Salvianolate injection ameliorates cardiomyopathy by regulating autophagic flux through miR-30a/becn1 axis in zebrafish

Figures

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  Figure 1. Salvianolate alleviated Dox/Azi-induced cardiotoxicity. (A–C) The zebrafish embryos treated with Dox or Dox + Salvianolate. (A) The representative image of zebrafish embryos.The red line depicts the pericardial edema of zebrafish (scale bar, 500 μm). (B) Proportions of heart edema among groups in (A) at 3 dpf. (C) Heart rate among groups in (A) at 3 dpf. (D–F) The zebrafish embryos treated with Azi or Azi + salvianolate. (D) The representative image of zebrafish embryos.The red line depicts the pericardial edema of zebrafish (scale bar, 500 μm). (E) Proportions of heart edema among groups in (D) at 3 dpf. (F) Heart rate among groups in (D) at 3 dpf. (G, H) The representative image and statistic diagram of fluorescence staining of TUNEL and EGFP (scale bar, 50 μm) of salvianolate treatment of zebrafish embryos with pre-administration of Azi. (G) Representative image and (H) statistic diagram. All data are showed as mean ± SEM, †P <0.01, ‡P <0.001. A: Atrium; Azi: Azithromycin; dpf: Day post fertilization; DMSO: Dimethyl sulfoxide; Dox: Doxorubicin; EGFP: Enhanced green fluorescent protein; SEM: Standard error of the mean; TUNEL: TdT-mediated dUTP nick end labeling; V: Ventricle.

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  Figure 2. Salvianolate regulates the autophagy–lysosome pathway in zebrafish. (A,B) The RNA-seq analysis of salvianolate-treated zebrafish embryos. (A) GO and (B) KEGG pathway analysis. (C,D) The representative images and statistic diagrams of autophagy–lysosome analysis by myl7:EGFP-LC3 system of zebrafish embryos treated with salvianolate. (C) Representative images of zebrafish heart of Tg(myl7:EGFP-LC3) line. The first line shows the Z-stack maximum projections (scale bar: 50 µm), and the second line shows the single slice (scale bar: 10 µm). Arrowhead represents the autophagosome. (D) Quantification of the density of autophagosomes in zebrafish cardiomyocytes treated with DMSO or salvianolate. (E,F) The representative image and statistic diagram of autophagic flux analysis by myl7:mRFP-EGFP-LC3 system of zebrafish embryos treated with salvianolate. (E) Representative images of zebrafish heart of Tg(myl7:mRFP-EGFP-LC3) line. The first line shows the Z-stack maximum projections (scale bar: 50 µm), and the second line shows the single slice with an EGFP signal, the third line shows the single slice with an mRFP signal, and the last line shows the single slice with merged EGFP and mRFP…

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  Figure 3. Autophagy played an important role in the cardioprotective effect of salvianolate. (A,B) Salvianolate followed by 3-methyladenine treatment of zebrafish embryos pre-administration of Dox. The heart edema rate at 3 dpf (A) and heart rate at 3 dpf per min (B). (C) Representative images of the single slice (scale bar: 10 µm) of zebrafish heart of Tg(myl7:EGFP-LC3) line. Arrowhead represents the autophagosome. Quantification of the density of autophagosomes in (C). (D) Representative images of zebrafish heart of Tg(myl7:mRFP-EGFP-LC3) line. The first column shows the single slice with an EGFP signal, the second column shows the single slice with an mRFP signal, and the last column shows the single slice with merged EGFP and mRFP signals (scale bar:10 µm). Quantification of the density of autophagosomes and autolysosomes in (D). All data are expressed as mean ± SEM, †P <0.01, ‡P <0.001. 3MA: 3-methyladenine; Azi: Azithromycin; Dox: Doxorubicin; dpf: Days post fertilization; EGFP: Efficiency green fluorescent protein; mRFP: Monomeric red fluorescent protein; ns: Not significant; SEM: Standard error of mean.

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  Figure 4. Salvianolate reduced cardiotoxicity by regulating cardiomyocyte autophagy through becn1. (A) The real-time PCR analysis of the becn1 level in Dox-treated zebrafish embryos followed by salvianolate or DMSO. (B) The Western blot analysis of becn1 level in Dox-treated zebrafish embryos followed by salvianolate or DMSO. (C) The structure of the constructed plasmid which co-expresses myl7-EGFP and becn1. (D,E) Treat Dox or DMSO to zebrafish embryos pre-injected becn1 or BFP plasmid. Heart edema rate at 3 dpf (D) and heart rate at 3 dpf per min (E). (F) Quantification of the density of autophagosomes in the myl7:EGFP-LC3 system. (G) Quantification of the density of autophagosomes and autolysosomes in the myl7:mRFP-EGFP-LC3 system. All data are expressed as mean ± SEM, *P <0.05, †P <0.01, ‡P <0.001. BFP: Blue fluorescent protein; DMSO: Dimethyl sulfoxide; Dox: Doxorubicin; dpf: Days post fertilization; EGFP: Efficiency green fluorescent protein; mRFP: Monomeric red fluorescent protein; ns: Not significant; PCR: Polymerase chain reaction; SEM: Standard error of mean.

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  Figure 5. The cardioprotective effects of salvianolate in the Dox/Azi-induced cardiotoxicity model was eliminated by becn1 knockdown. (A) The Western blot analysis of becn1 level in becn1-knockdown (becn1 MO) or control (Control MO) treated zebrafish embryos. (B,C) Becn1-knockdown (becn1 MO) or control (Control MO) treated Dox-induced cardiotoxicity zebrafish model pre-administrated with salvianolate or not. Heart edema rate at 3 dpf (B) and heart rate at 3 dpf per min (C). (D,E) Becn1-knockdown (becn1 MO) or control (Control MO) treated Azi-induced cardiotoxicity zebrafish model pre-administrated with salvianolate or not. Quantification of the density of autophagosomes in the myl7:EGFP-LC3 system (D). Quantification of the density of autophagosomes and autolysosomes in the myl7:mRFP-EGFP-LC3 system (E). All data are expressed as mean ± SEM, *P <0.05, †P <0.01, ‡P <0.001. EGFP: Efficiency green fluorescent protein; dpf: Days post fertilization; mRFP: Monomeric red fluorescent protein; MO: Morpholinos; ns: Not significant; SEM: Standard error of mean.

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  Figure 6. Salvianolate mediates cardiomyocyte autophagy through miR-30a regulation of becn1 and reduced Dox/Azi-induced cardiotoxicity. (A) The real-time PCR analysis of miR-30a level in Dox treated Dox-induced cardiotoxicity zebrafish model. (B) Luciferase reporter gene assay in HEK 293T cells showed that miR-30a directly binds to the becn1 3′UTR. (C–F) miR-30a mimic or NC mimic treated Dox-induced cardiotoxicity zebrafish model pre-administrated with salvianolate or not. Heart edema rate at 3 dpf (C) heart rate at 3 dpf per min (D). Quantification of the density of autophagosomes in the myl7:EGFP-LC3 system (E). Quantification of the density of autophagosomes and autolysosomes in the myl7:mRFP-EGFP-LC3 system (F). All data are expressed as mean ± SEM, *P <0.05, †P <0.01, ‡P <0.001. DMSO: Dimethyl sulfoxide; dpf: Days post fertilization; Dox: Doxorubicin; EGFP: Efficiency green fluorescent protein; mRFP: Monomeric red fluorescent protein; NC: Normal control; PCR: Polymerase chain reaction; SEM: Standard error of mean; UTR: Untranslated region.