NOX1 promotes myocardial fibrosis and cardiac dysfunction via activating the TLR2/NF-κB pathway in diabetic cardiomyopathy

Figures

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  FIGURE 1. Expression of NOX1 was upregulated in DCM. (A) NOX1 mRNA level in cardiac tissues (n = 3). (B) Expression of NOX1 protein in heart tissues was detected and quantified (n = 6). (C) mRNA level of NOX1 in cardiac fibroblasts with or without HG treatment (n = 3). (D) Expression of NOX1 protein in cardiac fibroblasts was detected and quantified (n = 4). (E) mRNA level of Col I and α-SMA in the heart (n = 3). (F) Expression of Col I and α-SMA was detected and quantified (n = 3). * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001.

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  FIGURE 2. NOX1 inhibition protected against diabetes-induced cardiac dysfunction in vivo. (A) Graph summarized body weight from each experimental group. (B) Blood glucose concentration. (C) Ratio of heart weight to body weight (HW/BW). (D) Ratio of heart weight to tibia length (HW/TL). (E) Representative echocardiography image of the four groups. (F–I) Echocardiographic parameters (n = 6 per group). * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001.

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  FIGURE 3. NOX1 inhibition suppressed myocardial fibrosis in DCM rats. (A) HE staining of the myocardium. (B–C) Masson’s trichrome staining of the myocardium and perivascular regions. (D–E) Quantification of collagen in the myocardium interstitial and perivascular regions. (F) Immunostaining of Col I protein expression. (G) Detection of expressed α-SMA protein in myocardial tissue was performed with a fluorescence microscope. (H) Gene expression levels of Col I, Col III, α-SMA, and TGF-β1 in each group. (I) Protein levels of Col I, Col III, α-SMA, and TGF-β1, and quantitative analysis in cardiac tissues (n = 3 per group). * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001.

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  FIGURE 4. NOX1 inhibition mitigated the TLR2/NF-κB-mediated response in diabetic myocardium. (A) STRING database showed the potential of candidate proteins acting downstream of NOX1. (B) Correlation of NOX1 expression with TLR2 expression in DCM rats’ cardiac tissues was analyzed by Pearson’s correlation coefficient (n = 6). (C) Gene expression levels of TLR2. (D) Protein levels of TLR2, n-NF-κB (p65), and the p-IKBα/IKBα ratio and quantitative analysis (n = 3). * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001.

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  FIGURE 5. NOX1 promoting fibrosis and oxidative stress in CFs. (A,G) Representative image of α-SMA expression (red) in myocardial cells by immunofluorescence. The nuclei were identified by DAPI (blue). (B,H) Gene expression levels of Col I, Col III, α-SMA, and TGF-β1. (C,I) Protein levels of Col I, Col III, α-SMA, and TGF-β1, and quantitative analysis in each group. (D,J) Intracellular ROS level was detected by DCFH-DA staining. (E,K) Quantitative result of the ROS level. (F,L) Level of MDA in the indicated group (n = 3 per group). * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001.

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  FIGURE 6. NOX1 regulated the activation of the TLR2/NF-κB pathway in the HG-induced CFs. (A,C,E) Gene expression levels of TLR2. (B,D,F,J) Protein levels of TLR2, n-NF-κB (p65), and the p-IKBα/IKBα ratio and quantitative analysis. (G) Representative image of α-SMA expression (red) in myocardial cells by immunofluorescence. The nuclei were identified by DAPI (blue). (H) Gene expression levels of Col I, Col III, α-SMA, and TGF-β1. (I) Protein levels of Col I, Col III, α-SMA, and TGF-β1, and quantitative analysis in each group (n = 3 per group). * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001.

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  FIGURE 7. Mechanism of NOX1 promoting myocardial fibrosis via activating the TLR2/NF-κB pathway in diabetic cardiomyopathy.