Cardiac energy metabolic disorder and gut microbiota imbalance: a study on the therapeutic potential of Shenfu Injection in rats with heart failure

Figures

• 

  Figure 1. Experimental flow chart (this figure was drawn using Figdraw 2.0).

• 

  Figure 2. Fingerprint of Shenfu Injection. (A–C) represent the results of three random sampling tests, respectively, (1)-negative; (2)-positive.

• 

  Figure 3. Cardiac indicators. (A) left ventricular ejection fraction, LVEF; (B) left ventricular fraction shortening, LVFS; (C) left ventricular posterior wall dimensions, LVPWd; (D) left ventricular posterior wall thickness in systole, LVPWs; (E) interventricular septum thickness at end-diastole, IVSd; (F) interventricular septum thickness at end-systole, IVSs; (G) left ventricular internal dimension in diastole, LVIDd; (H) left ventricular internal dimension in systole, LVIDs; (I) left ventricular end-diastolic volume, LVEDV; (J) left ventricular end-systolic volume, LVESV; (K) N-Terminal pro-Brain Natriuretic Peptide, NT-proBNP; (L–O) Echocardiography. (K) Normal group, (L) Model group, (M) SFI group, (N) TMZ group; (P–S) Myocardial tissue HE staining (The scale bar is 50 microns). (P) Normal group, (Q) Model group, (R) SFI group, (S) TMZ group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Normal, Model, SFI and TMZ represent the blank control group, HF control group, Shenfu Injection treatment group and positive drug control group, respectively.

• 

  Figure 4. Colonic indices. (A–D) Colon tissue HE staining. (A) Normal group, (B) Model group, (C) SFI group, (D) TMZ group. (1)-The scale bar is 200 microns; (2)-The scale bar is 50 microns; (E) Western Blot. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Normal, Model, SFI and TMZ represent the blank control group, HF control group, Shenfu Injection treatment group and positive drug control group, respectively.

• 

  Figure 5. Transmission electron microscopy and biochemical detection. (A–D) Cardiac transmission electron microscope (TEM). (A) Normal group, (B) model group, (C) SFI group, (D) TMZ group; (E) Pyruvic acid, PA; (F) Lactic acid, LA; (G) Non-Esterified Fatty Acid, NEFA; (H) Malondialdehyde, MDA; (I) Superoxide Dismutase, SOD; (J) Adenosine Triphosphate, ATP. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Normal, Model, SFI and TMZ represent the blank control group, HF control group, Shenfu Injection treatment group and positive drug control group, respectively.

• 

  Figure 6. Rarefaction curves and microbiota health indices. (A–C) Rarefaction curves, (A) Sobs index, reflecting species richness; (B) Shannon index, reflecting species diversity; (C) Heip index, reflecting species evenness; (D–F) Gut Microbiome Health Index (GMHI). A robust index based on the species-level taxonomic features of gut microbiome samples to assess health status, focusing on determining the likelihood of disease, independent of clinical diagnosis; compared to alpha indices, the GMHI significantly outperforms ecological indicators in differentiating between healthy and non-healthy populations and shows strong reproducibility in stratification of healthy versus non-healthy groups, generally considered a marker of gut health and dysbiosis. *p < 0.05. Normal, Model, SFI, and TMZ represent the blank control group (n = 4), HF control group (n = 4), Shenfu Injection treatment group (n = 4), and positive drug control group (n = 4), respectively.

• 

  Figure 7. Bioinformatics analysis. (A) intestinal microbiota dysbiosis index (MDI); (B) Venn diagram. The numbers in the overlapping parts represent the number of species common to multiple groups, while the numbers in the non-overlapping parts represent the number of species unique to the corresponding group; (C) neutral community model (NCM). The x-axis represents the log value of the mean relative abundance of species [log (Mean relative abundance)], and the y-axis represents the predicted occurrence frequency (occurrence frequency); the solid line represents the fit of the neutral model, and the dashed lines above and below represent the 95% confidence level of the model’s prediction; R2 represents the overall goodness of fit of the neutral community model, with a higher R2 indicating closer alignment with the neutral model, meaning that the assembly of the community is more influenced by random processes and less influenced by deterministic processes. N describes the size of the metacommunity, which is the total abundance of all OTUs in each sample. m quantifies the migration rate at the community level, which is uniform for each community member (species-independent). A lower m value…

• 

  Figure 8. Alpha diversity index. (A) ACE index diagram; (B) Chao index diagram; (C) Shannon index diagram; (D) Simpson index diagram. *p < 0.05; **p < 0.01. Normal, Model, SFI and TMZ represent the blank control group (n = 4), HF control group (n = 4), Shenfu Injection treatment group (n = 4) and positive drug control group (n = 4), respectively.

• 

  Figure 9. Species composition and differential analysis. (A,B) Community bar plots. These represent the bacterial composition at the phylum and genus levels, respectively. The vertical axis indicates the proportion of each species within the sample, with different colored bars representing different species. The length of the bars corresponds to the size of the proportion that each species occupies. (C–J) Community pie charts. Each pie chart represents the microbial community composition of a specific group at the selected taxonomic level. Different colors denote different species, and the area of the pie slices represents the percentage of each species. (K,L) Circos sample-species relationship diagrams. These visualizations use circular diagrams to depict the corresponding relationships between species and samples, aiding in understanding the distribution proportions of species among different groups and also showing the types of microorganisms present in each group along with their relative abundances. (M–O) LDA discriminant bar plots. These statistically represent microbial groups with significant effects across multiple groups. LDA scores are obtained through LDA analysis, with higher…

• 

  Figure 10. Targeted metabolomics analysis of SCFAs. (A) TIC (1)—standard TIC; (2)—sample TIC; (B) Cluster analysis of expression patterns. Each column in the figure represents a sample/group, each row represents a SCFA, and the color in the figure indicates the relative expression level of SCFAs within that sample/group. The left side shows a dendrogram of SCFA clustering, and the right side lists the names of SCFAs. The closer two SCFA branches are, the more similar their expression levels are. The top of the figure depicts a dendrogram of sample clustering, with different color blocks representing different samples/groups, and the bottom lists the names of the samples/groups. The closer the clustering branches of two samples/groups are, the more similar the expression patterns of all SCFAs in the two samples/groups, indicating a similar trend in the changes of SCFA expression levels. (1, 3, 5)—Heatmaps of expression pattern clustering for all samples; (2, 4, 6)—Heatmaps of expression pattern clustering for all groups; (1, 2)—Normal vs. Model, (3, 4)—Model vs. SFI, (5, 6)—Model vs. TMZ. (C) Metabolite Z-score Plot. The x-axis represents Z-score values, which are used to measure the…

• 

  Figure 11. Clinical factor correlation analysis and diagnostic model construction. (A) Heatmap of the correlation between genus-level bacteria and clinical indicators. The X-axis and Y-axis represent clinical factors and species, respectively. The correlation R values and p values are calculated. The R values are displayed in different colors in the figure, with the color range of different R values indicated in the legend on the right. The clustering trees of species and clinical factors are presented on the left and top sides, respectively. (B) Network analysis. The correlation network diagram illustrates the relationships between the top 50 species (bacteria color indicates Phylum level) in total abundance and clinical factors (correlation coefficient type: Spearman; absolute value of correlation coefficient ≥ 0.5; p < 0.05). The size of the nodes in the diagram reflects the abundance of species/clinical factor levels, with different colors representing different species/clinical factors. The color of the connecting lines indicates positive or negative correlations, with red for positive and green for negative. The thickness of the lines represents the magnitude of the correlation…

• 

  Figure 12. “Heart-gut” communication mechanism diagram (this figure was drawn using Figdraw 2.0).