NLRP3 inflammasome pathway activation is associated with cardiac remodeling, gut microbiota dysbiosis, and dietary protein restriction in a rat heart failure model: an integrative exploratory analysis

Figures

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  FIGURE 1. PCR analysis of NLRP3 inflammasome pathway genes. Bar graphs showing Ct values of TXNIP, NLRP3, and IL-18 in the upper row and the corresponding normalized ΔCt values relative to GAPDH in the lower row. Data presented as mean ± SD (n = 3 per group). Groups: Control, HF (heart failure + normal diet), HF + Pro (heart failure + normal diet + probiotics), LHF (heart failure + low-protein diet), LHF + Pro (heart failure + low-protein diet + probiotics). Lower Ct or ΔCt values indicate higher gene expression. Statistical analysis by one-way ANOVA with P values indicated. Error bars represent standard deviation.

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  FIGURE 2. NLRP3 inflammasome gene expression profiling across experimental groups (exploratory, n = 3 per group). (A–F) mRNA relative expression (ΔCt normalized to GAPDH) of NLRP3, TXNIP, ASC, IL-1β, IL-18, and Caspase-1, respectively. No statistically significant differences were detected across groups by one-way ANOVA (NLRP3: P = 0.371; TXNIP: P = 0.424; IL-18: P = 0.929). Directional trends are shown for descriptive purposes only. (G) Pearson correlation matrix of gene expression values (presented as descriptive visualization; interpret with caution given small sample size). (H) Principal component analysis (PCA) of gene expression profiles shown as a descriptive tool; each dot represents one animal, colored by group. Data shown as mean ± SD (n = 3/group).

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  FIGURE 3. NLRP3 inflammasome pathway protein quantification and gene-protein concordance analysis. Densitometric quantification of (A) NLRP3 (118 kDa), (B) TXNIP (53 kDa), (C) ASC (22 kDa), (D) IL-1β (35 kDa), and (E) IL-18 (22 kDa) protein expression normalized to GAPDH (37 kDa). (F) Representative Western blot band composite image for all five targets and GAPDH loading control across the five experimental groups. (G) Pearson correlation scatter plots of mRNA vs. protein expression for each inflammasome component (n = 15–30 data points per plot; regression line shown with 95% CI). (H) Integrated gene-protein expression heatmap with hierarchical clustering of all 30 animals; rows = gene/protein analytes, columns = individual animals grouped by treatment. Data in A–E shown as mean ± SD (n = 4–6/group). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control; #P < 0.05, ##P < 0.01 vs. LHF.

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  FIGURE 4. Gut microbiota 16S rRNA gene sequencing analysis and microbiome-inflammasome gene correlations. (A) Rarefaction curves for all samples showing adequate sequencing depth. (B) Alpha diversity: Shannon index across groups. (C) Alpha diversity: Chao1 species richness index. (D) Beta diversity: PCoA plot based on Bray-Curtis dissimilarity; each symbol represents one animal; ellipses show 95% confidence regions per group. (E) Firmicutes/Bacteroidetes ratio across groups as a marker of dysbiosis. (F) LEfSe linear discriminant analysis (LDA) score bar plot showing group-discriminating bacterial taxa (LDA >2.0, P < 0.05; positive = enriched in labeled group). (G) Spearman correlation heatmap between relative abundance of top 15 bacterial genera and myocardial NLRP3 inflammasome gene expression levels (NLRP3, TXNIP, ASC, IL-1β, IL-18, Caspase-1); color scale: red = positive, blue = negative; *P < 0.05, **P < 0.01, ***P < 0.001. (H) Scatter plots of serum TMAO levels vs. Firmicutes/Bacteroidetes ratio and vs. Akkermansia relative abundance with Pearson regression lines and 95% CI. All data mean ± SD (n = 4–6/group). *P < 0.05, ***P < 0.001 vs. Control.

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  FIGURE 5. Integrated correlation analysis and NLRP3 pathway protein network in heart failure. (A) Scatter plot of NLRP3 mRNA expression vs. left ventricular ejection fraction (LVEF, %) with Pearson regression line (exploratory; n = 3 per group for PCR). (B) TXNIP gene expression vs. myocardial collagen volume fraction (CVF, %). (C) IL-18 mRNA relative expression vs. serum IL-18 protein concentration (pg/mL). (D) Gut dysbiosis index (Shannon diversity reduction from control mean) vs. myocardial NLRP3 protein expression. (E) Serum TMAO levels vs. five inflammasome protein levels shown as a paired bar-and-scatter correlation panel. (F) Serum BNP vs. NLRP3 protein expression and vs. composite inflammasome protein score. (G) Multi-variable correlation heatmap: five inflammasome protein expressions (rows) vs. LVEF, LVFS, LVEDD, LVESD, CVF, BNP, TMAO, and albumin (columns); Pearson r values shown in cells; color scale from −1 (blue) to +1 (red). (H) Hypothesis-generating pathway diagram (not directly tested): low-protein diet → ROS/TXNIP axis → NLRP3 assembly → IL-1β/IL-18 maturation → cardiac remodeling, interconnected with gut dysbiosis → TMAO/LPS → NF-κB-driven NLRP3 priming; probiotic…

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  FIGURE 6. Serum biochemical parameters across experimental groups. Bar graphs showing serum albumin, TMAO, BNP, IL-18, IL-1β, and TNF-α levels across the five experimental groups. Data as mean ± SD. Groups: Control (n = 6), HF (n = 5), HF + Pro (n = 5), LHF (n = 4), LHF + Pro (n = 6). Key findings: decreased albumin in LHF, paradoxical TMAO elevation in LHF, elevated BNP in HF groups, and markedly increased IL-18 in LHF with probiotic protection.

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  FIGURE 7. Gut microbiota composition analysis across experimental groups. (A) Heatmap of bacterial genera abundance. Color intensity indicates relative abundance (red = high, blue = low). Groups: A (LHF), B (LHF + Pro), C (HF + Pro), D (HF), E (Control). n = 4-6 per group. (B) LEfSe cladogram showing taxonomic differences. Colored nodes indicate bacteria significantly enriched in each group (LDA >2.0, P < 0.05). Red (A), green (B), purple (C), orange (D), yellow (E). Taxonomic levels from center to periphery: kingdom to species. The analysis reveals group-specific bacterial signatures and the impact of dietary intervention and probiotics on gut microbiota in heart failure rats.

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  FIGURE 8. Western blot analysis of NLRP3 inflammasome pathway proteins in myocardial tissue. Representative Western blot images showing the expression of NLRP3 (118 kDa), IL-1β (35 kDa), ASC (22 kDa), IL-18 (22 kDa), and TXNIP (53 kDa) proteins across five experimental groups. GAPDH (37 kDa) was used as an internal loading control. Lanes from left to right: Control, LHF (heart failure + low-protein diet), LHF + Pro (heart failure + low-protein diet + probiotics), HF + Pro (heart failure + normal diet + probiotics), and HF (heart failure + normal diet). Band intensity reflects relative protein expression levels, with darker bands indicating higher expression.

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  FIGURE 9. Western blot protein expression analysis of NLRP3 inflammasome pathway proteins. Bar graphs showing the relative protein expression levels of NLRP3, IL-1β , ASC, IL-18, and TXNIP across the experimental groups. Protein expression was normalized to GAPDH. Groups: Control, HF + LP (LHF), HF + LP + Pro ( LHF + Pro), HF + Pro, and HF-NC (HF). Dashed red lines indicate the control baseline.

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  FIGURE 10. Transmission electron microscopy of myocardial ultrastructure across experimental groups. Representative transmission electron microscopy images showing myocardial ultrastructural changes in different groups. (A) Group (LHF): Heart failure with low-protein diet showing severe myofibril disruption and mitochondrial damage. (B) Group (LHF + Pro): Heart failure with low-protein diet plus probiotics showing relatively preserved myofibril structure. (C) Group (HF + Pro): Heart failure with normal diet plus probiotics showing mild mitochondrial swelling. (D) Group (HF): Heart failure with normal diet showing moderate mitochondrial swelling and aggregation. (E) Group (Control): Control group displaying normal myocardial ultrastructure with well-organized sarcomeres and intact mitochondria. Red arrows indicate representative pathological features. Scale bar = 2 μm. Magnification: ×8,000.

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  FIGURE 11. Histopathological analysis of myocardial tissue across experimental groups. Representative histological images of myocardial tissue stained with three different methods (×400 magnification). HE staining (upper panel): Shows myocardial cell morphology and inflammatory cell infiltration. Masson trichrome staining (middle panel): Blue areas indicate collagen fiber deposition, reflecting the degree of myocardial fibrosis. Sirius red staining (lower panel): Red areas represent collagen fibers, providing enhanced visualization of myocardial fibrosis. A4 (LHF): Heart failure with low-protein diet showing the most severe myocardial damage and extensive fibrosis. B1 ( LHF + Pro): Heart failure with low-protein diet plus probiotics showing reduced fibrosis. C6 (HF + Pro): Heart failure with normal diet plus probiotics showing moderate fibrosis. D5 (HF): Heart failure with normal diet showing moderate to severe fibrosis. E6 (Control): Control group displaying normal myocardial structure with minimal collagen deposition. Scale bar = 50 μm.