Fig. 3: ω-6 PUFAs activate autophagy and antioxidation through the AMPK signaling pathway.
AMPK activity was evaluated in hepatocytes incubated for 24 h in the control or 250 μM LA-containing medium (n = 3) (a). The levels of total and phosphorylated AMPKα and ULK1 and the levels of nuclear Nrf2 were examined by western blot analysis and quantitated after treatment with an AMPK pathway inhibitor (5 μM CC) or activator (500 μM AICAR) (n = 3). 10 gels were run and 10 blots were made. All of the blots were not stripped and re-probed. The blots of T-AMPKα, P-AMPKαThr172, T-ULK1, P-ULK1Ser467, P-ULK1Ser555, P-ULK1Ser637, and P-ULK1Ser757 were used for the GAPDH loading controls and the blots of n-Nrf2 was used for the LaminB loading controls (b, c). Representative confocal microscopic image of hepatocytes stained with LysoTracker after treatment with an AMPK pathway inhibitor or activator. Scale bars, 50 μm (d, f). The presence of LysoTracker-stained intracellular autolysosomes was demonstrated by flow cytometry, and the numbers of the autolysosomes were calculated by flow cytometric analysis of the mean red (FL4) fluorescence intensity after treatment with an AMPK pathway inhibitor or activator (e, g). The mRNA expression levels of key antioxidation-related genes (Nrf2, Sod1, Sod3, CAT, and Gpx) were analyzed after treatment with a pathway inhibitor or activator (n = 6) (h, i). The results are presented as the mean with SEM and were analyzed using independent t-tests and Tukey’s test. Bars bearing the same letters are not significantly different among treatments (*P ≥ 0.05).
