摘要
目的探讨不同浓度二十碳五烯酸(eicosapentaenoic acid,EPA)和二十二碳六烯酸(docosahexaenoic acid,DHA)对C2C12肌管细胞IL-6表达与分泌的调节作用和机制。方法采用25、50、100、200、400μmol/L EPA或DHA处理C2C12肌管细胞12、24、36 h,设立BSA对照组。MTT检测细胞活性,PCR与Western blot分别检测IL-6的mRNA与蛋白表达水平,ELISA测定IL-6分泌水平,检测TRPV1、PKC的mRNA表达水平及TRPV1、PKC、p-PKC蛋白表达水平,Fluo-4 AM钙离子荧光探针检测胞内Ca^(2+)浓度。结果IL-6表达与分泌方面,100μmol/L EPA或DHA干预24h可显著提高肌管细胞IL-6 mRNA表达;100μmol/LEPA或DHA干预24h、36h可显著促进IL-6分泌;100μmol/L EPA或DHA可呈时间梯度上调IL-6蛋白表达水平。各浓度EPA或DHA干预肌管细胞24h后,IL-6 mRNA表达水平与分泌均呈浓度梯度增加,其中200、400μmol/L效果最为显著;50、100、200μmol/L EPA与25、50、100、200μmol/L DHA干预组IL-6蛋白质表达水平较对照组均显著增加,100μmol/L EPA和DHA增加IL-6蛋白表达最为显著。信号通路方面,100、200、400μmol/L EPA与DHA显著提高肌管细胞TRPV1 mRNA表达水平;200、400μmol/L EPA与100、200、400μmol/L DHA干预组TRPV1蛋白表达水平较对照组显著升高。400μmol/L EPA与DHA可显著上调PKC mRNA表达水平;100、200、400μmol/L EPA干预组p-PKC蛋白表达水平与p-PKC/PKC比值显著增高,而200、400μmol/L DHA可显著增加肌管细胞PKC、p-PKC蛋白表达水平以及p-PKC/PKC比值。各浓度EPA与DHA干预组均显著增加肌管细胞胞内Ca^(2+)含量。结论EPA与DHA在适宜浓度可以促进C2C12肌管细胞IL-6的表达与分泌,EPA和DHA可能通过TRPV1/PKC/Ca^(2+)信号通路刺激肌源性IL-6的表达与分泌。[营养学报,2023,45(2):148-156]
Objective To explore the effects and mechanism of different concentrations of eicosapentaenoic acid(EPA)or docosahexaenoic acid(DHA)on the expression and secretion of IL-6 in C2C12 myotubes.Methods Myotubes were treated with 25,50,100,200,400μmol/L EPA or DHA for 12,24,and 36 h,and MTT was used to detect cell viability.IL-6 secretion was detected by ELISA.The mRNA expression of IL-6,TRPV1,PKC was detected by PCR.The protein levels of IL-6,TRPV1,PKC,p-PKC were detected by Western blot.The concentration of Ca^(2+)in myotubes was detected by calcium ion fluorescence probe.Results In terms of the expression and secretion of IL-6,100μmol/L EPA or DHA intervention for 24h significantly increased the expression of IL-6 mRNA in myotubes;100μmol/L EPA or DHA intervention for 24h and 36h significantly promoted the secretion of IL-6.The expression of IL-6 protein was up-regulated in a time dependent manner.After EPA or DHA intervention for 24 h,the mRNA expression and secretion of IL-6 were increased in a concentration dependent manner in which 200 and 400μmol/L had the most significant effects.Compared with the control group,50,100,200μmol/L EPA and 25,50,100,200μmol/L DHA intervention significantly increased the expression of IL-6 protein in which 100μmol/L EPA and DHA intervention had the most significant effect.In terms of signaling pathway,100,200,400μmol/L EPA and DHA significantly increased the TRPV1 mRNA expression in myotubes.Compared with the control group 200,400μmol/L EPA and 100,200,400μmol/L DHA significantly increased TRPV1 protein expression.400μmol/L EPA and DHA significantly up-regulated the mRNA expression of PKC,100,200,and 400μmol/L EPA significantly increased the p-PKC protein expression and p-PKC/PKC ratio,200,400μmol/L DHA significantly increased the PKC,p-PKC and p-PKC ratios in myotubes.EPA and DHA intervention significantly increased the intracellular Ca^(2+)content of myotubes.Conclusion Appropriate concentrations of EPA and DHA promoted the expression and secretion of IL-6 in C2C12
作者
曾欢婷
杨娴
毛联智
魏文婷
毛丽梅
ZENG Huan-ting;YANG Xian;MAO Lian-zhi;WEI Wen-ting;MAO Li-mei(Department of Nutrition and Food Hygiene,School of Public Health,Southern Medical University,Guangzhou 510515,China)
出处
《营养学报》
CAS
CSCD
北大核心
2023年第2期148-156,共9页
Acta Nutrimenta Sinica
基金
国家自然科学基金(No.82073542)。
