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

Abstract

The present study was conducted to evaluate the mechanism by which n-3 PUFA regulates the inhibitor of B (IκB)/NF-B/muscle RING finger 1 (MuRF1) pathway in C2C12 myotubes. After treatment with 150, 300 or 600m - linolenic acid (ALA) or -EPA for 24h in C2C12 myotubes, the levels of phosphorylated IB (p-IB) and total IB were measured by Western blot. Compared with the bovine serum albumin (BSA) control, 150 and 300m-ALA and -EPA, respectively, did not affect the total IB protein level (P>005). However, 600m-EPA, but not 600m-ALA, prevented IB phosphorylation and increased the total IB levels (P<001). Furthermore, total nuclear protein was isolated and analysed by the electrophoretic mobility shift assay for NF-B DNA-binding activity after treatment with 600m-ALA or -EPA for 24h. EPA (600m), but not ALA (600m), decreased the NF-B DNA-binding activity when compared with BSA (P<001). It was further observed that 600m-EPA caused a 338-fold reduction in the levels of MuRF1 mRNA expression compared with BSA (P<001). Additionally, 600m-EPA resulted in a 23-fold induction of PPAR mRNA expression (P<001). In C2C12 myotubes, PPAR knockdown by RNA interference significantly decreased PPAR mRNA and protein expression to approximately 50 and 60% (P<001), respectively. Interestingly, in C2C12 myotubes with PPAR knockdown, 600m-ALA and -EPA did not affect the levels of p-IB and total IB, NF-B DNA-binding activity or MuRF1 mRNA expression when compared with BSA (P>005). These results revealed that EPA, but not ALA, inhibited the IκBα/NF-κB/MuRF1 pathway in C2C12 myotubes in a PPAR-dependent manner. © 2010 The Authors.