Functional components of Chinese rice wine can ameliorate diabetic cardiomyopathy through the modulation of autophagy, apoptosis, gut microbiota, and metabolites

Figures

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  Figure 1. RWPH and RWPE ameliorate cardiac function and alleviate cardiac fibrosis and cardiomyocyte hypertrophy in DCM mice. (A) M-mode echocardiography and corresponding images of each group mice hearts. (B–E) Results of (B) left ventricular ejection fraction (LVEF), (C) left ventricular fractional shortening (LVFS), (D) left ventricular end-diastolic volume (LVEDV) and (E) left ventricular end-systolic volume (LVESV) in each group mice. (F–H), Results of (F) serum creatine kinase-MB (CK-MB), (G) serum lactate dehydrogenase (LDH) and (H) serum low-density lipoprotein cholesterol (LDL-C) in each group mice. (I) Hematoxylin and eosin (H&E) staining of the left ventricular tissue section in each group mice. (J) Masson trichrome stain of the left ventricular tissue section in each group mice. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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  Figure 2. RWPH and RWPE rescue impaired autophagy in high-glucose cultured H9C2 cells. (A–D) Western blot analysis of the relative protein expression of ATG5, P62, and LC3B in H9C2 cells. (E–G) stubRFP-sensGFP-LC3 fluorescence was used to determine the autophagic activity in HG group and treatment groups. GFP fluorescence represents autophagosomes, RFP fluorescence represents autophagosomes and autolysosomes, the two merged to form yellow dots represent autophagosomes, and the remaining red dots represent autolysosomes. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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  Figure 3. RWPH and RWPE clear dysfunctional mitochondria through enhanced autophagy in DCM mice. (A–D) Western blot analysis of the relative protein expression of ATG5, P62, and LC3B in myocardial tissue. (E) Transmission electron microscopy of left ventricular specimens showing mitochondria with different pathological forms. (F) the autophagosomes of each group under transmission electron microscopy, which are pointed out by red arrows. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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  Figure 4. RWPH and RWPE relieved apoptosis in DCM mice and high-glucose cultured H9C2 cells. (A–E) Western blot analysis of the relative protein expression of P53, Bcl-2, and Bax in H9C2 cells. (F–J) Western blot analysis of the relative protein expression of P53, Bcl-2, and Bax in myocardial tissue. (K,L) TUNEL assay was used to determine the apoptotic rate in myocardial tissue of different groups. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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  Figure 5. Autophagy inhibitor was able to counteract the anti-apoptotic effects of RWPH and RWPE. (A–D) Western blot analysis of the relative protein expression of ATG5, P62, and LC3B in each group. (E–I) Western blot analysis of the relative protein expression of Bcl-2, P53 and Bax in each group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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  Figure 6. RWPH and RWPE prevented gut microbiota dysbiosis in DCM mice (n=4 per group). (A,B) The Chao1 and Shannon diversity index of intestinal bacteria was examined by 16S-rRNA high-throughput sequencing. (C) The β-diversity of intestinal bacteria was compared by the weighted unifrac principal coordinates analysis (PCoA). (D) At the phylum level, the Bray-Curtis distance clustering tree and the relative abundance of the gut bacterial in each group. (E) At the genus level, the Bray-Curits distance clustering tree and the relative abundance of the gut bacterial in each group.

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  Figure 7. Linear discriminant analysis effect size (LEfSe), linear discriminant analysis (LDA) and abundance comparison of representative gut bacterial taxa. (A) Comparison of taxonomic abundances using LEfSe. The circles represent the taxonomic levels from phyla to species. The dots, which size is proportional to its taxonomic abundance, located on individual circles represent different classification levels of bacteria. (B) linear discriminant analysis (LDA) represented significant difference in abundance of gut bacteria in the DCM/CON or DCM/DCM + treatment groups. (C–F) abundances of representative significantly changed gut taxa among groups. *P < 0.05; **P < 0.01.

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  Figure 8. RWPH and RWPE modulated the gut microbiota metabolites in DCM mice. (A) Human Metabolome Database (HMDB) super class identification classification and annotation diagram of first-level metabolites. (B) Principal component analysis (PCA) analysis of metabolites for 5 groups.

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  Figure 9. Heat map analysis of significantly different metabolites between groups. (A–D) Comparison of significantly altered serum metabolites between the DCM/CON or DCM/DCM + treatment groups by heat map analysis.

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  Figure 10. Indensity comparison of representative significantly different metabolites between groups. (A–G) Indensity comparison of significantly changed metabolites among groups. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

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  Figure 11. The correlation analysis of gut microbiota, metabolites, cardiac function and myocardial injury parameters. (A) Spearman correlation between altered phylum bacterial taxa and altered metabolites. (B) Spearman correlation between altered genus bacterial taxa and altered metabolites. (C) Spearman correlation between altered bacterial taxa at the phylum level and echocardiographic and myocardial injury parameters. (D) Spearman correlation between altered bacterial taxa at the genus level and echocardiographic and myocardial injury parameters. (E) Spearman correlation between altered metabolites and echocardiographic and myocardial injury parameters. *P < 0.05, **P < 0.01, and ***P < 0.001.