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.

中文翻译：

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脂多糖和硫辛酸刺激的C2C12肌管的白细胞介素6反应。

我们先前的研究探讨了硫辛酸对C ₂ C ₁₂肌管中IL-6表达的调节的双重作用。但是，具体机制仍不清楚。为了评估骨骼肌中的IL-6信号传导，pCMV6-IL-6在C ₂ C _12中过表达肌管。实时定量聚合酶链反应用于检测mRNA表达。免疫组织化学和DeadEnd比色法TUNEL系统分别用于检测IL-6的定位和分析过表达IL-6的细胞的凋亡。使用caspase-3 / CPP32比色法和Western印迹法分别分析caspase-3活性和蛋白表达。我们的结果表明，过表达的IL-6不仅位于细胞质中，而且位于细胞膜的细胞内侧。此外，细胞核未显示IL-6过表达。DeadEnd比色法凋亡检测分析结果表明，过表达IL-6的细胞中存在凋亡核。但是，过表达的IL-6未能促进caspase-3活性。值得注意的是 外源热原脂多糖（LPS）显着促进IL-6 mRNA表达和caspase-3活性，但不诱导凋亡细胞的形成。此外，硫辛酸显着上调IL-6，IL-6Ra和gp130 mRNA表达，并显着增加caspase-3活性，但不诱导凋亡细胞的形成。硫辛酸可显着提高未处理细胞的p-Akt水平，而在LY294002处理细胞中则不会。两者合计，我们的结果表明，过表达的IL-6诱导的细胞凋亡可能不是由caspase-3介导的。LPS诱导的IL-6 mRNA表达可能不参与C中的IL-6经典信号传导或反信号传导 和gp130 mRNA表达并显着增加caspase-3活性，但不诱导凋亡细胞的形成。硫辛酸可显着提高未处理细胞的p-Akt水平，而在LY294002处理细胞中则不会。两者合计，我们的结果表明，过表达的IL-6诱导的细胞凋亡可能不是由caspase-3介导的。LPS诱导的IL-6 mRNA表达可能不参与C中的IL-6经典信号传导或反信号传导 和gp130 mRNA表达并显着增加caspase-3活性，但不诱导凋亡细胞的形成。硫辛酸可显着提高未处理细胞的p-Akt水平，而在LY294002处理的细胞中则不会。两者合计，我们的结果表明，过表达的IL-6诱导的细胞凋亡可能不是由caspase-3介导的。LPS诱导的IL-6 mRNA表达可能不参与C中的IL-6经典信号传导或反信号传导₂ C ₁₂肌管。硫辛酸诱导的IL-6 mRNA表达可能由C ₂ C ₁₂肌管中的IL-6经典信号传导介导。