation, Tokyo, Japan).
Figure S1 Human skeletal muscle cells were identified by the expression of PAX3 and PAX7.
Notes: Primary cultured human skeletal muscle cells have exhibited the expression of PAX3 (green, upper panel) and PAX7 (red, lower panel), which have been known as human myoblast marker. Each of the stained cells was merged with nuclei.
Figure S2 SF increased phosphorylation of FOXO1.
Notes: SF (0.5 μg/mL) was treated with differentiation medium. HSkMCs’ differentiation was induced for 1 day, 2 days, and 4 days. Phosphorylation of FOXO1 was observed by Western blot. p-FOXO1 levels were normalized to the levels of GAPDH.
Abbreviations: HSkMCs, human skeletal muscle cells; SF, Schisandrae fructus; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Acknowledgments
This study was supported by the R&D program of MOTIE/KEIT (10040391; Development of Functional Food Materials and Device for Prevention of Aging-associated Muscle Function Decrease).
Disclosure
The authors report no conflicts of interest in this work.