Citri Reticulatae Pericarpium protects against isoproterenol-induced chronic heart failure via activation of PPARγ

Figures

• 

  Figure 1. CRP attenuates ISO-induced cardiac dysfunction and pathological hypertrophy. (A) Left ventricular function, including ejection fraction (LVEF) and fraction shortening (LVFS) of mice 21 days after ISO and/or CRP treatment (n=8 per group) was measured by Echocardiography. (B) Representative images of HE staining of heart samples from ISO and/or CRP-treated mice. Cardiomyocyte cross-sectional area was calculated with Image J software. Scale bar, 100 µm, n=8 per group. (C) Total RNA was isolated from the ISO and/or CRP-treated mice heart samples. The mRNA expression levels of ANP and BNP were determined by qRT-PCR analysis, and GAPDH was used as an internal control, n=8 per group. (D) Representative images of immunofluorescent staining of NRVMs 48 hours after incubation with ISO and/or CRP (blue indicates nuclear stained with DAPI, green indicates cells stained with α-actinin antibody and FITC labeled secondary IgG antibody). Surface area of NRVMs was measured using Image J software. Scale bar, 50 µm, n=6 per group. (E) qRT-PCR analysis of the mRNA levels of ANP and BNP of NRVMS treated with ISO and/or CRP, and GAPDH as the internal control, n=6 per group. One-way ANOVA followed by…

• 

  Figure 2. CRP reduces ISO-induced cardiac fibrosis and apoptosis. (A) Representative images of Masson’s trichrome staining of ISO-infused mice myocardium samples with or without CRP treatment (blue indicates the scared fibrotic area red indicates the area of normal myocardium). Scale bar, 50 µm, n=8 per group. Cardiac fibrosis (the ratio of fibrotic area to total myocardial area) was measured using Image J software. (B,C) Total proteins were extracted from control saline-treated, CRP-treated, ISO-treated, and ISO plus CRP-treated mice heart samples, n=6 per group. The deposition of fibrosis-related proteins including collagen type I, collagen type III and α-SMA (B) and the expression of apoptosis-related proteins including Bax, Bcl-2, cleaved caspase-3 and caspase-3 (C) were evaluated by western blot analysis, housekeeping gene β-Tubulin was used as the loading control. The relative band density of protein signals was measured by Image J software. One-way ANOVA followed by Bonferroni’s post hoc test was used to analyze the data. Results were presented as the mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001. CRP, Citri Reticulatae Pericarpium; ISO, isoproterenol; ANOVA, one-way analysis of…

• 

  Figure 3. CRP protects against ISO-induced cardiac remodeling via activation of PPARγ. (A,B) The expression of PPARγ and PGC-1α in the extracts of control saline-treated, CRP-treated, ISO-treated, and ISO plus CRP-treated heart samples (A, n=6 per group) or NRVMs (B, n=6 per group) was determined by immunoblotting analysis, GAPDH was used as the loading control. The relative band density of PPARγ and PGC-1α to GAPDH was measured by Image J software. One-way ANOVA followed by Bonferroni’s post hoc test was used to analyze the data. Results were presented as the mean ± SD. *, P<0.05; **, P<0.01; ***, P<0.001. CRP, Citri Reticulatae Pericarpium; ISO, isoproterenol; ANOVA, one-way analysis of variance.

• 

  Figure 4. The protective efficacy of CRP on ISO-induced myocardial dysfunction and pathological hypertrophy was abolished by PPARγ inhibitor. (A) Western blot analysis of the expression of PPARγ in the extracts of heart samples from ISO and CRP-treated mice, with or without PPARγ inhibitor intervention, GAPDH used as the loading control, n=6 per group. Image J software was used to evaluated the relative band density of PPARγ to GAPDH signals. (B) Left ventricular function, including ejection fraction (LVEF) and fraction shortening (LVFS) of mice, 21 days after control saline-treated, ISO-treated, ISO plus CRP-treated and ISO, CRP plus PPARγ inhibitor-treated (n=8 per group) was measured by echocardiography. (C) Cardiomyocyte cross-sectional area was determined by HE staining analysis. Scale bar, 100 µm, n=8 per group. (D) The surface area of NRVMs was evaluated by immunofluorescent staining (green indicates cells stained with α-actinin marker, blue indicates DAPI staining). Scale bar, 50 µm, n=6 per group. (E) The mRNA expression levels of ANP and BNP of ISO, CRP and PPARγ inhibitor treated mice hearts were determined by qRT-PCR and GAPDH as an internal control, n=8 per group. (F) qRT-PCR…

• 

  Figure 5. The protective efficacy of CRP on ISO-induced cardiac fibrosis and apoptosis was abrogated by PPARγ inhibitor. (A) Cardiac fibrosis was measured by Masson trichrome staining analysis (blue indicates the scared fibrotic area, red indicates the normal myocardium area). Scale bar, 50 µm, n=8 per group. (B) The deposition of fibrosis-related proteins including collagen type I, collagen type III and α-SMA was evaluated by western blot analysis, housekeeping gene β-tubulin used as the normalization control, n=6 per group. (C) The expression of apoptosis-related proteins including Bax, Bcl-2, cleaved caspase-3 and caspase-3 was measured by western blot analysis, β-tubulin as the loading control, n=6 per group. One-way ANOVA followed by Bonferroni’s post hoc test was used to analyze the data. Results were presented as the mean ± SD. **, P<0.01; ***, P<0.001. CRP, Citri Reticulatae Pericarpium; ISO, isoproterenol; ANOVA, one-way analysis of variance.