摘要
以C2C12肌管细胞为试验材料,探讨脂多糖(LPS)诱导的炎症反应对肌管蛋白降解相关基因及信号通路的影响。选取不同质量浓度(10、100、1 000ng·mL-1)的LPS分别刺激C2C12肌管细胞30min和3h,通过RTqPCR检测炎症因子TNF-α和IL-6的基因转录,确定LPS最佳刺激浓度以建立细胞炎症模型。用最佳浓度LPS分别刺激C2C12肌管细胞0min、30min、1h、3h、6h、12h、24h,RT-qPCR检测MAFbx基因和MuRF基因在不同时间点的转录量变化。此外,1 000ng·mL-1 LPS刺激C2C12肌管细胞12h后,Western blot检测AKT/FOXO1、mTOR和P38信号通路相关蛋白质的表达情况。结果表明:LPS刺激C2C12肌管细胞的最佳浓度为1 000ng·mL-1,在作用30min和3h时均能显著上调TNF-α、IL-6的基因转录,而在刺激12和24h时MuRF1、IL-1β、IL-6和TLR4的基因转录显著上调。此外,1 000ng·mL-1 LPS刺激C2C12肌管细胞12h时,仅AKT和FOXO1蛋白磷酸化水平明显降低。基于以上结果,推测LPS通过调控AKT/FOXO1信号通路诱导C2C12肌管细胞MuRF1转录。
This study was designed to investigate the effects of the inflammatory induced by lipopolysaccharide（LPS）on the genes and pathways controlling the protein degradation in C2C12 myotubes.In order to establish an inflammatory cell mode,the C2C12 myotubes were treated with graded concentrations of LPS（10,100 and 1 000ng·mL^-1）for 30 min and 3h,and then the mRNA transcription of TNF-αand IL-6were determined by qPCR method.The results showed that1 000ng·mL-1 LPS was the optimal concentration.After the C2C12 myotubes were treated with1 000ng·mL-1 LPS for 0min,30 min,1h,3h,6h,12 hand 24h,the mRNA transcription of MAFbxand MuRF1 were determined by qPCR.And the activation of AKT/FOXO1,mTOR and p38 MAPK signaling pathway was detected by western blot at 12 hafter LPS treatment.The results showed that 1 000ng·mL-1 LPS significantly induced the mRNA transcription of TNF-αand IL-6at 30 min and 3h.The transcription of MuRF1,IL-1β,IL-6and TLR4 were significantlyup-regulated at 12 hand 24hafter LPS treatment.In addition,the phosphorylation of AKT and FOXO1 in C2C12myotubes was significantly suppressed at 12 hafter LPS treatment.Therefore,our results suggest that LPS can induce MuRF1 expression in C2C12 myotubes through the regulation of AKT/FOXO1 signaling pathway.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2016年第2期374-380,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
湖北省自然科学基金(2013CFA029
2015CFB514)
