Research Paper Volume 12, Issue 6 pp 5384—5398

N-Propargyl caffeate amide (PACA) prevents cardiac fibrosis in experimental myocardial infarction by promoting pro-resolving macrophage polarization

Figure 4. Inhibitory effects of PACA on the expression of pro-fibrotic factors and the activation of cardiac fibroblasts. (A) Inhibitory effects of PACA on TGF-β1 and PDGF-a expression in macrophages. After the treatment with PACA ± LPS, the cellular proteins were analyzed by Western blotting with specific antibodies. Data were expressed as mean ± SD (n = 3), *, p<0.05; **, p<0.01 (PACA+LPS vs LPS alone). (B) qRT-PCR analysis of the mRNAs for MMP9, MMP12, MMP13 and TRAIL. After the treatment with PACA and LPS, the RNAs were isolated and analyzed by qRT-PCR technique. MMP12 and MMP13 activities in the culture medium were measured by ELISA kit. Data were expressed as mean ± SD (n = 3 or n = 4). *, p<0.05; **, p<0.01; ***, p<0.001 (PACA+LPS vs LPS alone); #, p<0.05, ###, p<0.001 (Ctrl vs LPS alone). (C) Disruption of macrophage-mediated signals against the activation and survival of cardiac fibroblasts. The conditioned medium was prepared by treating RAW264.7 macrophages with PACA ± LPS. Adult rat cardiac fibroblasts were treated with the conditioned macrophage medium. Cardiac fibroblasts were subsequently stained with antibodies against vimentin and α-SMA and followed by the detection with fluorophore-labelled secondary antibodies. The images were captured under a fluorescence microscope. Green, vimentin; Red, α-SMA; Blue, DAPI. Scale bar: 20 μm. α-SMA expression was also analyzed by Western blotting with specific antibody.