Danlou tablet inhibits high-glucose-induced cardiomyocyte apoptosis via the miR-34a-SIRT1 axis

Figures

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  Fig. 1. Generation of diabetic cardiomyopathy model using AC16 cells. a Flow cytometry analysis of AC16 cells cultured with normal or high glucose (HG) medium and stained with Annexin V-FITC and PI dyes (n = 4). b Flow cytometry analysis of AC16 cells cultured with normal or HG medium and stained with DHE-FITC-A dye (n = 4). c Western blot analysis for Bax, Bcl2, cleaved-Caspase3 and Caspase3 in AC16 cells cultured with normal or HG medium (n = 3). **, p < 0.01; ***, p < 0.001. Data were represented as mean ± SD.

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  Fig. 2. Dan treatment reduced the apoptosis rate and oxidative stress of HG-treated AC16 cells. a Flow cytometry analysis of AC16 cells treated with high glucose (HG) medium and Danlou tablet (Dan), and stained with Annexin V-FITC and PI dyes (n = 6). b Flow cytometry analysis of AC16 cells treated with HG medium and Dan, and stained with DHE-FITC-A dye (n = 4). c Western blot analysis for Bax, Bcl2, cleaved-Caspase3 and Caspase3 in AC16 cells treated with HG medium and Dan (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. Data were represented as mean ± SD.

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  Fig. 3. Dan prevented cardiomyocyte apoptosis via decreasing miR-34a levels. a qPCR analysis of miR-34a level in AC16 cells treated with high glucose (HG) medium and Danlou tablet (Dan) (n = 6). b Flow cytometry analysis of AC16 cells cultured with normal glucose medium and treated with NC mimic or miR-34a mimic, and stained with Annexin V-FITC and PI dyes (n = 3). c Western blot analysis for Bax, Bcl2, cleaved-Caspase3 and Caspase3 in AC16 cells cultured with normal glucose medium and treated with NC mimic or miR-34a mimic (n = 3). d Flow cytometry analysis of AC16 cells cultured with normal glucose medium and treated with NC inhibitor or miR-34a inhibitor, and stained with Annexin V-FITC and PI dyes (n = 3). e Western blot analysis for Bax, Bcl2, cleaved-Caspase3 and Caspase3 in AC16 cells cultured with normal glucose medium and treated with NC inhibitor or miR-34a inhibitor (n = 3).*, p < 0.05; **, p < 0.01; ***, p < 0.001. Data were represented as mean ± SD.

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  Fig. 4. miR-34a mimic counteracted the anti-apoptosis effect of Dan. a Flow cytometry analysis of AC16 cells cultured with normal glucose medium and treated with miR-34a mimic and Danlou tablet (Dan), and stained with Annexin V-FITC and PI dyes (n = 3). b Flow cytometry analysis of AC16 cells cultured with high glucose (HG) medium and treated with miR-34a mimic and Dan, and stained with Annexin V-FITC and PI dyes (n = 4). c Western blot analysis for Bcl2 and Bax in AC16 cells cultured with normal glucose medium and treated with miR-34a mimic and Dan (n = 3). d Western blot analysis for Bcl2, Bax, Caspase3 and cleaved-Caspase3 in AC16 cells cultured with HG medium and treated with miR-34a mimic and Dan (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001. Data were represented as mean ± SD.

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  Fig. 5. miR-34a increased cardiomyocyte apoptosis via downregulating SIRT1. a Western blot analysis for SIRT1 in AC16 cells treated with miR-34a mimic or miR-34a inhibitor (n = 3). b Luciferase reporter assays performed in 293T cells transfected with miR-34a mimic or NC mimic and luciferase reporter plasmids containing the binding site or the mutated binding site in the 3′ UTR of SIRT1 (n = 6). c Flow cytometry analysis of AC16 cells cultured with normal glucose medium and treated with miR-34a inhibitor and shSIRT1, and stained with Annexin V-FITC and PI dyes (n = 3). d Flow cytometry analysis of AC16 cells cultured with high glucose (HG) medium and treated with miR-34a inhibitor and shSIRT1, and stained with Annexin V-FITC and PI dyes (n = 4). *, p < 0.05; **, p < 0.01; ***, p < 0.001. Data were represented as mean ± SD.