Citri reticulatae Pericarpium attenuates Ang II-induced pathological cardiac hypertrophy via upregulating peroxisome proliferator-activated receptors gamma

Figures

• 

  Figure 1. CRP alleviates Ang II-induced hypertrophy in cardiomyocytes. (A) Immunostaining of NRCMs for α-actinin indicated that CRP treatment of NRCMs attenuated the Ang II-stimulated increase in cardiomyocyte size, scale bars =50 µm (n=6); (B) qRT-PCR results showed that CRP-treatment significantly prevented the Ang II-induced hypertrophic marker (Anp and Bnp) expression in NRCMs (n=6). Data are presented as mean ± SD. *, P<0.05; **, P<0.01. CRP, Citri reticulatae Pericarpium; Ang II, angiotensin II; NRCMs, neonatal rat cardiomyocytes; Anp, natriuretic peptide type A; Bnp, natriuretic peptide B.

• 

  Figure 2. CRP improves cardiac function and attenuates cardiac hypertrophy induced by Ang II infusion in vivo. (A) Echocardiography for mice indicated that CRP treatment prevented Ang II-induced cardiac dysfunction with recovery of the ejection fraction (EF) and fractional shortening (FS) (n=8, 8, 9, 10); (B) histological analysis with haematoxylin and eosin (H&E) staining revealed that the increased cross-sectional area induced by Ang II was dramatically attenuated in CRP treatment mice, scale bars =100 µm (n=8, 8, 9, 9); (C) qRT-PCR results showed that CRP alleviated the upregulation of hypertrophic markers (Anp and Bnp) expression induced by Ang II infusion in mice (n=8, 8, 9, 10). Data are presented as mean ± SD. *, P<0.05; **, P<0.01. CRP, Citri reticulatae Pericarpium; Ang II, angiotensin II; NRCMs, neonatal rat cardiomyocytes; Anp, natriuretic peptide type A; Bnp, natriuretic peptide B.

• 

  Figure 3. CRP alleviates cardiac fibrosis induced by Ang II infusion in vivo. (A) Masson’s trichrome staining revealed that CRP decreased cardiac fibrosis in Ang II infusion in mice, scale bars =50 µm (n=8, 8, 9, 9); (B) qRT-PCR analysis of α-SMA, Col1a1 and Col3a1 expression showed that CRP treatment attenuated the Ang II-induced upregulation of above fibrotic genes in mice (n=8, 8, 9, 10); (C) Western blot analysis of treatment with or without CRP indicated that the protein level of Collagen I and α-SMA were dramatically downregulated in CRP-treated mice after Ang II infusion (n=6). Data are presented as mean ± SD. *, P<0.05; **, P<0.01. CRP, Citri reticulatae Pericarpium; Ang II, angiotensin II.

• 

  Figure 4. CRP alleviates Ang II-induced hypertrophy via PPARγ. (A,B) Western blot analysis of treatment with or without CRP revealed that PPARγ and PGC-1α were positively regulated by CRP both in NRCMs and in mice hearts (n=6); (C) immunofluorescent staining of NRCMs showed that PPARγ inhibitor T0070907 (1 µM) abolished the preventing effects of CRP in Ang II-stimulated cardiomyocyte hypertrophy, whereas PPARγ agonist Rosiglitazone (1 µM) did not exert additional protective effect, scale bars =50 µm (n=6); (D) qRT-PCR analysis of hypertrophic marker (Anp and Bnp) expression indicated that PPARγ inhibitor T0070907 (1 µM) blocked the protective effects of CRP in Ang II-stimulated cardiomyocyte hypertrophy, though PPARγ agonist Rosiglitazone (1 µM) did not further enhance the preventing effects of CRP (n=6). Data are presented as mean ± SD. *, P<0.05; **, P<0.01. CRP, Citri reticulatae Pericarpium; Ang II, angiotensin II; NRCMs, neonatal rat cardiomyocytes; Anp, natriuretic peptide type A; Bnp, natriuretic peptide B; PPARγ, peroxisome proliferator-activated receptor gamma; PGC-1α, PPARγ coactivator-1α.

• 

  Figure 5. CRP increases cardiac function and ameliorates cardiac hypertrophy via PPARγ. (A) PPARγ inhibitor (1 mg/kg/d) abolished the prevention of CRP on Ang II-induced cardiac dysfunction evidenced by echocardiography for mice including decrease of ejection fraction (EF) and fractional shortening (FS) (n=8, 6, 6, 6); (B) Histological analysis with haematoxylin and eosin (H&E) staining revealed that PPARγ inhibitor (1 mg/kg/d) blocked the CRP-mediated inhibition of Ang II-induced pathological hypertrophy proved by enlargement of cell size in mice, scale bars =100 µm (n=8, 6, 6, 6); (C) qRT-PCR results showed that PPARγ inhibitor (1 mg/kg/d) blocked the prevention of CRP on Ang II-induced pathological hypertrophy evidenced by upregulation of hypertrophic markers (Anp and Bnp) expression in mice (n=8, 6, 6, 6). Data are presented as mean ± SD. *, P<0.05; **, P<0.01.CRP, Citri reticulatae Pericarpium; Ang II, angiotensin II; NRCMs, neonatal rat cardiomyocytes; Anp, natriuretic peptide type A; Bnp, natriuretic peptide B; PPARγ, peroxisome proliferator-activated receptor gamma.

• 

  Figure 6. CRP attenuates cardiac fibrosis induced by Ang II infusion via PPARγ in vivo. (A) Masson’s trichrome staining revealed that PPARγ inhibitor (1 mg/kg/d) blunted the anti-fibrotic effect of CRP in Ang II infusion hearts, scale bars =50 µm (n=8, 6, 6, 6); (B) qRT-PCR analysis of α-SMA, Col1a1 and Col3a1 expression showed that PPARγ inhibitor (1 mg/kg/d) abolished the protective effect of CRP on cardiac fibrosis induced by Ang II infusion (n=8, 6, 6, 6); (C) the results of western blot indicated that the protein level of collagen I and α-SMA were dramatically upregulated in PPARγ inhibitor-treated mice (1 mg/kg/d) (n=6). Data are presented as mean ± SD. *, P<0.05; **, P<0.01. CRP, Citri reticulatae Pericarpium; Ang II, angiotensin II; PPARγ, peroxisome proliferator-activated receptor gamma.