Interleukin-6 Response of C2C12 Myotubes Stimulated with Lipopolysaccharide and Lipoic Acid.

Our previous study explored the dual effect of lipoic acid on the regulation of IL-6 expression in C2C12 myotubes. However, the specific mechanism remains unclear. To evaluate IL-6 signaling in skeletal muscle, pCMV6-IL-6 was overexpressed in C2C12 myotubes. The real-time quantitative polymerase chain reaction was used to detect mRNA expression. Immunohistochemistry and a DeadEnd colorimetric TUNEL system were used to detect IL-6 localization and analyze the apoptosis in IL-6-overexpressing cells, respectively. A caspase-3/CPP32 colorimetric assay and Western blotting were used to analyze caspase-3 activity and protein expression, respectively. Our results showed the overexpressed IL-6 was not only located in the cytosol but also on the intracellular side of the cell membrane. Moreover, the nucleus did not demonstrate IL-6 overexpression. The DeadEnd colorimetric apoptosis detection assay results demonstrated that apoptotic nuclei were present in IL-6-overexpressing cells. However, the overexpressed IL-6 failed to promote caspase-3 activity. Notably, the exogenous pyrogen lipopolysaccharide (LPS) significantly promoted IL-6 mRNA expression and caspase-3 activity but did not induce apoptotic cell formation. Moreover, lipoic acid significantly upregulated IL-6, IL-6Ra, and gp130 mRNA expression and significantly increased caspase-3 activity but did not induce apoptotic cell formation. Lipoic acid significantly increased the p-Akt level in untreated cells but not in LY294002-treated cells. Taken together, our results suggesting that the overexpressed IL-6-induced apoptosis may not be mediated by caspase-3. LPS-induced IL-6 mRNA expression may not be involved in IL-6 classical signaling or trans-signaling in C2C12 myotubes. Lipoic acid-induced IL-6 mRNA expression may be mediated by IL-6 classical signaling in C2C12 myotubes.