(Pro)renin receptor aggravates myocardial pyroptosis in diabetic cardiomyopathy through AMPK-NLRP3 pathway

Figures

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  FIGURE 1. A flow chart of groups of animals and various treatments. (A) The flow chart of groups of animals and various treatments in the PRR overexpression experiment. (B) The flow chart of groups of animals and various treatments in the PRR silencing experiment.

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  FIGURE 2. Pyroptosis in diabetic cardiomyopathy is aggravated by the overexpression of PRR. (A–D) Immunohistochemistry staining was used to detect the level of PRR, Caspase-1, IL-1β, IL-18 and corresponding quantitative analysis. Scale bar: 100 μm. (E) Western blot was used to detect the level of PRR, Caspase-1, IL-1β, IL-18 and corresponding quantitative analysis. (F) The level of IL-1β and IL-18 in serum were detected by ELISA. * P < 0.05, **P < 0.01, compared with the Control group; # P < 0.05, ## P < 0.01, compared with the Ad-EGFP group.

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  FIGURE 3. The overexpression of PRR increases the activation of NLRP3 inflammasome, and decreases the phosphorylation of AMPK in DCM. (A) The level of NLRP3 in myocardial tissue was detected by immunohistochemistry staining and corresponding quantitative analysis. Scale bar: 100 μm. (B) The level of PRR, NLRP3, p-AMPK, t-AMPK in myocardial tissue were detected by Western blot and corresponding quantitative analysis. *P < 0.05, **P < 0.01, compared with the Control group; # P < 0.05, ## P < 0.01, compared with the Ad-EGFP group.

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  FIGURE 4. PRR overexpression aggravates the levels of fibrosis in myocardial tissue in DCM. (A–C) The immunohistochemical staining was used to assess the level of Collagen-Ⅰ, Collagen-Ⅲ and TGF-β in myocardial tissue. Scale bar: 100 μm. (D) The Masson staining was used to observe the level of collagen fiber of rats. *P < 0.05, **P < 0.01, compared with the Control group; # P < 0.05, ## P < 0.01, compared with the Ad-EGFP group.

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  FIGURE 5. The overexpression of PRR worsens cardiac function of DCM rats. (A) The HE staining was used to observe the morphological changes of myocardial tissue. Scale bar: 100 μm. (B) The echocardiography was used to detect the change of cardiac function of rats. (C–F) Quantitative analysis of LVEF, LVESD, LVEDD, E/A ratio in rats. *P < 0.05, **P < 0.01, compared with the Control group; # P < 0.05, ## P < 0.01, compared with the Ad-EGFP group.

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  FIGURE 6. Pyroptosis in diabetic cardiomyopathy is reduced by the silencing of PRR. (A–E) Immunohistochemistry staining was used to detect the level of PRR, NLRP3, Caspase-1, IL-1β, IL-18 and corresponding quantitative analysis. Scale bar: 100 μm. (F) Western blot was used to detect the level of PRR, NLRP3, Caspase-1, IL-1β, IL-18 and corresponding quantitative analysis. (G) The level of IL-1β and IL-18 in serum were detected by ELISA. *P < 0.05, **P < 0.01, compared with the Control group; # P < 0.05, ## P < 0.01, compared with the Ad-SC-shRNA group.

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  FIGURE 7. The silencing of PRR reduces heart damage in DCM rats. (A–C) Immunohistochemistry staining was used to detect the level of Collagen I, Collagen III, TGF-β and corresponding quantitative analysis. Scale bar: 100 μm. (D) The Masson staining was used to measure the deposition of collagen fiber of rats and corresponding quantitative analysis. Scale bar: 100 μm. (E) The HE staining was used to observe the morphological changes of myocardial tissue. Scale bar: 100 μm. (F) The echocardiography was used to detect the change of cardiac function of rats. (G–J) Quantitative analysis of LVEF, LVESD, LVEDD, E/A ratio in rats. *P < 0.05, **P < 0.01, compared with the Control group; # P < 0.05, ## P < 0.01, compared with the Ad-SC-shRNA group.

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  FIGURE 8. High glucose stimulates the elevation of PRR expression in primary cardiomyocyte. (A) Western blot was used to detect the expression of PRR under the high glucose stimulation after 0, 12, 24, 36, 48 h and the corresponding quantitative analysis. (B) The immunofluorescence was used to observe the expression of PRR in three groups and corresponding quantitative analysis. (C) Western blot was used to assess the level of PRR after 48 h of high glucose stimulation. (D) Real-Time PCR was used to assess the mRNA level of NLRP3, Caspase-1, IL-1β and IL-18 after 48 h of high glucose stimulation. *P < 0.05, **P < 0.01, compared with the Control group; # P < 0.05, ## P < 0.01, compared with the HP group.

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  FIGURE 9. Overexpression of PRR increases pyroptosis of primary cardiomyocyte. (A) Western blot was used to detect the expression of PRR, NLRP3, Caspase-1, IL-1β and IL-18 and the corresponding quantitative analysis. (B) The ELISA was used to detect the content of IL-1β in cell culture supernatant of four groups. (C) The ELISA was used to detect the content of IL-18 in cell culture supernatant of four groups. *P < 0.05, **P < 0.01, compared with the Control group; # P < 0.05, ## P < 0.01, compared with the Ad-EGFP group.

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  FIGURE 10. PRR participates in the pyroptosis of primary cardiomyocyte through AMPK-NLRP3 pathway. (A) Western blot was used to detect the expression of p-AMPK in four groups and the corresponding quantitative analysis. *P < 0.05, **P < 0.01, compared with the Control group; # P < 0.05, ## P < 0.01, compared with the HG group and Ad-EGFP group. (B) Western blot was used to detect the expression level of p-AMPK, NLRP3, Caspase-1, IL-1β and IL-18 in four groups. *P < 0.05, **P < 0.01, compared with the Ad-PRR group; # P < 0.05, ## P < 0.01, compared with the Ad-EGFP group.

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  FIGURE 11. Inhibition of PRR reduces the pyroptosis of primary cardiomyocyte under high glucose stimulation. (A) Western blot was used to detect the expression of NLRP3, Caspase-1, IL-1β and IL-18 in four groups and the corresponding quantitative analysis. (B) The ELISA was used to detect the content of IL-1β in cell culture supernatant of four groups. (C) The ELISA was used to detect the content of IL-18 in cell culture supernatant of four groups. *P < 0.05, **P < 0.01, compared with the Control group; # P < 0.05, ## P < 0.01, compared with the Ad-SC-shRNA group.