Rivaroxaban, a direct inhibitor of coagulation factor Xa, attenuates adverse cardiac remodeling in rats by regulating the PAR-2 and TGF-β1 signaling pathways

Figures

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  Figure 1. RIV and FSLLRY improves cardiac morphological changes in MI Rats. (A) Experimental scheme whereby rats were subjected to LAD surgery or a sham procedure for four weeks. Eight-week-old rats were treated with RIV or FSLLRY until harvesting. (B) The overall appearance of the rat heart. T scale =1 cm. (C) Representative images of H&E and Masson’s trichrome staining in hearts after four weeks, scale bar = 50 µm. (D) Quantitative analysis of collagen deposition in the left ventricle; n = 3; ***P < 0.001, Sham vs LAD; ###P < 0.001, LAD vs LAD+RIV; △△△P < 0.001, LAD vs LAD+FSLLRY. IG, intragastrical; IP, intraperitoneal; WB, western blotting; IHC, immunohistochemical.

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  Figure 2. RIV and FSLLRY improved cardiac function and hemodynamics in MI Rats. (A) Hemodynamic assessments of rats in each group. (B) Quantitative analysis of hemodynamic parameters. n = 3; ***P < 0.001, **P < 0.01, Sham vs LAD; ##P < 0.01, LAD vs RIV; △△△P < 0.001, △△P < 0.01, △P < 0.05, LAD vs FSLLRY. (C) M-mode echocardiography of rats in each group. (D) Quantitative analysis of left ventricular EF and FS determined by echocardiography. n = 4; ***P < 0.001, Sham vs LAD; ##P < 0.01, LAD vs LAD+RIV; △△P < 0.01, △P < 0.05, LAD vs LAD+FSLLRY.

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  Figure 3. RIV and FSLLRY attenuated myocardial fibrosis in MI Rats. (A) Representative images of immunohistochemistry of α-SMA. (B) Representative images of fibrosis-related proteins in the Sham and LAD group treated with RIV and FSLLRY detected by western blotting. n = 3; (C–E) Light density assessment of fibrosis-related proteins in the Sham and LAD group treated with RIV and FSLLRY detected by western blotting. n = 3; ***P < 0.001, **P < 0.01, Sham vs LAD; ##P < 0.01, #P < 0.05, LAD vs LAD+RIV; △△P < 0.01, LAD vs LAD+FSLLRY.

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  Figure 4. RIV inhibited Ang II-induced proliferation in CFs. (A) Schematic representation of in vitro experiments. (B) CCK-8 assay detected the CFs viability after induction with different concentrations of RIV. (C) CCK-8 assay detected the cell proliferation of Ang II-induced CFs after pretreatment with different concentrations of RIV. (D) CCK-8 assay detected the cardiomyocytes viability after induction with different concentrations of RIV. (E) CCK-8 assay detected the cardiomyocytes viability after induction with different concentrations of FSLLRY.

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  Figure 5. RIV inhibited Ang II-induced proliferation in CFs. (A) The expression of α-SMA was detected by immunofluorescence in the in vitro experiments. (B) Quantitative analyses of fluorescent signals for α-SMA. n = 3; ** P < 0.01, * P < 0.05, Control vs AngII; ##P < 0.01, #P < 0.05, AngII vs RIV+ AngII; △△P < 0.01, △P < 0.05, AngII vs FSLLRY+AngII. ††P < 0.01, †P < 0.05, RIV vs RIV+ PAR2-AP+AngII. (C) The expression of Ki-67 was detected by immunofluorescence in the in vitro experiments. (D) Quantitative analyses of fluorescent signals for Ki-67. n = 3; *** P < 0.01, ** P < 0.01, * P < 0.05, Control vs AngII; ## P < 0.01, # P < 0.05, AngII vs RIV+ AngII; △△P < 0.01, △P < 0.05, AngII vs FSLLRY+AngII. ††P < 0.01, †P < 0.05, RIV vs RIV+ PAR2-AP+AngII.

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  Figure 6. RIV attenuated Ang II-induced fibrosis in CFs. (A) Real-time quantitative PCR analysis of fibrosis-related genes in the vitro experiments. n = 3; **P < 0.01, *P < 0.05, Control vs AngII; ##P < 0.01, #P < 0.05, AngII vs RIV+ AngII; △ △P < 0.01, △P < 0.05, AngII vs FSLLRY+AngII. ††P < 0.01, †P < 0.05, RIV vs RIV+ PAR2-AP+AngII. (B) Representative images of fibrosis-related proteins in the vitro experiments detected by western blotting. n = 3; (C–F) Light density assessment of fibrosis-related proteins in the vitro experiments. detected by western blotting. n = 3; * P < 0.05, Control vs AngII; ##P < 0.01, #P < 0.05, AngII vs RIV+ AngII; △P < 0.05, AngII vs FSLLRY+ AngII. †P < 0.05, RIV vs RIV+ PAR2-AP+AngII.

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  Figure 7. RIV inhibited TGFβ-related signaling pathways in MI rats. (A) Representative images of immunohistochemistry of PAR-2 and TGF-β1. (B) Representative images of PAR-2, TGF-β1, p-Smad2, p-Smad3 proteins detected by western blotting. (C–F) Light density assessment of PAR-2, TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3 in the Sham and LAD group treated with RIV and FSLLRY detected by western blotting. n = 3; **P < 0.01, Sham vs LAD; ##P < 0.01, #P < 0.05, LAD vs LAD+RIV; △ △△P < 0.001, △△P < 0.01, △P < 0.05, LAD vs LAD+FSLLRY.

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  Figure 8. RIV inhibited TGFβ-related signaling pathways in CFs. (A) Representative images of PAR-2, TGF-β1, p-Smad2, p-Smad3 proteins in the in vitro experiments detected by western blotting. (B–E) Light density assessment of PAR-2, TGF-β1, p-Smad2/Smad2, p-Smad3/Smad3 in the in vitro experiments detected by western blotting. n = 3; ***P < 0.001, **P < 0.01, Control vs AngII; ##P < 0.01, AngII vs RIV+ AngII; △△△P < 0.001, △△P < 0.01, AngII vs FSLLRY+AngII. †P < 0.05, RIV vs RIV+ PAR2-AP+AngII.