Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan.
Translational Research Center of Neuromuscular Diseases, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
PLoS One. 2022 Jul 28;17(7):e0272231. doi: 10.1371/journal.pone.0272231. eCollection 2022.
Arecoline is known to induce reactive oxygen species (ROS). Our previous studies showed that arecoline inhibited myogenic differentiation and acetylcholine receptor cluster formation of C2C12 myoblasts. N-acetyl-cysteine (NAC) is a known ROS scavenger. We hypothesize that NAC scavenges the excess ROS caused by arecoline. In this article we examined the effect of NAC on the inhibited myoblast differentiation by arecoline and related mechanisms. We found that NAC less than 2 mM is non-cytotoxic to C2C12 by viability analysis. We further demonstrated that NAC attenuated the decreased number of myotubes and nuclei in each myotube compared to arecoline treatment by H & E staining. We also showed that NAC prevented the decreased expression level of the myogenic markers, myogenin and MYH caused by arecoline, using immunocytochemistry and western blotting. Finally, we found that NAC restored the decreased expression level of p-ERK1/2 by arecoline. In conclusion, our results indicate that NAC attenuates the damage of the arecoline-inhibited C2C12 myoblast differentiation by the activation/phosphorylation of ERK. This is the first report to demonstrate that NAC has beneficial effects on skeletal muscle myogenesis through ERK1/2 upon arecoline treatment. Since defects of skeletal muscle associates with several diseases, NAC can be a potent drug candidate in diseases related to defects in skeletal muscle myogenesis.
槟榔碱已知可诱导活性氧（ROS）。我们之前的研究表明，槟榔碱抑制 C2C12 成肌细胞的肌生成分化和乙酰胆碱受体簇形成。N-乙酰半胱氨酸（NAC）是一种已知的 ROS 清除剂。我们假设 NAC 可以清除槟榔碱产生的过量 ROS。在本文中，我们研究了 NAC 对槟榔碱抑制的成肌细胞分化的影响及其相关机制。我们发现，通过细胞活力分析，NAC 小于 2mM 对 C2C12 无细胞毒性。我们进一步证明，与槟榔碱处理相比，NAC 可减少 H&E 染色中每个肌管中的肌管数量和细胞核数量减少。我们还表明，NAC 可通过免疫细胞化学和 Western blot 防止槟榔碱引起的肌生成标志物肌球蛋白和 MYH 的表达水平降低。最后，我们发现 NAC 恢复了槟榔碱引起的 p-ERK1/2 表达水平降低。总之，我们的结果表明，NAC 通过 ERK 的激活/磷酸化减轻了槟榔碱抑制的 C2C12 成肌细胞分化的损伤。这是首次报道表明 NAC 通过 ERK1/2 对槟榔碱处理后的骨骼肌生成具有有益作用。由于骨骼肌缺陷与多种疾病有关，NAC 可能是与骨骼肌生成缺陷相关疾病的有效药物候选物。
