Pilose Antler Peptide-3.2KD Ameliorates Adriamycin-Induced Myocardial Injury Through TGF-β/SMAD Signaling Pathway

Figures

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  Figure 1. PAP-3.2KD reversed HW/BW index of ADR-induced rats. (A) Every 3 days, body weight (BW) was measured in four groups, includingcontrol, ADR, ADR+ PAP-3.2KD (100 mg/kg), ADR+ PAP-3.2KD (200 mg/kg). (B,C) Both Heart weight (HW) and HW/BW index were detected in four groups after the rats were euthanized. Data were presented as Mean ± standard error of mean (n = 10). #P < 0.05, ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the ADR group, ADR, Adriamycin; PAP-3.2KD, 3.2 KD pilose antler polypeptide.

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  Figure 2. PAP-3.2KD recovered heart rates and ST heights of ADR-induced rats. Both heart rate (A) and ST height (B) were determined by an eight-channel biological signal acquisition and processing system. Data were presented as Mean ± standard error of mean (n = 10). ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the ADR group.

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  Figure 3. PAP-3.2KD restored the levels of cTnT and cTnI in ADR-induced rats. Serum concentrations of both cTnT (A) and cTnI (B) were determined by ELISA. Data were presented as Mean ± standard error of mean (n = 10). ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the ADR group, Cardiac troponin T (cTnT); Cardiac troponin I (cTnI).

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  Figure 4. PAP-3.2KD ameliorated the histopathological damages of ADR-induced myocardial tissue. Histopathological damages of myocardial tissues were detected by HE staining. (A) Control group, (B) ADR group, (C) PAP-3.2KD group (100 mg/kg), (D) PAP-3.2KD group (200 mg/kg). (Magnification, ×200). (E) Mean histopathological scores in each group. Data were presented as Mean ± standard error of mean (n = 6). ##P < 0.01 vs. the control group; **P < 0.01 vs. the ADR group.

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  Figure 5. PAP-3.2KD inhibited ADR-induced myocardial fibrosis. Myocardial Fibrosis in each group were detected by Masson's trichrome staining. (A) Control group, (B) ADR group, (C) PAP-3.2KD group (100 mg/kg), (D) PAP-3.2KD group (200 mg/kg). (Magnification, ×200). (E) Collagen volume fraction (CVF) was calculated in each group. All the myocardial section were made by Left ventricular apex. The zone of endocardium (Endoc)-epicardium (Epic) was indicated. Data were presented as Mean ± standard error of mean (n = 6). ##P < 0.01 vs. the control group; **P < 0.01 vs. the ADR group.

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  Figure 6. PAP-3.2KD blocked the apoptosis of ADR-induced myocardial tissue. Apoptosis of myocardial tissues was detected by TUNEL staining. TUNEL-positive cells exhibited dark buffy nuclei staining. (A) Control group, (B) ADR group, (C) PAP-3.2KD group (100 mg/kg), (D) PAP-3.2KD group (200 mg/kg). (Magnification, ×400). (E) Statistical analysis of apoptotic index in each group. The independent experiments (n = 6) were performed in each group. ##P < 0.01 vs. the control group; **P < 0.01 vs. the ADR group.

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  Figure 7. PAP-3.2KD regulated the expression levels of Bcl-2, Bax, and caspase-3 in ADR-induced rats. The concentrations of Bcl-2 (A), Bax (B) and Caspase-3 (C) in cardiac tissue were determined by ELISA. Data were presented as Mean ± standard error of mean (n = 10). (D) The protein expression levels of Bcl-2, Bax and Caspase-3 were determined by western blotting. (E) Statistical analysis for expression levels of relative protein in (D), GAPDH protein levels as control. Data were presented as Mean ± standard error of mean (n = 3). ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the ADR group, ADR, Adriamycin; PAP-3.2KD, 3.2 KD pilose antler polypeptide; Bcl-2, B-cell lymphoma-2; Bax, BcL-2-Associated X.

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  Figure 8. PAP-3.2KD attenuated ACR-induced myocardial injury through TGF-β/SMAD pathway. (A) The protein expression levels of TGF-β1, SMAD4, and SMAD7 in heart tissues were determined by western blotting. (B) Statistical analysis of relative levels of target protein in (A), compared to GAPDH protein levels. The representation (C) and quantitative analysis (D) of western analysis results for SMAD2/SMAD3 and their phosphorylation. Coenzyme Q10 (CoQ10) as positive control group (CoQ10 has been proved to have protective roles for cardiomyocyte after myocardial injury through TGF-β pathway, Data not shown). Thus, splicing occurred between ADR group (Lane 2) and PAP-3.2KD (100 mg/kg) (Lane 3) group in all Western Blotting results. Data were expressed as mean ± standard error of mean (n = 3). ##P < 0.01 vs. the control group; **P < 0.01 vs. the ADR group. TGF-β1, transforming growth factor-β1; SMAD, drosophila mothers against the decapentaplegic protein.