Aerobic exercise inhibits oxidative stress and improves diabetic cardiomyopathy in rats by activating the PROC/PAR1/Nrf2/HO-1 signaling pathway

FIGURE 1. Differentially expressed genes in control and DCM samples. Normalized expression matrices for (A) GSE4745, (B) GSE5606, (C) GSE6880 and (D) GSE4616. Principal component analysis diagrams of the (E) GSE4745, (F) GSE5606, (G) GSE6880 and (H) GSE4616 datasets. Part of the hierarchical clustering heat maps of the (I) GSE4745, (J) GSE5606, (K) GSE6880 and (L) GSE4616 datasets showing the differentially expressed genes in the control and DCM samples. DCM, diabetic cardiomyopathy.

FIGURE 2. DEGs and OS-related genes of the GSE4745, GSE5606, GSE6880 and GSE4616 datasets. (A–D) Volcano plots of DEGs. The red nodes represent upregulated DCM-related DEGs with P < 0.05 and logFC>1; the blue nodes represent downregulated DCM-related DEGs with P < 0.05 and logFC<-1. (E,F) Venn diagrams of 224 upregulated OS-DEGs and 229 downregulated OS-DEG. (G,H) Protein-protein interaction network diagrams of OS-DEGs. Not sig, not significant; DEGs, differentially-expressed genes; OS, oxidative stress.

FIGURE 3. DEGs in DCM and DCM-trained samples. (A) Normalized expression matrices of the GSE4616 dataset. (B) Principal component analysis diagrams of the GSE4616 dataset. (C) Part of the hierarchical clustering heat map of the GSE4616 dataset DEGs in DCM and DCM-trained samples. Venn diagrams of (D) seven upregulated OS-DCM-trained DEGs and (E) 19 downregulated OS-DCM-trained DEGs. (F) Volcano plot of seven upregulated OS-DCM-trained DEGs and 19 downregulated OS-DCM-trained DEGs. (G,H) Protein-protein interaction network diagrams of OS-DCM-trained DEGs. (I) Nine hub genes of upregulated OS-DCM-trained DEGs using Cytoscape. DCM, diabetic cardiomyopathy; DEGs, differentially-expressed genes; FGG, fibrinogen γ chain; HGD, homogentisate 1,2-dioxygenase; PROC, protein C; TRADD, tumor necrosis factor receptor type 1-associated DEATH domain protein; GC, vitamin D-binding protein; CCNA1, cyclin A1; SPAG5, sperm-associated antigen 5; CASP8, caspase-8; TOP2A, DNA topoisomerase 2α; Not sig, not significant; LAC, local average connectivity.

FIGURE 4. GO and KEGG analyses. (A,B) GO and KEGG pathway enrichment analyses. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; logFC, log2 fold change; BP, biological process; MF, molecular function; CC, cellular component; FGG, fibrinogen γ chain; HGD, homogentisate 1,2-dioxygenase; PROC, protein C; TRADD, tumor necrosis factor receptor type 1-associated DEATH domain protein; GC, vitamin D-binding protein; SPAG5, sperm-associated antigen 5; CASP8, caspase-8; TOP2A, DNA topoisomerase 2α.

FIGURE 5. Changes in insulin sensitivity, body weight and blood glucose of rats with DCM. (A) FBG levels. (B) FINS levels. (C) ISI. (D) HOMA-IR. (E) Changes in rat body weight. (F) Changes in FBG. Data are presented as the mean ± SD (n = 5). *P < 0.05 vs. CON group. DCM, diabetic cardiomyopathy; FBG, fasting blood glucose; FINS, fasting plasma insulin; ISI, insulin sensitivity index; HOMA-IR, homeostatic model assessment of insulin resistance; CON, control; DCME, DCM plus exercise.

FIGURE 6. Effects of aerobic exercise on myocardial enzymes and oxidative stress indexes in rats with DCM. Representative (A) BNP, (B) CK-MB, (C) c-TnT and (D) LDH levels. (E) MDA content. (F) GSH and (G) SOD levels. Data are presented as the mean ± SD (n = 5). *P < 0.05 vs. CON group; #P < 0.05 vs. DCM group. BNP, brain natriuretic peptide; CK-MB, creatine kinase MB; c-TnT, cardiac troponin T; LDH, lactate dehydrogenase; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; CON, control; DCM, diabetic cardiomyopathy; DCME, DCM plus exercise.

FIGURE 7. Effects of exercise training on histopathological and echocardiographic abnormalities in myocardial tissue. (A) H&E staining (×200 magnification) showed irregular arrangement of myocardial cells, uneven distribution of cytoplasm and ruptured myocardial fibers in the DCM group, whereas exercise training attenuated these histopathological changes. (B) Masson’s trichrome staining (×200 magnification) showed irregular and noticeably increased interstitial collagen fibers (blue region) in the DCM group. (C) Echocardiography of rats in each group. (D) LVEF, LVFS, LVIDd and LVIDs in each group of rats. Data are presented as the mean ± SD (n = 3). *P < 0.05 vs. CON group; #P < 0.05 vs. DCM group. H&E, hematoxylin and eosin; CON, control; DCM, diabetic cardiomyopathy; DCME, DCM plus exercise; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVIDd, left ventricular diastolic dimension; LVIDs, left ventricular systolic dimension.

FIGURE 8. Effect of aerobic exercise on the expression levels of predicted genes in rats with DCM. Representative (A) FGG, (B) HGD, (C) PROC, (D) TRADD, (E) CASP8, (F) CCNA1, (G) SPAG5, (H) GC and (I) TOP2A mRNA expression levels. Data are presented as the mean ± SD (n = 3). *P < 0.05 vs. CON group; #P < 0.05 vs. DCM group. CON, control; DCM, diabetic cardiomyopathy; DCME, DCM plus exercise; FGG, fibrinogen γ chain; HGD, homogentisate 1,2-dioxygenase; PROC, protein C; TRADD, tumor necrosis factor receptor type 1-associated DEATH domain protein; GC, vitamin D-binding protein; CCNA1, cyclin A1; SPAG5, sperm-associated antigen 5; CASP8, caspase-8; TOP2A, DNA topoisomerase 2α.

FIGURE 9. Effect of aerobic exercise on the expression levels of predicted proteins and of proteins in the PROC/PAR1/Nrf2/HO-1 signaling pathway in rats with DCM. (A) Representative TRADD, cleaved-CASP8, CASP8 and PROC levels; GAPDH was used as an internal control. (B–E) Representative TRADD and PROC ratios to GAPDH protein, and cleaved-CASP8/CASP8 ratio. (F) Representative active PROC levels. (G) Representative PAR1, Nrf2 and HO-1 levels; GAPDH was used as an internal control. (H–J) Representative PAR1, Nrf2 and HO-1 to GAPDH protein ratios. Data are presented as the mean ± SD (n = 3). *P < 0.05 vs. CON group; #P < 0.05 vs. DCM group. PROC, protein C; PAR1, proteinase-activated receptor 1; Nrf2, nuclear factor (erythroid-derived 2)-like 2; HO-1, heme oxygenase-1; TRADD, tumor necrosis factor receptor type 1-associated DEATH domain protein; CASP8, caspase-8; CON, control, DCM, diabetic cardiomyopathy; DCME, DCM plus exercise.

FIGURE 10. Schematic diagram of aerobic exercise alleviating oxidative stress and diabetic cardiomyopathy (DCM) via the PROC/PAR1/Nrf2/HO-1 signalling pathway. Aerobic exercise alleviates DCM by targeting the hub gene PROC, as identified through bioinformatics and animal models. Mechanistically, aerobic exercise upregulates PROC expression and activates the PROC/PAR1/Nrf2/HO-1 axis, leading to enhanced antioxidant capacity, reduced myocardial injury, and improved cardiac function. PROC, protein C; PAR1, proteinase-activated receptor 1; Nrf2, nuclear factor (erythroid-derived 2)-like 2; HO-1, heme oxygenase-1; DCM, diabetic cardiomyopathy.