Roxadustat improves diabetic myocardial injury by upregulating HIF-1α/UCP2 against oxidative stress

Figures

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  Fig. 1. Construction of a mouse model of T2DM combined with myocardial injury. a Flowchart of mouse modeling. b–c Changes in body weight and blood glucose in control and diabetic mice at weeks 0–10. n = 5–6 mice per group. d Changes in blood pressure in control and diabetic mice at weeks 0–10. n = 5–6 mice per group. e Serum BNP levels in control and diabetic mice at week 10. n = 5 mice per group. f Mitochondrial structure of mouse cardiac muscle tissues under transmission electron microscopy; magnification 20000X, scale bar = 1 μm. n = 3 mice per group. g–i Changes in LVEF and LVFS, indicators of cardiac function in mice. n = 3 mice per group. P values were determined by t-test. **P < 0.01, *P < 0.05

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  Fig. 2. FG-4592 pretreatment improves myocardial injury in T2DM mice. a FG-4592 pretreatment modeling process. b–c Changes in body weight and blood glucose of mice at week 10. n = 6 mice per group. d Changes in blood pressure of mice at week 10. n = 5–6 mice per group. e Serum BNP level of mice at week 10. n = 5 mice per group. f–h Results of echocardiography of mice as well as the index of LVEF and LVFS Changes. n = 3 mice per group. i Mitochondrial structure of mouse cardiac muscle tissues under transmission electron microscopy; magnification 20000X, scale bar = 1 μm. n = 3 mice per group. P values were determined by one-way ANOVA. **P < 0.01, *P < 0.05

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  Fig. 3. FG-4592 pretreatment attenuates oxidative stress injury and regulates mitochondrial respiratory efficiency in myocardial tissue of diabetic mice. a-b HIF-1α expression detected by immunohistochemistry in myocardial tissues, magnification 400x, scale bar = 50 μm. n = 5 mice per group. c–d HIF-1α expression detected by Western Blot. n = 5 mice per group. e HIF-1α mRNA level in mice detected by RT-qPCR. n = 4 mice per group. f–h Expression of T-GSH, GSSH and GSH in myocardial tissues. n = 4 mice per group. i Expression of superoxide dismutase expression. n = 5 mice per group. j MDA expression in myocardial tissues. n = 4 mice per group. k–l Mitochondrial complex I and IV activities in myocardial tissues. n = 5 mice per group. m Mitochondrial ATP levels in myocardial tissues. n = 5 mice per group. P values were determined by one-way ANOVA. ***P < 0.001, **P < 0.01, *P < 0.05, ns represents no statistical significance

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  Fig. 4. Transcriptome sequencing screening for differentially expressed genes and functional enrichment analysis. a Heatmap of transcriptome sequencing screening for genes related to mitochondria. b KEGG enrichment analysis of gene expression data. c Volcano map of differentially expressed genes, blue represents down-regulation and red represents up-regulation. n = 3 mice per group

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  Fig. 5. Expression of HIF-1α and UCP2 in high sugar-induced H9c2 cardiomyocyte model. a-b Expression of Bax at different sugar concentrations. n = 3 experiments per group. c CCK8 detection of the effect of different sugar concentrations on the proliferation of H9c2 cells. n = 4 experiments per group. d–e Western Blot detection of the expression of HIF-1α and UCP2 in H9c2 cells. n = 3 experiments per group. f Quantitative statistical results of HIF-1α Western Blot in H9c2 cells. g RT-qPCR detection of HIF-1α mRNA level in H9c2 cells. n = 3 experiments per group. h Quantitative statistical results of Western Blot for UCP2. P values were determined by one-way ANOVA. **P < 0.01, *P < 0.05

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  Fig. 6. FG-4592 pretreatment improves high glucose-induced oxidative stress and mitochondrial respiratory function in H9c2 cells. a-d siRNA knockdown of HIF-1α and UCP2 expression. n = 3 experiments per group. e MDA content assay. n = 4 experiments per group. f GSH content assay. n = 4 experiments per group. g MitoSox Red staining to detect mitochondrial superoxide level, scale bar = 50 μm. n = 3 experiments per group. h–i O2k to detect mitochondrial oxygen consumption. n = 3 experiments per group. P values were determined by one-way ANOVA. **P < 0.01, *P < 0.05

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  Fig. 7. Up-regulation of HIF-1α and UCP2 by FG-4592 pretreatment affects PI3K/AKT/Nrf2 pathway. a-j Western Blot for expression of Nrf2, HO-1, SOD2, PI3K, P-PI3K, AKT, P-AKT. n = 3 experiments per group. k The mechanism cascade of FG-4592 against oxidant stress. Figure created with BioRender.com. P values were determined by one-way ANOVA. **P < 0.01, *P < 0.05

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  Fig. 8. Roxadustat Improves Diabetic Myocardial Injury by Upregulating HIF-1α/UCP2 Against Oxidative Stress. Roxadustat improves diabetic myocardial injury by inhibiting PHD activity increasing the expression of HIF-1α and UCP2, then reduces ROS production in mitochondria. Figure created with BioRender.com