AFC1 Compound Attenuated MI/R-Induced Ventricular Remodeling via Inhibiting PDGFR and STAT Pathway

Figures

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  Figure 1. Administration of AFC1 contributed to the recovery of cardiac function after MI/R in murine models. Mice were injected with low (7 mg/kg), high-dosage (14 mg/kg) of AFC1 compound or TSA (5 mg/kg) intraperitoneally for 7 days after MI/R (n = 5). (A) Lung, liver, spleen, and kidney sections were harvested on day 14 for H&E staining. Figures showed the representative data. Magnification was ×50. (B) Body weight change of mice after AFC1 administration was analyzed on day 14. (C) HUVECs were seeded at a concentration of 1×104/well in 96-well plate and treated with DMSO, AFC1 compound, or TSA for 24 h. Then cell viability was evaluated by MTT assay. (D) EF, FS, and LVIDs were measured by echocardiography on day 14 following MI/R. (E) Average values for EF, FS, and LVIDs. Each experiment was repeated for at least three times and results indicated mean ± SEM of one independent experiment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus sham or selected group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus MI/R. *represent the significant different between MI/R group vs the sham group; #represent the significant different between treatment group and MI/R group.

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  Figure 2. AFC1 Compound attenuated MI/R-induced cardiac remodeling. Heart sections were harvested as mentioned above (n = 5). (A) Heart to body weight ratio (HW/BW%) was measured on day 14. (B) Representative photomicrographs illustrating ventricular myocyte cross-sections stained WGA. (C) Cross-sectional area quantification was shown in statistic graph. (D) Hearts were sliced and stained with Masson’s trichrome to assess fibrosis. (E) Percent of fibrosis area in each group was shown in statistics. Each experiment was repeated for at least three times and results indicated mean ± SEM of one independent experiment. **p < 0.01, ****p < 0.0001 versus sham; ##p < 0.01, ###p < 0.001, ####p < 0.0001 versus MI/R.

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  Figure 3. Antioxidant and anti-inflammatory Effects of AFC1 compound on infarcted myocardium following MI/R in vivo. Heart sections were harvested and frozen as mentioned above (n = 5). (A) Reactive oxygen species production was assessed by dihydroethidium (DHE) conversion to red fluorescent ethidium. (B) Fluorescence intensity was evaluated to determine ROS level. (C) Immunofluorescence staining of CD45 in transverse section of heart (400×). (D) CD45+ cells were quantified in the field. (E) Hearts were homogenized and q-PCR was performed to quantify IL-1β, TNF-α, and IL-6 mRNA levels. Results indicated mean ± SEM of one representative experiment and the experiment was repeated three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus sham; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 versus MI/R.

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  Figure 4. AFC1 inhibited the expression of PDGFR in heart following MI/R (n = 5). Total RNA was extracted from hearts in each group and q-PCR was performed to quantify PDGFRα, PDGFRβ (A), PDGF-A, and PDGF-B (B) mRNA levels. (C) Immunofluorescence staining of PDGFRα in transverse section of heart (400×). Each experiment was repeated for at least three times and results indicated mean ± SEM of one representative experiment. *p < 0.05, **p < 0.01, ****p < 0.0001 versus sham; #p < 0.05, ##p < 0.01 versus MI/R.

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  Figure 5. Effect of PDGF stimulation on PDGFR expression in NCM and NCF in vitro. PDGFRα (A) and PDGFRβ (B) mRNA expression in NCM were assessed by q-PCR. NCM hypertrophy (C) was measured by 3H-leucine incorporation and NCF collagen synthesis (D) measured by 3H-proline incorporation on different PDGF isoform stimulation. Each experiment was repeated for four times and results indicated mean ± SEM of one representative experiment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control.

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  Figure 6. AFC1 reversed the elevation of PDGFR induced by PDGF-AB in both NCM and NCF in vitro. PDGFR expression in NCM (A, B) and NCF (C) were quantified by q-PCR. (D) Viability of NCF stimulated with PDGF-AB or AFC1 compound (0.1, 1.0, 3.0, 10.0 µM) was evaluated by MTT assay. This experiment was repeated four times and results indicated mean ± SEM of one independent experiment and representative pictures. **p < 0.01 versus control; #p < 0.05, ##p < 0.01 versus PDGF-AB.

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  Figure 7. AFC1 suppressed PDGF-AB induced NCM hypertrophy and NCF collagen synthesis. NCM hypertrophy (A) was measured by 3H-leucine incorporation and NCF collagen synthesis (C) measured by 3H-proline incorporation on different concentration of AFC1 after PDGF-AB (10 ng/ml) stimulation. mRNA levels of ANP, β-MHC, α-SKA in NCM (B), and Col I, Col III in NCF (D) were measured by q-PCR. This experiment was repeated four times and results indicated mean ± SEM of one independent experiment. *p < 0.05, **p < 0.01, ****p < 0.0001 versus control; #p < 0.05, ##p < 0.01, ###p < 0.001 versus PDGF-AB.

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  Figure 8. Potential mechanism of the protective function of AFC1 treatment in vitro and in vivo n = 5. After being treated with JI1 (A) or 979 (B) and then stimulated with PDGF-AB, NCM hypertrophy was measured by 3H-leucine incorporation and NCF collagen synthesis measured by 3H-proline incorporation. (C, D) Protein levels of p-JAK2, p-STAT3 in NCM and p-p38 in NCF were determined via Western blot analysis. (E) Infarct area of heart in each group was lysed and p-STAT3, STAT3 protein levels were determined via Western blot. (F) p-STAT3/STAT3, p-STAT3/GAPDH, and STAT3/GAPDH were analyzed with image J software. Two or three samples were randomly selected from each group for Western blot experiment. Each experiment was repeated for at least three times and results indicated mean ± SEM of one independent experiment and the representative pictures. *p < 0.05, **p < 0.01, ****p < 0.0001 versus control or sham; #p < 0.05, ##p < 0.01, ###p < 0.001 versus PDGF-AB or MI/R.