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. 2020 Apr 2;12(7):5590–5611. doi: 10.18632/aging.102977

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Copyright © 2020 Zhao et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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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.