小麦肽及其与大豆肽、豌豆肽复配肽的体外组合对TNF-α刺激C2C12骨骼肌细胞蛋白表达的影响

Effects of wheat oligopeptides and its combination with soybean oligopeptides and pea oligopeptides on protein expression in C2C12 skeletal muscle cells stimulated by TNF-αin vitro

通过细胞实验探讨小麦肽、大豆肽、豌豆肽复合物对TNF-α刺激C2C12骨骼肌细胞蛋白表达的影响,并初步探明作用通路。采用终质量浓度为25 ng/mL的TNF-α处理C2C12细胞4 d建立TNF-α刺激模型,CCK-8法确定小麦肽、大豆肽、豌豆肽对C1C12细胞的安全剂量;检测不同复配比例的小麦肽、大豆肽、豌豆肽实验组的肌球蛋白相关指标(MyoD、MyoG、MyHC)、泛素-蛋白酶体系统肌肉萎缩相关指标(TRIM63和MAFbx)的蛋白表达、Akt、mTOR和其磷酸化蛋白表达情况。结果表明,与对照组相比,TNF-α刺激的C2C12细胞中MyoD、MyoG和MyHC的蛋白表达量均有下降,其中MyHC显著降低;MAFbx和TRIM63表达量显著升高;Akt和mTOR的磷酸化水平显著降低,而经过肽样品处理后,各指标均有缓解,且以小麦肽与大豆肽+豌豆肽质量比1∶3效果最好。小麦肽与大豆肽+豌豆肽以1∶3的质量比进行复配在几种配比中可以最有效地改善由TNF-α造成的C2C12细胞肌蛋白含量降低,可通过调节E3泛素连接酶的表达和提高Akt/mTOR的蛋白磷酸化水平的通路,从而降低细胞中蛋白降解和促进蛋白合成。

This study aimed to investigate the effects of the complex of wheat oligopeptides,soybean oligopeptides,and pea oligopeptides on the protein expression of C2C12 skeletal muscle cells stimulated by TNF-αand preliminarily explore the mechanism.C2C12 cells were treated with a final concentration of 25 ng/mL TNF-αfor 4 days to establish the TNF-αstimulation model.The safe dose of wheat oligopeptides,soybean oligopeptides,and pea oligopeptides on C1C12 cells was determined by the CCK-8 method.The protein expressions were detected with different proportions of complex peptides,including myosin-related indexes(MyoD,MyoG,and MyHC),the muscle atrophy-related indexes(TRIM63 and MAFbx)of the proteasome system,Akt,mTOR,and their phosphorylated proteins.Results showed that compared with the control group,the protein expressions of MyoD,MyoG,and MyHC in TNF-α-stimulated C2C12 cells were decreased,MyHC was significantly decreased,and the expressions of MAFbx and TRIM63 were significantly increased.The phosphorylation levels of Akt and mTOR were significantly reduced,but all indexes were alleviated after treatment with peptides,and the ratio of wheat oligopeptides to soybean oligopeptides+pea oligopeptides of 1∶3 was the most effective.The combination of wheat oligopeptides to soybean oligopeptides+pea oligopeptides at the ratio of 1∶3 can effectively improve the reduction of myoprotein content in C2C12 cells caused by TNF-α,reduce protein degradation,and promote protein synthesis by regulating the expression of E3 ubiquitin ligase and improving the protein phosphorylation level of Akt/mTOR.