Fig. 1. L. plantarum-derived EVs (L-EVs) increased BDNF expression in HT22 cells. (A) Expression levels of tBdnf, Bdnf1, Bdnf4, and Ngf in cultured HT22 cells in the presence of GC (400 ng/ml), L-EV (20 µg/ml) or GC+L-EV for 24 h (n=8~12 per group). Two-way ANOVA followed by Bonferroni post hoc test (tBdnf, F(1,37)=0.0052 and p=0.9429 for GC main effect, F(1,37)=11.19 and p=0.0019 for L-EV main effect, and F(1,37)=14.75 and p=0.0005 for GC×L-EV; Bdnf1, F(1,32)=1.390 and p=0.2471 for GC main effect, F(1,32)=13.60 and p=0.0008 for L-EV main effect, and F(1,32)=7.166 and p=0.0116 for GC×L-EV; Bdnf4, F(1,35)=0.2697 and p=0.6068 for GC main effect, F(1,35)=22.60 and p<0.0001 for L-EV main effect, and F(1,35)=12.49 and p=0.0012 for GC×L-EV; Ngf, F(1,34)=27.95 and p<0.0001 for GC main effect, F(1,34)=24.33 and p<0.0001 for L-EV main effect, and F(1,34)=2.639 and p=0.1135 for GC×L-EV). (B) Western blot data showing the expression level of proBDNF in HT22 cells treated with GC (400 ng/ml), L-EV (20 µg/ml) or GC+L-EV for 24 h (n=6 per group). Two-way ANOVA followed by Bonferroni post hoc test (F(1,20)=14.19 and p=0.0012 for GC main effect, F(1,20)=3.819 and p=0.0648 for L-EV main effect, and F(1,20)=3.819 and p=0.0648 for GC×L-EV). (C) Expression levels of Sirt1, Hdac2, and Creb1 in HT22 cells treated with GC (400 ng/ml), L-EV (20 µg/ml) or GC+L-EV for 24 h (n=6~10 per group). Two-way ANOVA followed by Bonferroni post hoc test (Sirt1, F(1,27)=0.0168 and p=0.8979 for GC main effect, F(1,27)=65.48 and p<0.0001 for L-EV main effect, and F(1,27)=22.96 and p<0.0001 for GC×L-EV; Hdac2, F(1,22)=5.034 and p=0.0353 for GC main effect, F(1,22)=0.2970 and p=0.5913 for L-EV main effect, and F(1,22)=3.793 and p=0.0643 for GC×L-EV; Creb1, F(1,26)=0.0513 and p=0.8227 for GC main effect, F(1,26)=4.846 and p=0.0368 for L-EV main effect, and F(1,26)=0.0056 and p=0.9412 for GC×L-EV). (D, E) Expression levels of Sirt1(D), tBdnf, Bdnf1, Bdnf4, and Creb1 (E) in HT22 cells treated with siRNA-CON (50 pmol), siRNA-Sirt1(50 pmol), L-EV (20 µg/ml) or siRNA-Sirt1+L-EV for 24 h (n= 5~10 per group). Two-way ANOVA followed by Bonferroni post hoc test (Sirt1, F(1,23)=22.48 and p<0.0001 for siSirt1 main effect, F(1,23)=0.5684 and p=0.4586 for L-EV main effect, and F(1,23)=0.3164 and p=0.5792 for siSirt1×L-EV; Hdac2, F(1,28)=1.194 and p=0.2839 for siSirt1 main effect, F(1,28)=0.4969 and p=0.4867 for L-EV main effect, and F(1,28)=2.108 and p=0.1576 for siSirt1×L-EV; Creb1 , F(1,19)=47.84 and p<0.0001 for siSirt1 main effect, F(1,19)=0.0141 and p=0.9069 for L-EV main effect, and F(1,19)=0.1306 and p=0.7218 for siSirt1×L-EV; tBdnf, F(1,24)=3.681 and p=0.0670 for siSirt1 main effect, F(1,24)=1.632 and p=0.2137 for L-EV main effect, and F(1,24)=1.446 and p=0.2409 for siSirt1×L-EV; Bdnf1, F(1,21)=5.607 and p=0.0276 for siSirt1 main effect, F(1,21)=0.7048 and p=0.4106 for L-EV main effect, and F(1,21)=0.7991 and p=0.3815 for siSirt1×L-EV; Bdnf4, F(1,23)=25.64 and p<0.0001 for siSirt1 main effect, F(1,23)=0.3326and p=0.5697 for L-EV main effect, and F(1,23)=0.1315 and p=0.7202 for siSirt1×L-EV). GC, glucocorticoid; veh, vehicle. Data are presented as mean±SEM. *p<0.05; **p<0.01.
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