Brite Adipocyte FGF21 Attenuates Cardiac Ischemia/Reperfusion Injury in Rat Hearts by Modulating NRF2

Figures

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  Figure 1. Identification of differentially expressed genes (DEGs) by transcriptome sequencing and functional analysis in IR rat hearts. (A) Hierarchical cluster analysis of RNA sequencing data. (B) Volcano plot depicting the differential RNAs, with the vertical lines corresponding to 2-fold up- and downregulation and the horizontal line representing a p value of 0.05. (C) GO analysis of differentially expressed RNAs. (D) GSEA enrichment plots for two hallmark gene sets enriched in IR rat hearts.

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  Figure 2. Beige adipocyte differentiation of rat adipose-derived stem cells. (A) Representative microscopic images of adipocytes stained with ORO (magnification, ×100, Scale bar = 100 μm). (B) Assessment of the mRNA expression of browning markers by qPCR. (C) Validation of differentially regulated browning markers by immunoblot analysis. The intensity of each band was quantified with ImageJ software. The data are shown as the mean ± standard error of the mean (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. ASC. (D) Assessment of the expression of browning markers by immunofluorescence staining. Scale bar = 50 μm. UD means “undifferentiated”.

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  Figure 3. Regulation of antioxidant and apoptotic factors by beige adipocyte secretions. (A) Cell viability and cytotoxicity in H9c2 cells under H2O2 stimulation. (B) Cell viability and cytotoxicity in H9c2 cells under H2O2 stimulation with CM. H9c2 cells were treated with 300 μM H2O2 for 3 h and then exposed to CM for 1 h. The data are shown as the mean ± standard error of the mean (n = 5). (C) Annexin V/PI staining as detected by flow cytometry. (D,E) Differential expression of antioxidant/apoptotic factors in H9c2 cells by ASC, LBAC, or HBAC-derived CM treatment. The factors were determined using western blot analysis (D) and qPCR (E). The data are shown as the mean ± standard error of the mean (n = 3). * p < 0.05 and ** p < 0.01 vs. non-treated (A) or H2O2-treated group (B–E). cl means ”cleaved form”.

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  Figure 4. Regulation of multiple cell death signals by beige adipocyte secretions under oxidative stress. (A) Hierarchical cluster map of gene expression determined by PCR array. (B) Apoptosis signals. (C) Necroptosis signals. (D) Autophagy signals. The relative expression levels of each gene are plotted for each group comparison (nontreated vs. H2O2-stimulated sample, H2O2-stimulated sample vs. CM-treated sample). The diagonals show the same representation in both groups with a 2-fold change boundary. Upregulated genes with greater than 2-fold changes are marked above the middle line, and downregulated genes are marked below the line in each graph.

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  Figure 5. Brite adipocyte FGF21 attenuates cardiac IR injury by modulating NRF2. (A) Elevated FGF21 secretion from beige adipocytes in the adipokine array. (B) Validation of FGF21 protein expression in ASC, LBAC, or HBAC-derived CM by western blot analysis (up) and FGF21 gene expression in ASC, LBAC, or HBAC using qPCR (down). (C) Oxidative stress is alleviated by NRF2 translocation. * p < 0.05 and ** p < 0.01 vs. H2O2-treated group. (D) Representative images of NRF2 immunofluorescence and DAPI staining. Scale bar = 200 μm. (E) Effect of Nrf2 knockdown on the expression of oxidant and apoptosis-related proteins. The data are shown as the mean ± standard error of the mean (n = 3). * p < 0.05, ** p < 0.01. cl means “cleaved form”.

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  Figure 6. Effect of FGF21 treatment on H2O2-induced oxidative stress and apoptosis in H9c2 cells. Assessment of the differential expressed antioxidant/apoptotic factors by western blot (A) and qPCR (B). The data are shown as the mean ± standard error of the mean (n = 3). * p < 0.05 and ** p < 0.01 vs. non-treated group; # p < 0.05, ## p < 0.01 and ### p < 0.001 vs. H2O2-treated group. cl means “cleaved form”.

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  Figure 7. Effect of beige adipocyte secretions on IR rat hearts. (A) Triphenyl tetrazolium chloride (TTC) staining showing the infarct areas in transverse sections. Representative images of rat heart slices stained with TTC. (B) Histological analysis of IR rat hearts after beige adipocyte secretion injection. Cardiac fibrosis was evaluated by Masson’s trichrome staining. Scale bar = 2 mm (upper panel images), 200 μm (down panel images). (C) Representative images of TUNEL staining and NRF2 immunofluorescence. Scale bar = 50 μm. (D) Cardiac function was valuated with a Millar catheter in rat heart at three weeks after IR and treatment of CMs. EF, ejection fraction; ESV, end-systolic volume; V@dP/dt min, volume at dP/dt min. The data are shown as the mean ± standard error of the mean (n = 5). * p < 0.05 and ** p < 0.01 vs. IR group.