EPA inhibits the inhibitor of κBα (IκBα)/NF-κB/muscle RING finger 1 pathway in C2C12 myotubes in a PPARγ-dependent manner

The present study was conducted to evaluate the mechanism by which n-3 PUFA regulates the inhibitor of kappa B alpha (I kappa B alpha)/NF-kappa B/muscle RING finger 1 (MuRF1) pathway in C2C12 myotubes. After treatment with 150, 300 or 600 mu M-alpha-linolenic acid (ALA) or -EPA for 24 h in C2C12 myotubes, the levels of phosphorylated I kappa B alpha (p-I kappa B alpha) and total I kappa B alpha were measured by Western blot. Compared with the bovine serum albumin (BSA) control, 150 and 300 mu M-ALA and -EPA, respectively, did not affect the total I kappa B alpha protein level (P>0.05). However, 600 mu M-EPA, but not 600 mu M-ALA, prevented I kappa B alpha phosphorylation and increased the total I kappa B alpha levels (P<0.01). Furthermore, total nuclear protein was isolated and analysed by the electrophoretic mobility shift assay for NF-kappa B DNA-binding activity after treatment with 600 mu M-ALA or -EPA for 24 h. EPA (600 mu M), but not ALA (600 mu M), decreased the NF-kappa B DNA-binding activity when compared with BSA (P<0.01). It was further observed that 600 mu M-EPA caused a 3.38-fold reduction in the levels of MuRF1 mRNA expression compared with BSA (P<0.01). Additionally, 600 mu M-EPA resulted in a 2.3-fold induction of PPAR gamma mRNA expression (P<0.01). In C2C12 myotubes, PPAR gamma knockdown by RNA interference significantly decreased PPAR gamma mRNA and protein expression to approximately 50 and 60% (P<0.01), respectively. Interestingly, in C2C12 myotubes with PPAR gamma knockdown, 600 mu M-ALA and -EPA did not affect the levels of p-I kappa B alpha and total I kappa B alpha, NF-kappa B DNA-binding activity or MuRF1 mRNA expression when compared with BSA (P>0.05). These results revealed that EPA, but not ALA, inhibited the I kappa B alpha/NF-kappa B/MuRF1 pathway in C2C12 myotubes in a PPAR gamma-dependent manner.