Exposure to TBBPA and polystyrene nanoplastics induces cardiac fibrosis via the activation of the ESR/H19/Wnt pathway

Figures

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  Fig. 1. Characterization of PS-NPs and TBBPA, and their effects on cardiac function in mice. (A) Morphological characterizations of PS-NPs. (B–C) Diameters of PS-NPs. (D) Zeta potential of PS-NPs and PS-NPs + TBBPA. (E) Representative M-mode echocardiographic images of the left ventricle and measurements of (F) ejection fraction (EF), (G) fractional shortening (FS), (H) left ventricular systolic internal dimensions (LVIDs), (I) interventricular septum thickness in systole (IVSs), (J) left ventricular posterior wall thickness in systole (LVPWs) and (K) heart rate in mice exposed to TBBPA (1 mg/kg·bw), PS-NPs (50 mg/kg·bw) and co-exposure (1 mg/kg·bw TBBPA + 50 mg/kg·bw PS-NPs) for 2 months, administered every two days. n = 12 per group. Data are represented as the means ± SEM. Significant differences were determined using One-way ANOVA followed by Bonferroni post hoc test. “*” indicates statistically significant differences with the control (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). “#” indicates statistically significant difference between two treatment groups. (#p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001).

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  Fig. 2. Effects of TBBPA, PS-NPs and co-exposure on cardiac histopathology and serum inflammatory levels. Mice were exposed to TBBPA (1 mg/kg·bw), PS-NPs (50 mg/kg·bw) and co-exposure (1 mg/kg·bw TBBPA + 50 mg/kg·bw PS-NPs) for 2 months, administered every two days. (A) Representative micrographs of heart sections stained with H&E, Masson's trichrome, and Sirius red, showing interstitial and perivascular areas (scale bar, 50 μm). The green stars represent inflammatory cell infiltration; the green triangles represent cellular edema. (B) Quantification of myocardial fibrosis area from Masson's trichrome-stained sections. (C) Quantification of collagen deposition area from Sirius red-stained sections. n = 5 per group. (D–E) Quantitative analysis of IL-6 and TNF-α in serum. n = 9 per group. Data are represented as the means ± SEM. Significant differences were determined using One-way ANOVA followed by Bonferroni's post hoc test. “*” indicates statistically significant differences with the control (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

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  Fig. 3. Expression of cardiac fibrosis markers in mouse hearts following an exposure to TBBPA, PS-NPs and co-exposure. Mice were exposed to TBBPA (1 mg/kg·bw), PS-NPs (50 mg/kg·bw) and co-exposure (1 mg/kg·bw TBBPA + 50 mg/kg·bw PS-NPs) for 2 months, administered every two days. (A–B) Representative immunofluorescence images and quantification of α-SMA expression in heart sections (scale bar, 50 μm). (C–D) Representative immunofluorescence images and quantification of Col1a1 expression in heart sections (scale bar, 50 μm). n = 6 per group. Data are represented as the means ± SEM. Significant differences were determined using One-way ANOVA followed by Bonferroni's post hoc test. “*” indicates statistically significant differences with the control (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

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  Fig. 4. Expression of cardiac remodeling markers in H9C2 cells after exposure to TBBPA, PS-NPs, and co-exposure. H9C2 cells were exposed to TBBPA, PS-NPs, and co-exposure (5 μg/mL) for 24h. (A-B) Representative fluorescent images (scale bar, 100 μm) and quantification of α-SMA expression in H9C2 cells. (C–D, H) The mRNA expression of cardiac fibrosis biomarkers: Col1a1, Col3a1 and α-SMA. (E–F) Representative images (red: phalloidin; blue: DAPI) and relative cell surface area (scale bar, 100 μm). (G) The mRNA expression of cardiac hypertrophy biomarkers: ANF and BNP. (I) The mRNA expression of cardiac inflammation biomarkers: IL-6. n = 3 per group. Data are represented as the means ± SEM. Significant differences were determined using One-way ANOVA followed by Bonferroni's post hoc test. “*” indicates statistically significant differences with the control (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

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  Fig. 5. RNA-Seq analysis of mouse hearts exposed to TBBPA, PS-NPs and co-exposure. Mice were exposed to TBBPA (1 mg/kg·bw), PS-NPs (50 mg/kg·bw) and mixed samples (1 mg/kg·bw TBBPA + 50 mg/kg·bw PS-NPs) for 2 months, administered every two days. (A) Bar plots showing the number of DEGs in comparison of three groups. (B) Venn diagram presenting the overlapping DEGs in TBBPA vs CON, PS-NPs vs CON, co-exposure vs CON. (C) Heatmap of clustering analysis on top 50 smallest p-value genes expression pattern comparison (Complete linkage; Z-score). (D–F) Volcano plot showing the expression of DEGs in three groups. (G–I) GO enrichment analysis of DEGs in three groups. (J–L) KEGG enrichment analysis of DEGs in three groups. n = 5 per group.

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  Fig. 6. Identification of H19 as a key mediator in cardiac fibrosis induced by an exposure to TBBPA and co-exposure. Mice were exposed to TBBPA (1 mg/kg·bw), PS-NPs (50 mg/kg·bw) and mixed samples (1 mg/kg·bw TBBPA + 50 mg/kg·bw PS-NPs) for 2 months, administered every two days. (A–C) Gene-disease enrichment analysis in three groups revealing the relationship between target genes and predicted diseases in CTD. The color and size of the circles represent inference scores between predicted diseases and target genes. (D–E) The mRNA expression of H19 in TBBPA and co-exposure group. n = 3 per group. (F) Mechanism roadmap of cardiac remodeling induced by co-exposure to TBBPA and PS-NPs. (G–I) GSEA enrichment plots of Wnt/β catenin pathway in three groups. n = 5 per group. (J) Representative fluorescent images of β-catenin expression in heart sections (scale bar, 50 μm). n = 5 per group. Data are represented as the means ± SEM. Significant differences were determined using Student's t-test. “*” indicates statistically significant differences with the control (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

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  Fig. 7. ESRs mediate TBBPA- and PS-NPs-induced fibrotic responses in H9C2 cells. H9C2 cells were treated with TBBPA (5 μg/mL) and/or PS-NPs (5 μg/mL) for 24 h, with or without ICI 182,780 (0.1 μM), in the following groups: control, TBBPA, PS-NPs, TBBPA + PS-NPs, ICI, TBBPA + ICI, PS-NPs + ICI, and TBBPA + PS-NPs + ICI. (A–C) The mRNA expression of ESR1, ESR2 and H19. (D–G) The mRNA expression of four genes in Wnt/β-catenin pathway (Ctnnb1, Myc, Axin, Gsk3β). (H–I) The mRNA expression of Col1a1 and Col3a1. n = 3 per group. (J) Representative immunofluorescence images of β-catenin expression in H9C2 cells (scale bar, 100 μm). n = 5 per group. Data are represented as the means ± SEM. Significant differences were determined using One-way ANOVA followed by Bonferroni's post hoc test. “*” indicates statistically significant differences with the control (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). “#” indicates statistically significant difference between two treatment groups. (#p < 0.05, ##p < 0.01, ###p < 0.001 and ####p < 0.0001).

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  Fig. 8. H19 knockdown promotes cardiomyocyte fibrosis through Wnt/β-catenin activation. H9C2 cells were exposed to TBBPA (5 μg/mL), PS-NPs (5 μg/mL), or their co-exposure for 24 h. (A) The protein levels of β-catenin, Myc, Axin, Col1a1, and Col3a1 in the TBBPA, PS-NPs and their co-exposure groups. (B–C) Densitometric quantification of the protein levels shown in (A). H9C2 cells were transfected with shRNA-H19 or negative control (NC) for 48 h. (D–I) The mRNA expression of H19, Ctnnb1, Myc, Axin, Col1a1, and Col3a1. (J) The protein expression of β-catenin, Myc, Axin, Col1a1 and Col3a1. (K–L) Densitometric quantification of the protein levels shown in (J). Note: Ctnnb1 encodes β-catenin. Data are represented as the means ± SEM. Significant differences were determined using One-way ANOVA followed by Bonferroni's post hoc test. “*” indicates statistically significant differences with the control (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001).

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  Fig. 9. Overexpression of H19 inhibits cardiomyocyte fibrosis induced by TBBPA and PS-NPs by suppressing the Wnt/β-catenin pathway. H9C2 cells were transfected with H19 overexpression plasmid or NC for 48 h, followed by 24 h exposure to TBBPA (5 μg/mL), PS-NPs (5 μg/mL), or their co-exposure. (A–C) The mRNA levels of H19, Ctnnb1, Axin, Myc, Col1a1, and Col3a1 in the TBBPA groups (Note: Ctnnb1 encodes β-catenin). (D) The protein levels of β-catenin, Myc, Axin, Col1a1, and Col3a1 in the TBBPA groups. (E–F) Densitometric quantification of the protein levels shown in (D). (G–I) The mRNA levels of H19, Ctnnb1, Myc, Axin, Col1a1, and Col3a1 in the PS-NPs groups. (J) The protein levels of β-catenin, Myc, Axin, Col1a1, and Col3a1 in the PS-NPs groups. (K–L) Densitometric quantification of the protein levels shown in (J). (M − O) The mRNA levels of H19, Ctnnb1, Myc, Axin, Col1a1, and Col3a1 in the co-exposure groups. (P) The protein levels of β-catenin, Myc, Axin, Col1a1, and Col3a1 in the co-exposure groups. (Q–R) Densitometric quantification of the protein levels shown in (P). Data are presented as mean ± SEM. Significant differences were determined using one-way ANOVA followed by Bonferroni's…