Priority Research Paper Volume 12, Issue 7 pp 5590—5611

Down-regulation of AMPK signaling pathway rescues hearing loss in TFB1 transgenic mice and delays age-related hearing loss

Figure 7. AMPK knockout interrupts and decreases apoptosis in the cochlea. (A) Immunoblots analyses of the four genotype mice aged 10-12 months show proteins from the cochleae related to the apoptotic pathway, including Bcl-2, Bax, Caspase-3, and Cytochrome C. GAPDH serves as the loading control. (B) The histograms summarized the expression levels of proteins related to the apoptosis pathway. The apoptosis signaling (cleaved caspase-3) in Tg-B1 mice was significantly stronger than AMPK KO mice and WT controls (Tg-B1 vs. AMPK+/−/Tg-B1, p<0.0001; Tg-B1 vs. WT, p<0.0001; AMPK+/−/Tg-B1 vs. WT, p=0.1807). The expression of pro-apoptotic protein (Bax) in Tg-B1 mice is significantly higher than the other three groups (Tg-B1 vs. AMPK+/−/Tg-B1, p<0.0001; Tg-B1 vs. WT, p<0.001; Tg-B1 vs. AMPK+/−, p<0.0001; WT vs. AMPK+/−, p<0.0001; AMPK+/−/Tg-B1 vs. AMPK+/−, p=0.0001). Bcl-2/Bax ratio in the Tg-B1 group is significantly lower than the other three groups (Tg-B1 vs. AMPK+/−/Tg-B1, p<0.0001; Tg-B1 vs. WT, p=0.0072; Tg-B1 vs. AMPK+/−, p<0.0001), so does the wild type to AMPK+/− group (p=0.008). Experiments were performed in triplicate, and p-values were determined by one-way ANOVA followed by Bonferroni post-test. n=3 per group. (C) Western blot results show changes in autophagy-related proteins in the cochleae of aging mice. There is a remarkable decline of mTOR signaling (Tg-B1 vs. AMPK+/−/Tg-B1, p<0.0001; Tg-B1 vs. WT, p=0.0001) and more Beclin-1 (Tg-B1 vs. AMPK+/−/Tg-B1, p<0.0001, one-way ANOVA followed by Bonferroni post-test) expressed in the cochleae of Tg-B1 mice. (D) The histograms of western blot analyses show knockouts of AMPK relieve the ROS-induced autophagic stress in Tg-B1 mice. Analysis performed by using Image J software and one-way ANOVA followed by Bonferroni post-test. * P<0.05, ** P<0.01, ***P<0.001, **** P<0.0001; n=3 per group.