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

Abstract

Diabetic cardiomyopathy (DCM) is highly prevalent and increases the risk of heart failure and sudden death. Therefore, proper and effective treatments for DCM are in urgent demand. Danlou tablet (Dan) is reported to confer protective effects on several heart diseases. However, to our knowledge, whether Dan provides protection against DCM is unclear. In this study, we explored the effect of Dan on DCM with the in vitro DCM model using AC16 cardiomyocytes. We found that Dan treatment significantly reduced cardiomyocyte apoptosis and oxidative stress in high-glucose (HG)-treated cardiomyocytes, as evidenced by decreased Annexin V-FITC+ cardiomyocytes, intracellular reactive oxygen species (ROS) levels, Bax/Bcl2 ratio, and cleaved-Caspase3/Caspase3 ratio. Interestingly, Dan treatment caused a decreased level of microRNA-34a (miR-34a), which could enhance cardiomyocyte apoptosis. Furthermore, miR-34a mimic blocked Dan's effect in apoptosis prevention. Finally, we observed that the miR-34a mimic effectively decreased the level of sirtuin 1 (SIRT1), while the miR-34a inhibitor increased the level of SIRT1. And downregulation of SIRT1 effectively reversed the effect of miR-34a inhibitor on cardiomyocyte apoptosis. Taken together, our study showed that Dan prevented HG-induced cardiomyocyte apoptosis through downregulating miR-34a and upregulating SIRT1. Our study has provided experimental support for the potential use of Dan in treating DCM. Further detailed study of Dan and the underlying mechanisms may shed light on the prevention and treatment of DCM.

Keywords: Cardiomyocyte apoptosis; Danlou tablet; Diabetic cardiomyopathy; SIRT1; miR-34a.

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.

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.

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.

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.

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.