Protection of Sacubitril/Valsartan against Pathological Cardiac Remodeling by Inhibiting the NLRP3 Inflammasome after Relief of Pressure Overload in Mice

Figures

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  Fig. 1. Sacubitril/valsartan preserved cardiac function in a mouse model of pressure unloading. (a) Schedule of the 12-wk experiment. The mice underwent DB surgery after 8 wk of AB through tightening and loosening of ligatures around the aorta. Subsequently, all mice were given Sac/Val (60 mg/kg) or equivalent doses of PBS each day by oral gavage for another 4 wk. (b) EF, FS (d), LVESD (e), and LVEDD (f) of the mice from the sham, AB, DB + PBS, and DB + Sac/Val groups were measured and calculated at 12 wk (n = 8 per group). (c) Representative M-mode images of mouse hearts from the four groups are shown. Values are expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; NS, no significance

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  Fig. 2. Sacubitril/valsartan attenuated cardiac hypertrophy after pressure unloading. (a) Heart weight/body weight (HW/BW) ratios and (b) LV mass of the mice from the sham, AB, DB + PBS, and DB + Sac/Val groups were measured and analyzed at 12 wk (n = 8 per group). (c) Representative images of WGA staining (green) and DAPI (blue) and quantification of myocyte size (e) are shown for mouse hearts from the four groups (n = 3 per group). Scale bar =20 μm. (d) Representative images of TUNEL staining (red) and calculation (f) of the apoptotic index (the ratio of TUNEL-positive nuclei to the total number of cells) are shown for cardiac sections from the four groups at 12 wk (n = 3 per group). Scale bar =20 μm. Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001; NS, no significance

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  Fig. 3. Sacubitril/valsartan ameliorated cardiac fibrosis after pressure unloading. (a) Representative images of Masson’s staining, immunohistochemical staining for collagen type I (Col I) (b) and immunofluorescence staining (c) for α-SMA (green) and DAPI (blue) in mouse hearts from the sham, AB, DB + PBS, and DB + Sac/Val groups are shown. Scale bar =20 μm. (d) Quantification of the percentage of collagen volume fraction, Col I area (e) and α-SMA-positive cells (f) were analyzed and shown (n = 3 per group). (g-h) Western blotting showed the protein expression levels of Col I, MMP-2, and TGF-β and quantified and normalized them to the levels of β-actin (n = 3 per group). Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 4. Sacubitril/valsartan impaired the cardiac inflammatory process after pressure unloading. (a) Representative images of hematoxylin and eosin (H&E) staining and immunofluorescence staining (b) for CD45 (red) and DAPI (blue) in mouse hearts from the sham, AB, DB + PBS, and DB + Sac/Val groups are shown. Scale bar =20 μm. (c) Quantification of the percentage of inflammatory infiltration and CD45-positive cells (d) were analyzed and presented for mouse cardiac sections among the different groups. (e) qRT-PCR analysis of TNF-α and IL-1β mRNA expression levels in heart tissues is shown. (n = 3 per group). Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

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  Fig. 5. Sacubitril/valsartan inhibited NLRP3 inflammasome activation after pressure unloading. (a) Western blotting shows the protein expression levels of NLRP3, ASC, pro-IL-1β, IL-1β, caspase-1, and cleaved caspase-1 from the sham, AB, DB + PBS, and DB + Sac/Val mouse hearts. (b) The protein levels of NLRP3, ASC, and cleaved/caspase-1 and the ratio of IL-1β to pro-IL-1β were quantified and normalized to the levels of β-actin. (c) Immunofluorescence images for NLRP3 (red) and (d) NF-κB (red) as well as DAPI (blue) in mouse hearts from the 4 groups are shown. Scale bar =20 μm. (e, f) Quantification of the percentage of NLRP3- and NF-κB-positive cells was analyzed and presented in mouse cardiac sections among different groups. (g, h) NF-κB and p-NF-κB levels in heart tissues are shown by Western blotting, and the ratio of p-NF-κB to NF-κB is quantified and normalized to the levels of β-actin. (n = 3 per group). Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001