Fig 2: Metrnl in macrophages is downregulated after MI/RI, and overexpression of Metrnl attenuates MI/RI. A Immunofluorescence co-localization of F4/80 (red) and AAV9 (GFP) in mouse hearts at 4 weeks post-injection of AAV9-F4/80-Metrnl virus. Scale bar = 50 μm, magnification = 400x (n = 5). B qRT-PCR was employed to detect the expression of Metrnl in the infarct zone of mice in each group following I/R surgery (n = 5). C WB was used to compare the expression of Metrnl protein in the infarct zone of mice (n = 4). D Quantification of panel C.The Metrnl protein expression is the ratio of Metrnl protein to β-actin (n = 4). E Serum cTnT levels of mice in each group were measured by ELISA kits (n = 6). F Evans blue-triphenyltetrazolium chloride (TTC) staining was utilized to determine the infarct area. Area-at-risk area is red and infarct area is white (n = 5). G Infarct area (%) was expressed as the percentage of white area to total area (n = 5). H Echocardiography was performed to assess cardiac function. I Left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVFS) were compared between groups of mice (n = 5). J H&E staining revealed the morphological changes of myocardial tissue in each group of mice (n = 6). Scale bar = 20 μm, magnification = 400x. Data are presented as means ± SD, ns not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 3: Overexpression of Metrnl in macrophages attenuates I/R-induced inflammatory response and cardiomyocyte apoptosis. A–C qRT-PCR was performed to detect the expression levels of inflammatory factors IL-1β, TNF-α, IL-6, MCP-1 and anti-inflammatory factors IL-10 and TGF-β in each group, respectively (n = 5). D Immunohistochemistry was used to compare the expression levels of p-NF-κB in each group (n = 6). Scale bar = 20 μm, magnification = 400x. E WB was employed to detect the expression of anti-apoptotic protein BCL-2 and its quantitative comparison (n = 6). F WB was used to detect the expression of apoptotic protein Bax and its quantitative comparison (n = 6). G TUNEL staining was conducted to compare the apoptosis rate of each group (n = 6). Scale bar = 50 μm, magnification = 400x. H Quantification of panel G. The apoptosis rate (%) was calculated as the number of nuclei stained by green fluorescence divided by the number of all nuclei (n = 6). Data are presented as means ± SD, ns not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 4: Metrnl attenuates H/R-induced cardiomyocyte apoptosis via AMPK-mediated M1-to-M2 macrophage polarization. A Immunofluorescence staining detected CD86 (green) expression in each group. Scale bar = 50 μm, magnification = 200 × . B Quantification of panel A. Mean fluorescence intensity of CD86 = CD86 gray value/fluorescence area. C Immunofluorescence staining detected CD206 (red) expression in four groups. Scale bar = 50 μm, magnification = 200 × . D Quantification of panel C. CD206 mean fluorescence intensity = CD206 gray value/fluorescence area. E Flow cytometry detected macrophage polarization in four groups. F Quantification of panel E. G WB experiments verified p-AMPK and AMPK protein expression in four groups. H Quantification of panel G. Relative expression of p-AMPK = p-AMPK/AMPK. I WB detected BCL-2 and BAX protein expression in AC16 cells in each group. J Quantification of panel L. Relative expression of BCL-2 = BCL-2/GAPDH; relative expression of BAX = BAX/GAPDH. All tests were repeated independently at least three times. Data are presented as means ± SD, ns not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 5: Molecular mechanism diagram. It revealed that Metrnl alleviated MI/RI by activating AMPK-mediated M2 macrophage polarization to attenuate inflammatory response and cardiomyocyte apoptosis