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The aim is to study the effects and underlying mechanisms of astragalus polysaccharide (APS) on the peroxide-induced injury in C2C12 myoblasts in vitro. Cell viability in the presence or absence of APS was detected by the methyl thiazolyl tetrazolium colorimetric assay. The autophagosomes were observed by electron microscopy to examine the influence of APS on autophagy caused by H2O2 in C2C12 cells, and the percentage of apoptosis cells was measured by flow cytometry. To further confirm the effect of H2O2 on C2C12 cells, the protein expression of LC3 and RARP, which are the markers of autophagy and apoptosis, respectively, was analyzed by Western blot, as well as the expression levels of p-p70S6K, p70S6K, Bcl-2, Bax, cyto-C, and Caspase-3, to reveal the underlying mechanisms. We observed multiple effects of APS on C2C12 functionality. APS treatment of C2C12 cells at 1 mg/mL reduced cell viability to less than 70 %, and analysis by electron microscopy revealed that APS also reduced the number of H2O2-induced autophagosome formation. Similarly, APS abated the H2O2-mediated increase in cell apoptosis, which was accompanied by the inhibition of LC3 II and RARP that are normally upregulated by H2O2. The expression of p-p70S6K and p70S6K, however, remained unchanged in C2C12 cells in the Control, H2O2 and H2O2 + APS groups. In addition, APS promoted the expression of protein Bcl-2 in H2O2-treated C2C12 cells, but did not change Bax, thus reducing the Bax/Bcl-2 ratio that in turn prevented the release of cytochrome c and the activation of caspase-3. APS inhibits the autophagy and apoptosis induced by peroxide injury in C2C12 myoblasts through two independent signaling pathways: the mTOR-independent pathway for the inhibition of autophagy, and the caspase-3-dependent pathway for the suppression of apoptosis.

Muscle atrophy poses a serious concern to patients inflicted with inflammatory diseases. There is now increasing evidence which suggests a vital role for tumor necrosis factor alpha (TNF-α) in muscle pathology associated with impairment of differentiation and muscle wasting. Resveratrol has been an ascribed inhibitory effect on glucocorticoid-induced muscle atrophy in vitro, but the influence of resveratrol on the growth of C2C12 myotubes exposed to TNF-α remains unclear. The present study aimed to investigate the involvement of TNF-α in the regulation of skeletal muscle hypertrophy and atrophy, and the possibility to interfere with such modulations by means of resveratrol supplementation. For this purpose, C2C12 myotubes were treated with TNF-α in the presence or absence of resveratrol. Myotube treatment with TNF-α contributes to both hyperexpression of the muscle-specific ubiquitin ligase MAFbx and MuRF1, and these alterations are linked to a decrease of anabolic targets (Akt, mTOR, p70S6k and 4E-BP1) and an increase of catabolic targets (FoxO1, FoxO3a, MAFbx and MuRF1). Resveratrol supplementation effectively counteracts TNF-α induced muscle protein loss and reverses declining expression of Akt, mTOR, p70S6K, 4E-BP1and FoxO1, but exerts no influence of FoxO3a expression. Our study demonstrates that resveratrol can reverse the muscle cell atrophy caused by TNF-α through regulation of the Akt/mTOR/FoxO1 signaling pathways, followed by inhibition of the atrophy-related ubiquitin ligase. Our findings suggested that resveratrol could represent a possible strategy to improve muscle mass.