Effect of rapamycin on the phosphorylation of the mTOR signalling pathway and CD36 translational efficiency under inflammatory stress.

<p>HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium containing 25 ng/mL TNF-α (TNF-α) or 25 ng/mL TNF-α plus 10 ng/mL rapamycin (TNF-α+Rapa) or 20 ng/mL IL-6 (IL-6) or 20 ng/mL IL-6 plus 10 ng/mL rapamycin (IL-6+ Rapa). C57BL/6J mice were fed a normal chow diet and received a casein injection (Casein) or casein and rapamycin injection (Casein+Rapa) for 14 weeks. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total p70S6K, p-4E-BP1 (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (A) and livers (B). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. A polysomal analysis was performed to detect CD36 translational efficiency in the cells (C) and livers (D). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. The results are depicted as the mean ± SD from three separate experiments. * denotes a significant difference at <i>P</i><0.05, and ^# denotes a significant difference at <i>P</i><0.01.