Figure 5
Luteolin inhibits glutamate-induced excessive autophagy and mitophagy activation. HT-22 cells were pre-treated with luteolin at different concentrations (5–50 µM) for 24 h and 10 µM quercetin was used as a positive control, followed by 5 mM glutamate for 18 h. Chloroquine (CQ, 50 µM) served as the positive control for autophagy induction. (A) The protein expression level of LC3B, Beclin-1 (autophagy) and BNIP3L/NIX (mitophagy) were analyzed by Western blot, and β-actin served as the loading control. (B) Relative protein levels of LC3B, Beclin-1 (autophagy) and BNIP3L/NIX (mitophagy) were quantified by densitometry and the mean data from at least three independent experiments were normalized to the results (n = 3). (C) The co-localization of lysosome and mitochondria. HT-22 cells were stained with the mitochondria (Mitotracker: red) Lysosome (Lysotracker: green) and nucleus (Hoechst: blue). They were observed under the confocal laser scanning microscope with 40X objective magnification (scalebar is 10 µm). (D) The bar graph of co-localization was considered with Pearson’s correlation coefficient analyzed using ImageJ JACoP plugin. Data represent the means ± SEM and represent averages of results from at least 50 cells (n = 3). (E) Autophagy inhibitor, ammonium chloride (NH4Cl), inhibits glutamate-induced HT-22 cell death. The morphology of HT-22 cells was visualized under the inverted light microscope. A bar graph shows the MTS cell viability results (n = 3). The data represent the means ± SEM collected from at least three independent experiments and. *p value < 0.05, **p value < 0.01, ****p value < 0.001 compared with only glutamate-treated group, #p value < 0.05 compared with untreated control group. CQ:chloroquine, L:luteolin, Q:quercetin.
