Sacubitril/Valsartan Reduces Fibrosis and Alleviates High-Salt Diet-Induced HFpEF in Rats

FIGURE 1. The animal model of HFpEF was successfully constructed after feeding the rats with high-salt feed (8% NaCl) until 19 weeks. (A) Body weight was measured to evaluate general condition of rats (n = 10 in each group). (E) Echocardiographic analysis was performed to assess the cardiac function of rats in different weeks. (B–D, F–K) Different parameters such as HR, LVIDd, EF%, FS%, SBP, IVSd, LVPWd, LA, and LV mass corrected were obtained via many measurements (echocardiography and detection of blood pressure). (L–O) The ratios of E/A and E/E′ were collected through color Doppler ultrasound. *p < 0.05 means statistic significance; NS means no statistic significance. All these data represent the mean ± SD of at least three times of the experiment. HR, heart rate; LVIDd, left ventricle internal dimensions at the end of diastole; EF, left ventricular ejection fraction; FS, left ventricular fractional shortening; SBP, systolic blood pressure; IVSd, interventricular septum thickness at the end of diastole; LVPWd, left ventricular posterior wall thickness at the end of diastole; LA, left atrial internal dimensions; E, maximum peak blood flow velocity at early phase of diastole; A, maximum…

FIGURE 2. Treatment of Sac/Val in rats accompanied with HFpEF shows an obvious improvement of cardiac function of the rats in 23 weeks. (I, K) Measurements of cardiac function of rats were conducted by M-mode ultrasound and color Doppler ultrasound of the echocardiography (n = 10 in each group). (Q, R) ELISA analysis of BNP and NT-ProBNP levels in serum of rats. (A–H, J, L, M–P, S, T, V) The parameters (body weight, HR, SBP, LV/BW, (Wet lung-Dry lung)/BW, LA/BW, IVSd, LVPWd, LA, LV mass corrected, EF%, FS%, E/A, E/E′, Tau, −dp/dtmax, and LVEDP) were collected to evaluate the treatment of Sac/Val in rats of HFpEF. (U) H&E and Masson’s trichrome staining of heart tissues. (W) Assessment of cardiac fibrosis by Masson’s trichrome staining. Quantification of fibrotic areas: *p < 0.05 means statistic significance; NS means no statistic significance. All these data represent the mean ± SD of at least three times of the experiment. Tau, left ventricular relaxation time constant; −dp/dtmax, maximum left ventricular pressure decreasing rate; LVEDP, left ventricular end-diastolic pressure.

FIGURE 3. Treatment of Sac/Val in rats accompanied with HFpEF significantly alleviated the vascular injury of HFpEF induced by high-salt diet. (A) The primary procedures of evaluating the vascular endothelium diastolic function (n = 10 in each group). (B, C) Acetylcholine (Ach) is used to measure endothelium-dependent vasodilation and sodium nitroprusside (SNP) to measure endothelium-independent vasodilation. (D) The levels of NO in serum of rats were detected to evaluate the vascular injury. (E) H&E and Masson’s trichrome staining were used to assess the pathological changes of blood vessel. (G) Assessment of vascular fibrosis by Masson’s trichrome staining; quantification of fibrotic areas. (F, H, I) Immunohistochemical staining of Collagen 1 and Collagen 3 in blood vessel. Quantification of Collagen 1 and Collagen 3 areas: *p < 0.05 means statistic significance; NS means no statistic significance. All these data represent the mean ± SD of at least three times of the experiment.

FIGURE 4. Treatment of Sac/Val can inhibit cardiac fibrosis via TGF-β1/Smad3 signaling pathway potentially. (A, L) Western blot analysis of protein expression levels in rats heart tissues (n = 10 in each group). The proteins include Collagen 1, Collagen 3, MMP2, TIMP2, Smad 7, TGF-β1, and Smad3. (B–E, G) Statistic analysis of protein expression levels of Collagen 1, Collagen 3, MMP2, TIMP2, and Smad 7. (F) The ratio of MMP2/TIMP2 was calculated. (H–J) qRT-PCR analysis of Collagen 1, Collagen 3, and Smad 7 mRNA expression levels in heart tissues. (K) Immunohistochemical staining of Collagen 1 and Collagen 3 in heart tissues. (M, N) Statistic analysis of protein expression levels of TGF-β1 and Smad3. (O) qRT-PCR analysis of TGF-β1 mRNA expression level in heart tissues. *p < 0.05 means statistic significance; NS means no statistic significance. All these data represent the mean ± SD of at least three times of the experiment.