| BackgroundLipid metabolism disorder is the core mechanism of atherosclerosis,obesity,diabetes and other chronic non communicable diseases(NCDs),and is the initial key link of NCDs.Muscle-derived IL-6 has an outstanding regulatory effect on lipid metabolism disorder.Eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA)to improve lipid metabolism disorders continues to receive attention,however,the mechanism have mainly focused on fat and liver tissues,and less involved endocrine function of skeletal muscle.Our research aims to explore the effects of EPA and DHA on IL-6 expression and secretion in C2C12 myotube cells,and to elucidate the molecular mechanism of EPA and DHA stimulation and regulation of myogenic IL-6 via TRPV1/PKC/Ca2+signaling pathway.Our research can provide a new theory for rational intake of EPA and DHA to improve lipid metabolism disorder from the perspective of myokines.Methods1.In vitro culture and induction of differentiated C2C12 myotube cells,cells treated with different concentrations(25,50,100,200,400 μM)of EPA or DHA for 12,24,36 h,MTT to determine cell activity,Real-time PCR and Western-blot to determine the expression level of IL-6 mRNA and protein respectively,ELISA to determine IL-6 secretion level.2.Select the intervention time that can best affect IL-6 expression,treat cells with EPA or DHA at different concentrations(25,50,100,200,400 μM),and measure the TRPV1,PKC,p-PKC,NFATc1,CaN expression levels,and Fluo-4 AM calcium ion fluorescent probe detected intracellular Ca2+concentration to unveil the underlying mechanism.3.After 1 hour of pretreatment with TRPV1 agonist N-Arachidonoyl Dopamine(NADA)and inhibitor Capsazepine(CPZ),the cells were treated with an appropriate concentration(200 μM)EPA or DHA for 24 hours,detected the expression level of IL-6,TRPV1 and p-PKC.Results1.Effects of EPA and DHA on IL-6 expression and secretion of C2C12 myotubular cells25,50,100,200,400 μM EPA and DHA intervention did not produce obvious cytotoxicity within 24 hours.100 μM EPA or DHA upregulates IL-6 protein expression in a time gradient.After 24 hours of intervention by EPA or DHA in myotube cells,the expression and secretion of IL-6 increased in a concentration gradient,among which 200 and 400 μM had the most significant effects.The expression of IL-6 protein in the 50,100,and 200 μM EPA and 25,50,100 and 200μM DHA intervention groups were significantly increased compared with the control group,and the expression of IL-6 protein was increased by 100μM EPA and DHA.2.Effects of EPA and DHA on C2C12 myotube inflammatory factorThere was no significant change in the expression levels of NFκB and IKKβafter EPA and DHA intervention,but DHA could significantly reduce the expression of TNF-α mRNA in myotubes,while EPA could significantly increase the mRNA expression of anti-inflammatory factor IL-10.3.Effects of EPA and DHA on TRPV1/PKC/Ca2+ signaling pathway in C2C12 myotubular cellsThe expression of p-TRPV1 and TRPV1 protein and p-TRPV1/TRPV1 ratio in the 200 μM and 400 μM EPA intervention groups increased significantly compared with the control group.The expression of p-TRPV1 protein was significantly increased in all concentrations of DHA intervention group.The ratio of TRPV1 protein expression to p-TRPV1/TRPV1 in the 100 μM and 200 μM DHA intervention groups was significantly higher than that in the control group.The expression level of p-PKC protein and the p-PKC/PKC ratio in the 100 μM,200 μM and 400 μM EPA intervention groups were significantly increased,while the expression level of PKC and p-PKC protein and p-PKC ratio of 200 μM and 400 μM DHA significantly increased the expression of p-PKC protein and p-PKC/PKC ratio in myotubular cells.The concentrations of EPA and DHA in the intervention groups significantly increased the content of Ca2+ in myotucules,CaN and NFATc1 protein expression.NAD A further improve the expression of EPA-mediated IL-6,TRPV1 and p-PKC,and CPZ attenuates the expression of EPA-mediated IL-6 and TRPV1 and p-PKC;NADA further increase the exp ression of DHA-mediated IL-6 and p-PKC,and CPZ attenuates the expression of EPA-mediated IL-6 and p-PKC.Conclusions1.EPA and DHA at appropriate concentrations can promote the expression and secretion of IL-6 in C2C12 myotube cells,of which 200 μM intervention for 24 h has a significant effect.2.EPA and DHA may stimulate the expression and secretion of myogenic IL-6 through the TRPV1/PKC/Ca2+ signaling pathway. |
PDF Full Text Request