Caffeic acid mitigates myocardial fibrosis via suppressing MAPKs/CREB-mediated inflammation in myocardial ischemia–reperfusion injury mice

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  Fig. 1. Caffeic acid improved cardiac dysfunction and fibrosis in MIRI-induced mice. A Two-dimensional chemical structure of CA. B Schematic diagram of the experimental protocol. MIRI models or control models were established by LAD ligation or by the same surgical procedure without ligation. Starting 10 min before MIRI modeling (day 0) and continuing until day 14, different doses of CA (12.5 mg/kg, 25 mg/kg, 50 mg/kg), pirfenidone (300 mg/kg) or an equivalent volume of normal saline were administered once daily by oral gavage. Inflammatory markers, cardiac function, and pathological changes were assessed at designated time points. C Representative short-axis M-mode echocardiographic images at 2 weeks after MIRI. D Corresponding quantitative measurements of LVEF and LVFS. n = 6 mice per group). E Morphological change of cardiomyocytes as shown by staining with WGA. Scale bar = 20 μm. F Measurements of cardiomyocytes size (green-stained) based on WGA staining. Cardiomyocytes were outlined in transversely sectioned images using ImageJ software, and the mean area was calculated from at least 20 cells per sample. n = 5 mice per group. G Images of the hearts from mice when the study…

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  Fig. 2. Caffeic acid improved myocardial injury and inflammatory response in MIRI-induced mice. A H&E staining of myocardial sections. View fields were randomly selected in the infarct border zone. Scale bar = 100 μm; n = 3 mice per group. B Relative mRNA levels of TNF-α, IL-1β, and IL-6 in heart tissue. n = 8 mice per group. C, D Flow cytometry to detect macrophage frequency. n = 10 mice per group. E, F Flow cytometry to detect the phenotypic changes of macrophages. n = 10 mice per group. G Representative immunofluorescence images showing CD86 and CD206 expression on cardiac tissue. Green, CD86; red, CD206; blue, cell nuclei. Scale bar = 50 μm; n = 3 mice per group. H Relative mRNA levels of IL-6, TNF-α, Arg1, and iNOS in RAW 264.7 following LPS or CA treatment. n = 6 independent biological replicates per group. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analyses were performed using one-way ANOVA followed by Tukey's post hoc test. Results are presented as mean ± SD

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  Fig. 3. Caffeic acid mitigates inflammation-induced activation of myocardial fibroblasts. A Immunofluorescence staining for CD86 and α-SMA on mouse myocardial tissues. Green, CD86; red, α-SMA; blue, cell nuclei. Scale bar = 50 μm; n = 3 mice per group. B Schematic of the experimental procedure. The CM was collected from RAW 264.7 cells after culture under different conditions: Control, LPS, LPS+CA (32 μM), LPS+CA (64 μM). This CM was then applied to cardiac fibroblast cultures to assess its effects on proliferation and migration. C, D Representative images and quantification of western blot analysis to detect the protein levels of PCNA and CCND1 in MCFs. Vinculin served as a loading control. n = 4 independent biological replicates per group. E EdU assay for evaluating the proliferation rate of MCFs. Red, proliferating cells; blue, cell nuclei. Scale bar = 50 μm. F EdU-positive rate quantification. In randomly selected view fields, the number of EdU-positive cells (red) and the total number of cells (blue + red) were counted using ImageJ software. The ratio of EdU-positive cells to total cells was then calculated. n = 5 independent biological replicates per group. G Migratory activation…

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  Fig. 4. The effects of CA on macrophage-induced inflammation and myocardial fibrosis via MAPKs. A Principal component analysis (PCA) confirming similar response phenotypes between sham+veh and MIRI+CA group that was partially distinct from those in the MIRI+veh group. n = 3 mice per group. B, C Illustration of gene differential expression between MIRI+veh versus sham+veh groups and MIRI+H-CA versus MIRI+veh groups, respectively. n = 3 mice per group. D Scattered plot showing the differentially expressed genes that overlapped between MIRI+veh versus sham+veh groups and MIRI+H-CA versus MIRI+veh groups. n = 3 mice per group. E Bubble plots showing the signaling pathways of the differentially expressed genes (marked with blue in panel D) identified by KEGG. F, G Representative immunoblots of p38 MAPK14, phospho-p38 MAPK, JNK, phospho-JNK, ERK, and phospho-ERK in myocardial tissue of mice. Each protein level was assessed (Vinculin as loading control), and statistical comparisons were made for the phospho-p38 MAPK/p38 MAPK14, phospho-JNK/JNK and phospho-ERK/ERK ratios, respectively. n = 4 mice per group. H SPR analysis showing the binding affinity between CA and p38 MAPK14. The sensorgram was…

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  Fig. 5. Effects of CA on inflammation and cardiac fibroblast activity via the MAPKs/CREB pathway. A, B The protein expression of CREB and phospho-CREB in cardiac tissues determined by western blot analysis. Protein levels of phospho-CREB and CREB were assessed (Vinculin as loading control), and statistical comparison was made for the phospho-CREB/CREB ratio. n = 4 mice per group. C Representative immunofluorescence images showing subcellular localization of phospho-CREB in RAW 264.7 cells treated with vehicle or CA (64 μM) in the presence of LPS or not. Red, phospho-CREB; blue, cell nuclei. n = 3 independent biological replicates per group. D, E Western blot analysis for detecting CREB, phospho-CREB, p38 MAPK14, and phospho-p38 MAPK in RAW 264.7 cells treated with forskolin or CA in the presence of LPS. Vinculin served as a loading control. n = 4 independent biological replicates per group. F Relative mRNA levels of IL-6, IL-1β, TNF-α, Arg1, and iNOS in RAW 264.7 following forskolin or CA (64 μM) treatment. n = 7 independent biological replicates per group. G, H Representative images of transwell migration assay and quantitative analysis of migrated cell numbers. n = 6 independent…

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  Fig. 6. The roles of MAPKs and CREB in the amelioration of inflammation and myocardial fibrosis by CA. A, B Western blot analysis used to detect the protein expression of CREB, phospho-CREB, p38 MAPK14, and phospho-p38 MAPK in the myocardial tissues of mice. Vinculin served as a loading control. n = 4 mice per group. C, D Representative images and quantitative analysis of echocardiography to reflect the cardiac function of mice. n = 6 mice per group. E ELISA for evaluating the serum contents of IL-6, IL-1β, and TNF-α. n = 4 mice per group. F H&E staining of myocardial tissues from mice. View fields were randomly selected in the infarct border zone. Scale bar = 100 μm; n = 3 mice per group. G, H Representative images and quantitative analysis of Masson's trichrome staining in myocardial tissue of mice. Red, myofibers; blue, collagen. Scale bar = 100 μm; n = 3 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test. Results are presented as mean ± SD