Gastrodin alleviates cardiomyocyte senescence by regulating autophagy through the AMPK/mTOR/4EBP1 pathway

Figures

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  FIGURE 1. Effects of gastrodin on SA-β-gal staining and senescence-associated proteins. (A) Representative images of SA-β-gal staining (B). Quantification of SA-β-gal-positive cell percentage (C). Immunofluorescence detection of P16, P21, and P53 expression (D). Quantitative analysis of immunofluorescence intensity of P16, P21, and P53; (E). Western blot analysis of P16, P21, P53, and γ-H2AX expression levels; (F). Quantification of grayscale values of P16, P21, P53, and γ-H2AX protein bands. Compared with the Control group: ###p < 0.001; compared with the Model group: *p < 0.05, ***p < 0.001; magnification ×10, scale bar = 200 μm.

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  FIGURE 2. Effects of the autophagy inhibitor 3-MA on the protective effects of gastrodin against senescence-related indicators. (A) Apoptosis analysis by flow cytometry (B). Immunofluorescence staining for detection of P16, P21, P53, and γ-H2AX expression (C). Quantitative analysis of immunofluorescence intensity (D). Western blot analysis of P16, P21, P53, and γ-H2AX expression (E). Quantitative analysis of P16, P21, P53, and γ-H2AX protein expression levels and corresponding grayscale values. Compared with the Control group: #p < 0.05, ###p < 0.001; compared with the Model group: *p < 0.05, **p < 0.01, ***p < 0.001; compared with the Gas group: &p < 0.05, &&p < 0.01, &&&p < 0.001; ns, no statistically significant difference, p > 0.05; magnification ×10, scale bar = 200 μm.

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  FIGURE 3. Effects of the autophagy inhibitor 3-MA on gastrodin-mediated regulation of autophagy. (A) Immunofluorescence staining for detection of LC3 expression (B). Western blot analysis of P62, Beclin-1, and LC3-II/I expression (C). Quantitative analysis of P62, Beclin-1, and LC3-II/I protein expression levels and corresponding grayscale values. Compared with the Control group: ###p < 0.001; compared with the Model group: *p < 0.05, **p < 0.01, ***p < 0.001; compared with the Gas group: &p < 0.05, &&p < 0.01, &&&p < 0.001; ns, no statistically significant difference, p > 0.05; magnification ×10, scale bar = 200 μm.

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  FIGURE 4. Effects of the AMPK inhibitor CC on the anti-senescence effects of gastrodin. (A) Apoptosis analysis by flow cytometry (B) Immunofluorescence staining for detection of P16, P21, and P53 expression; (C). Quantitative analysis of immunofluorescence intensity (D) Western blot analysis of P16, P21, P53, and γ-H2AX expression (E). Quantitative analysis of P16, P21, P53, and γ-H2AX protein expression levels and corresponding grayscale values. Compared with the Control group: ###p < 0.001; compared with the Model group: **p < 0.01, ***p < 0.001; compared with the Gas group: &p < 0.05, &&&p < 0.001; ns, no statistically significant difference, p > 0.05; magnification ×10, scale bar = 200 μm.

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  FIGURE 5. Effects of the AMPK inhibitor CC on gastrodin-mediated regulation of autophagy. (A) Immunofluorescence staining for detection of LC3 expression (B). Western blot analysis of P62, Beclin-1, and LC3-II/I expression (C). Quantitative analysis of P62, Beclin-1, and LC3-II/I protein expression levels and corresponding grayscale values. Compared with the Control group: ###p < 0.001; compared with the Model group: *p < 0.05, **p < 0.01, ***p < 0.001; compared with the Gas group: &p < 0.05, &&p < 0.01, &&&p < 0.001; ns, no statistically significant difference, p > 0.05; magnification ×10, scale bar = 200 μm.

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  FIGURE 6. Effects of gastrodin on the AMPK/mTOR/4EBP1 signaling pathway. (A) Western blot analysis of the ratios of p-AMPK/AMPK, p-mTOR/mTOR, and p-4EBP1/4EBP1 (B). Quantitative analysis of grayscale values of p-AMPK/AMPK, p-mTOR/mTOR, and p-4EBP1/4EBP1 protein ratios. Compared with the Control group: ###p < 0.001; compared with the Model group: **p < 0.01, ***p < 0.001; compared with the Gas group: &p < 0.05, &&&p < 0.001; ns, no statistically significant difference, p > 0.05; magnification ×10, scale bar = 200 μm.

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  FIGURE 7. Schematic diagram of the potential mechanism by which gastrodin alleviates Dox-induced cardiomyocyte senescence. Dox treatment inhibits AMPK activity and activates the mTOR/4EBP1 signaling pathway, accompanied by abnormalities in autophagy-related markers, elevated expression of senescence markers, and increased apoptosis. Gas intervention partially restores AMPK activity and suppresses excessive phosphorylation of mTOR/4EBP1, while ameliorating abnormalities in autophagy-related markers and attenuating senescence and apoptosis phenotypes, ultimately mitigating Dox-induced cardiomyocyte injury.