Regulation of TLR4 expression mediates the attenuating effect of erythropoietin on inflammation and myocardial fibrosis in rat heart

Figures

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  Figure 1. Target gene expression in left ventricular wall. Microscopy analysis was performed in order to determine whether the target genes were stably expressed following intramyocardial administration of recombinant lentivirus for 1 month. The left ventricular wall of randomly selected rats was stripped, frozen, sectioned by a microtome, and visualized using immunofluorescence analysis. (A) EPO expression in the left ventricular wall of AAC rats, injected with EPO-expressing lentivirus (magnification, ×200). The sequence element contained EGFP (green signal). EPO displayed a homogenous expression in the entire left ventricular wall. (B) EPO expression levels in the left ventricular wall of AAC rats treated with EPO + TLR4 (magnification, ×200). (C) TLR4 expression levels in the left ventricular wall of AAC rats treated with EPO + TLR4. The sequence element of the TLR4-expressing lentiviral vector contained cherry fluorescent protein (magnification, ×200; red signal). (D) Merged image of B and C. EPO and TLR4 demonstrated a homogenous expression in the left ventricular wall. EPO, erythropoietin; AAC, abdominal aortic constriction; EGFP, enhanced green fluorescent protein; TLR4, Toll-like…

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  Figure 2. Histopathological analysis of the heart tissue in each experiment group. (A) Representative images of H&E staining. (B) Representative images of Masson's trichrome staining. (C) Quantification of the proportion of myocardial fibrosis area using Masson's trichrome staining. The data are expressed as the mean ± standard error of the mean (n=6 per group). *P<0.05 vs. the control group; #P<0.05 vs. the AAC only group; and &P<0.05 vs. the AAC + EPO group. H&E, hematoxylin and eosin; AAC, abdominal aortic constriction; EPO, erythropoietin; TLR4, Toll-like receptor 4.

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  Figure 3. Effects of EPO overexpression on the serum levels of inflammatory mediators in rats that underwent AAC. ELISA was employed to determine the levels of inflammatory mediators (A) TGF-β1, (B) TNF-α, (C) IL-1β, (D) IL-6 and (E) IL-17A in the rat serum in each group. The data are expressed as the mean ± standard error of the mean (n=6 per group). *P<0.05 vs. the control group; #P<0.05 vs. the AAC only group; and &P<0.05 vs. the AAC + EPO group. EPO, erythropoietin; AAC, abdominal aortic constriction; TGF, transforming growth factor; TNF, tumor necrosis factor; IL, interkleukin; TLR4, Toll-like receptor 4.

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  Figure 4. Effects of EPO overexpression on gene expression of TLR4, PI3K and Akt1 in different groups. Relative mRNA expression levels were examined in the heart tissues in each group. The data are expressed as the mean ± standard error of the mean (n=6 per group). *P<0.05 vs. the control group; #P<0.05 vs. the AAC only group; and &P<0.05 vs. the AAC + EPO group. EPO, erythropoietin; TLR4, Toll-like receptor 4; PI3K, phosphoinositide 3-kinase; Akt1, AKT serine/threonine kinase 1; AAC, abdominal aortic constriction.

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  Figure 5. Effects of EPO overexpression on the protein levels of TLR4 in hearts of rats subjected to AAC. The protein expression levels of TLR4 were determined by western blot analysis. The data are expressed the mean ± standard error of the mean (n=6 per group). *P<0.05 vs. the control group; #P<0.05 vs. the AAC only group; and &P<0.05 vs. the AAC + EPO group. EPO, erythropoietin; TLR4, Toll-like receptor 4; AAC, abdominal aortic constriction.

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  Figure 6. (A and B) Effects of EPO overexpression on the protein levels of PI3K, Akt and p-Akt in hearts of rats subjected to AAC. Western blot analysis was performed to determine the protein levels of PI3K, Akt and p-Akt. The data are expressed the mean ± standard error of the mean (n=6 per group). *P<0.05 vs. the control group; #P<0.05 vs. the AAC only group; and &P<0.05 vs. the AAC + EPO group. EPO, erythropoietin; Akt, AKT serine/threonine kinase 1; p-, phosphorylated; AAC, abdominal aortic constriction; TLR4, Toll-like receptor 4.

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  Figure 7. Effects of EPO overexpression on the protein expression levels of TGF-β1 in hearts of rats subjected to AAC. Western blot analysis was performed to determine the protein expression levels of TGF-β1. The data are expressed the mean ± standard error of the mean (n=6 per group). *P<0.05 vs. the control group; #P<0.05 vs. the AAC only group; and &P<0.05 vs. the AAC + EPO group. EPO, erythropoietin; TGF, transforming growth factor; AAC, abdominal aortic constriction; TLR4, Toll-like receptor 4.

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  Figure 8. (A and B) Effects of EPO overexpression on the protein expression levels of MMP-9 and MMP-2 in the hearts of rats who underwent AAC. The protein expression levels of MMP-9 and MMP-2 were determined by western blot analysis. The data are expressed the mean ± standard error of the mean (n=6 per group). *P<0.05 vs. the control group; #P<0.05 vs. the AAC only group; and &P<0.05 vs. the AAC + EPO group. EPO, erythropoietin; MMP, matrix metal-loproteinase; AAC, abdominal aortic constriction; TLR4, Toll-like receptor 4.