Anti‐Inflammatory Effect of Curcumin on the Mouse Model of Myocardial Infarction through Regulating Macrophage Polarization

Figures

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  Figure 1. Curcumin attenuates adverse remodeling after MI: (a) 3-month overall survival analysis after MI; (b) quantitative analysis of infract size; (c) cardiomyocyte width; (d) myocardial fibrosis in border; (e) remote area; (f) qPCR analysis of ANP, BNP, and COL1A1 mRNA expression in the myocardium at 3 months post-MI. Data were shown as the mean ± SEM. ∗ was P < 0.05 vs. sham group, # was P < 0.05, and ns was P > 0.05 vs. MI group. And P value was calculated by the long-rank test in (a), Student's t-test in (b), and post hoc comparisons in (c–f).

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  Figure 2. Analysis of inflammation-related cytokine mRNA and protein expression in homogenized hearts. Data were shown as the mean ± SEM. # was P < 0.05, and ns was P > 0.05 vs. the MI group. And P value was calculated by Student's t-test. Baseline was defined as the gene expression in a normal mouse myocardium.

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  Figure 3. Curcumin decreases M1 macrophage marker expression (iNOS, CCL2, and CD86) and increases M2 marker mRNA expression (Arg1, CD163, and CD206) in homogenized hearts. Data were shown as the mean ± SEM. # was P < 0.05, and ns was P > 0.05 vs. the MI group. And P value was calculated by Student's t-test. Baseline was defined as the gene expression in a normal mouse myocardium.

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  Figure 4. Effect of curcumin on M1 and M2 macrophage marker protein expression in the myocardium at 7 days post-MI. (a) Representative immunohistochemistry detection of M1 macrophage marker (iNOS) expression; (b) representative immunohistochemistry detection of M2 macrophage marker (Arg1) expression; (c) quantification of iNOS-positive cells; (d) quantification of Arg1-positive cells. Seven mice in each group. Data were shown as the mean ± SEM; ∗ was P < 0.05 vs. the sham group, and # was P < 0.05 vs. the MI group. And P value was calculated by Student's t-test.

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  Figure 5. Curcumin decreases M1 macrophage and increases M2 macrophage in homogenized hearts. (a–d) Representative flow cytometry data of activated macrophages in the heart tissues of different groups (a), quantification of M1 (b) and M2 (c) macrophages, and calculation of the ratio of M1 and M2 macrophages (d). Data were shown as the mean ± SEM. # was P < 0.05 vs. the MI group. P value was calculated by Student's t-test.

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  Figure 6. Effect of curcumin on LPS/IFNγ-induced M1 and M2 macrophage marker expression in BMMs. mRNA expression of iNOS (a), CD86 (b), Arg1 (c), and CD163 (d) in BMMs using qPCR after being stimulated with different stimuli. Representative protein bands (e) and quantification of the grayscale value of iNOS (f), CD86 (g), Arg1 (h), and CD163 (i) protein. Each experiment was repeated 3 times independently. Data were shown as the mean ± SEM. ∗ was P < 0.05 vs. the LPS+IFNγ stimulation group, and # was P < 0.05 vs. the unstimulated group. P value was calculated by post hoc comparisons.

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  Figure 7. Effect of curcumin on LPS/IFNγ-induced macrophage polarization in BMMs. Concentration of TNF-α (a), IL-1β (b), IL-6 (c), and IL-10 (d) in cell culture medium of BMMs after being stimulated with different stimuli. Representative flow cytometry data of activated macrophages after being stimulated with different stimuli (e) and quantification of M1 (iNOS+F4/80+) (f) and M2 (Arg+F4/80+) (g) macrophages. Each experiment was repeated 3 times independently. Data were shown as the mean ± SEM. ∗ was P < 0.05 vs. the LPS+IFNγ stimulation group, and # was P < 0.05 vs. the unstimulated group. P value was calculated by post hoc comparisons.

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  Figure 8. Curcumin modulates M1/M2 macrophage polarization partly via AMPK. (a) The expression of p-AMPK and AMPK protein in homogenized hearts at 7 days after MI: representative protein bands (left) and quantification of protein gray values (right), 7 mice in each group. Data were shown as the mean ± SEM. ∗ was P < 0.05 vs. the sham group, and # was P < 0.05 vs. the MI group. And P value was calculated by Student's t-test. (b) After being stimulated with curcumin for different times, p-AMPK and AMPK proteins in BMMs were expressed: representative protein bands (left) and quantification of protein gray values (right). Representative protein bands (c) and quantification of the grayscale value of iNOS (d), CD86 (e), Arg1 (f), and CD163 (g) protein. Each experiment was repeated 3 times independently. Data were shown as the mean ± SEM. ∗ was P < 0.05 vs. the LPS+IFNγ stimulation group, and # was P < 0.05 vs. the LPS+IFNγ+Cur stimulation group. P value was calculated by post hoc comparisons.

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  Figure 9. Schematic diagram of curcumin regulating macrophage polarization to inhibit MI-induced inflammation.