Role of IRF4 in the Protection of Metformin-Mediated Sepsis Myocarditis

Figures

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  Figure 1.. Effect of metformin on mice. A, The survival curves. Each line represents survival of mice in a group, 8 mice were in each group. Kaplan-Meier analysis was performed to test the difference. The mice injected with the lethal dose of E coli (3 × 108 CFU), then treated with metformin at different time points, *versus saline. B, ELISA assay of TGF-β/IL-10 in peripheral blood of mice at different time point. **P < .01 (Student t test). C, Heart tissue of mice detected by H&E staining. D, Expression of IRF4 in total cellular lysates of mice cardiomyocyte. Representative Western blots of total cellular from 4 independent experiments. IRF4 indicates interferon regulatory factor 4.

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  Figure 2.. Effect of metformin on PKCε translocation to membrane and mitochondrial. A, Rat cardiomyocytes were treated with LPS and metformin treatment group (MF) after LPS. Double labelling confocal images of adult rat ventricular myocytes with anti-PKCε and anti-Cav-3 antibodies. Punctate areas of colocalization (represented by yellow) are apparent along the plasma membrane (n = 20). B, H9c2 cells were cotransfected with PKCε-YFP/Mito-CFP cDNAs. The images were captured from the same cell before and after treatment with metformin treatment group (MF). Representative images of PKCε-YFP, Mito-CFP, and FRETC. Metformin led to FRET signals due to energy transfer from CFP of mitochondrial to YFP of PKCε. Scale bar: 1μM (n = 20). C, Western blot shows MF promotes translocation of PKCε from cytosol to membrane in rat cardiomyocytes, n = 3. D, Western blot shows MF promotes translocation of PKCε from cytosol to mitochondria in rat cardiomyocytes, n = 3. cDNA indicates complementary DNA; FRETC, corrected fluorescence resonance energy transfer; LPS, lipopolysaccharide; PKCε indicates protein kinase C eplison.

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  Figure 3.. Effect of metformin on IRF4 translocation and association of IRF4 with PKCε. A, The effect of metformin on the translocation of IRF4 in H9c2 cells. IRF4 is in the cytosol under normal condition, and IRF4 translocates from cytosol to mitochondria in the cells treated with LPS for 2 hours. B, Effect of metformin on coprecipitation of IRF4 and PKCε in H9c2 cells. The mitochodria fractions from H9c2 cells treated with or without metformin. Co-IP were immunoprecitated with anti-PKCε antibody, followed by immunoblotting with antibodies against IRF4. IgG denotes immunoprecipitation with control IgG from the protein lysates of MF-treated cells. The mitochodria-rich fractions from H9c2 cells treated with or without Co-IP were immunoprecitated with anti-IRF4 antibody, followed by immunoblotting with antibodies against PKCε, n = 3. IgG indicates immunoglobulin G; IRF4, interferon regulatory factor 4; LPS, lipopolysaccharide; PKCε, protein kinase C eplison.