Glucose Fluctuations Aggravate Myocardial Fibrosis via the Nuclear Factor-κB-Mediated Nucleotide-Binding Oligomerization Domain-Like Receptor Protein 3 Inflammasome Activation

Figures

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  FIGURE 1. Glucose fluctuations promoted rat left ventricular systolic dysfunction. (A) Levels of blood glucose in the CBG, UBG, and FBG groups (n = 8 per group). (B) Body weight in the three groups (n = 8 per group). (C,D) Representative echocardiographic images of rats and heart weight/body weight (HW/BW) in the three groups (n = 8 per group). (E–H) Representative echocardiographic images of rats, left ventricular ejection fraction (% EF), left ventricular fractional shortening (% FS), left ventricular internal diameter at end-diastole (LVIDd), and left ventricular internal diameter at end-systole (LVIDs) examined by echocardiography (n = 8 per group). (I) IL-1β level in the three groups (n = 6 per group). N.S.: No significance.

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  FIGURE 2. Glucose fluctuations promoted myocardial fibrosis. (A–C) Representative images of hematoxylin and eosin (HE) staining for myocardial tissue in the CBG, UBG, and FBG groups (n = 3 per group). Representative images of myocardial fibrosis and fibrosis area (%) in the CBG, UBG, and FBG groups (n = 3 per group). Representative images of immunohistochemistry staining of α-SMA and positive area (%) in the three groups (n = 3 per group). (D,E) The mRNA levels of TGF-β1 and collagen I in rat hearts of the three groups (n = 4 per group). (F–H) The protein expressions of TGF-β1 and collagen I in rat hearts of the three groups (n = 4 per group). (I,J) The protein expressions of TGF-β1 in NRCFs of the NG, HG and GF groups (n = 4 per group). (K) Immunofluorescence staining of intracellular TGF-β1 in the NG, HG, and GF groups (n = 3 per group).

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  FIGURE 3. Glucose fluctuations promoted the activation of NLRP3 inflammasome. (A) Immunofluorescence staining of NLRP3-ASC interaction in rat hearts. The heart sections were labeled with anti-NLRP3 (red), anti-ASC (green), and DAPI (blue) (n = 3 per group). (B–D) The mRNA levels of NLRP3, IL-1β and IL-18 in rat hearts of the three groups (n = 4 per group). (E–H) The protein expressions of NLRP3, ASC and cleaved caspase-1 in rat hearts of the three groups (n = 4 per group).

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  FIGURE 4. Role of inflammasome in glucose fluctuation-induced myocardial fibrosis. (A) Immunofluorescence staining of intracellular NLRP3–ASC interaction examined by confocal microscopy. The NRCFs were labeled with anti-NLRP3 (red), anti-ASC (green), and DAPI (blue) (n = 3 per group). (B–G) After siRNA targeting NLRP3 transfection, the protein expressions of NLRP3, cleaved caspase-1 and collagen I were measured by western blot (n = 3 per group). (H) Representative images of immunofluorescence staining of intracellular α-SMA in the NG, HG, GF, and GF + siRNA groups (n = 3 per group).

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  FIGURE 5. Role of NF-κB in glucose fluctuation-induced inflammasome-related myocardial fibrosis. (A) Immunofluorescence staining of NF-κB/p65 nucleation (n = 3 per group). (B,C) The phosphorylation level of NF-κB/p65 in rat hearts of the three groups (n = 4 per group). (D) Immunofluorescence staining of intracellular NLRP3–ASC interaction in the GF and GF + TPCA-1 groups. The NRCFs were labeled with anti-NLRP3 (red), anti-ASC (green), and DAPI (blue) (n = 3 per group). (E–G) After application of TPCA-1 (0.5 μM), the protein expressions of cleaved caspase-1 and collagen I were measured by western blot (n = 3 per group).

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  FIGURE 6. NLRP3 inhibition with MCC950 reversed rat left ventricular systolic dysfunction induced by glucose fluctuations. (A,B) Levels of blood glucose in the FBG + NaCl and FBG + MCC950 groups (n = 6 per group). (C,D) Body weights in the FBG + NaCl and FBG + MCC950 groups (n = 6 per group). (E–G) Body weights, heart weights and heart weight/body weight ratios of rats in the FBG + NaCl and FBG + MCC950 groups (n = 6 per group). (H–L) Representative echocardiographic images of rats, LVEF (%), LVFS (%), LVIDd and LVIDs in the FBG + NaCl and FBG + MCC950 groups (n = 6 per group). (M,N) Relative mRNA levels of IL-1β and IL-18 in the FBG + NaCl and FBG + MCC950 groups (n = 6 per group). N.S.: No significance.

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  FIGURE 7. NLRP3 inhibition with MCC950 reversed myocardial fibrosis induced by glucose fluctuations. (A) Representative images of hematoxylin and eosin (HE) staining for myocardial tissue in the FBG + NaCl and FBG + MCC950 groups (n = 3 per group). (B–C) Representative images of myocardial fibrosis and fibrosis area (%) in the FBG + NaCl and FBG + MCC950 groups (n = 4 per group). (D–K) The protein expressions of NLRP3, cleaved caspase-1, TGF-β1, collagen I and collagen III in the FBG + NaCl and FBG + MCC950 groups (n = 6 per group).