| Part 1: Effect of rivaroxaban on clinical outcomes in patients with heart failure at high risk of MACEs: a retrospective case-control studyPurpose: Patients with heart failure(HF)and left ventricular thrombus(LVT)face Significantly increased risk of major cardiovascular adverse events(MACEs).This part aims to explore the impact of rivaroxaban and vitamin K antagonists(VKA)on the clinical outcomes of such HF patients with high MACEs risk.Methods: 2818 HF patients admitted to the China-Japan Union Hospital of Jilin University from January 2017 to June 2021 were included.Use echocardiography to screen patients with HF and LVT.The primary endpoint was a composite of major adverse cardiovascular events(MACEs),including a composite assessment of all-cause mortality,systemic embolism,and rehospitalization for cardiovascular events after LVT diagnosis.Secondary endpoints included LVT dissolution and the effect of rivaroxaban and VKA on the incidence of bleeding events.Results: Among 2818 HF patients,198 patients(7.0%)were diagnosed with LVT,among which 82.8% of LVT patients had acute myocardial infarction(AMI).There were 109 patients treated with rivaroxaban,78 patients in the VKA treatment group,and 11 patients received other anticoagulants.After a median follow-up of 17.0 months(interquartile range: 6.0-24.0 months),the incidence of MACEs was higher in both the rivaroxaban and VKA groups(49.5% vs.57.7%).However,the rivaroxaban group had lower rates of MACEs(adjusted HR: 0.636;95% CI: 0.418-0.970;P = 0.035)and systemic embolic events(4.6% vs.12.8%;adjusted HR: 0.318;95% CI: 0.108-0.933;P = 0.037)were significantly lower than those in the VKA group,and the difference was statistically significant.There was no significant difference in LVT dissolution rate between the two groups(59.6% vs.70.6%;adjusted HR: 1.303;95% CI: 0.898-1.890;P = 0.163;Gray test P = 0.073).In addition,there was no significant difference in the incidence of bleeding events defined by ISTH between the two groups(7.3% vs.6.4%;adjusted HR: 1.124;95% CI: 0.368-3.437;P = 0.083;Gray test P = 0.567).Conclusion: In this observational cohort study,patients with HF and LVT had a higher incidence of MACEs.Compared with VKA,rivaroxaban showed significant advantages in reducing the incidence of MACEs and systemic embolic events.At the same time,rivaroxaban has a similar LVT dissolution rate to VKA without increasing bleeding events.Part 2: Rivaroxaban reduces myocardial pyroptosis and inflammatory response through PAR2 inhibition of ROS/TXNIP/NLRP3 pathway in early stage of heart failure after myocardial infarction.Purpose: The results of the first part of the study suggest that the coagulation factor Xa(Factor Xa,FXa)inhibitor rivaroxaban has clinical benefits other than anticoagulation,but the mechanism of this benefit is unclear.Adverse ventricular remodeling is a sign of HF progression.Myocardial pyroptosis caused by inflammation after AMI is one of the important factors in early ventricular remodeling in HF.This part of the study aims to explore whether rivaroxaban can reduce ROS/TXNIP/NLRP3-mediated cardiomyocyte pyroptosis and inflammatory response through protease-activated receptor 1(PAR2)and improve adverse ventricular remodeling in the early stage of HF after AMI.Methods: Permanent ligation of the anterior descending coronary artery was used to establish HF post MI rat model.Randomly divided into sham operation group(Sham group),sham operation + rivaroxaban group(Sham + Riv group),early heart failure after myocardial infarction group(MI-HFE group),myocardial infarction + rivaroxaban group(MI-HFE + Riv Group).Immediately after surgery,rivaroxaban(3 mg/kg)or normal saline was administered intragastrically for 3 days.H&E staining,transmission electron microscopy,ROS and LDH level detection,immunohistochemistry and western blotting were used to evaluate the pathological morphology,inflammatory response and myocardial cell pyroptosis of myocardial tissue.In vitro experiments were conducted on H9C2 cells stimulated by hypoxia to observe the effect of rivaroxaban on cardiomyocyte pyroptosis and explore its potential mechanism.Results: Rivaroxaban effectively improves the pathological morphology of myocardial tissue in the early stage(3 days)of HF after AMI in rats,as shown in: myocardial fibers are arranged more orderly,swelling is reduced,and pathological phenomena such as myocardial cell degeneration and cytoplasmic vacuolization are alleviated.,inflammatory cell infiltration is reduced.Rivaroxaban effectively improves the myocardial injury and inflammatory response in the early stage(3 days)of HF in rats after AMI.The performance is as follows: rivaroxaban inhibits the inflammatory factors IL-1β,IL-1β,and inflammatory factors in the serum of rats in the MI-HFE group(3 days).IL-18 release,and reduced inflammatory cell infiltration in infarcted myocardial tissue.Rivaroxaban significantly reduced LDH levels in serum and primary cardiomyocytes stimulated by hypoxia in rats in the MI-HFE group,reduced ROS levels and improved oxidative stress.Western blot results showed that rivaroxaban significantly inhibited PAR2 and pyroptosis-related proteins(NLRP3,ASC,GSDMDN,Cleaved caspase-1,Cleaved IL-18,Cleaved IL-The expression of 1β)was upregulated.The results of immunofluorescence double staining of myocardial tissue and CO-IP experiment of cardiomyocytes showed that rivaroxaban can promote the dissociation of TXNIP/NLRP3 complex.In vitro experiments,overexpression of PAR2 in cardiomyocytes reversed the inhibitory effects of rivaroxaban on the above pyroptosis markers and the TXNIP/NLRP3 pathway.Rivaroxaban and the reactive oxygen species inhibitor can improve the accumulation of intracellular ROS levels caused by hypoxia,while PAR2 overexpression reverses this phenomenon.Conclusion: In the early stage of HF after AMI,rivaroxaban decreased ROS levels by inhibiting the expression of PAR2 protein,thereby inhibiting the activation of TXNIP/NLRP3 complex and down-regulating the expression and activation of Caspase-1 and GSDMD.Reducing the expression of pro-inflammatory factors IL-18 and IL-1β alleviates myocardial pyroptosis and inflammation to exert anti-ventricular remodeling effect.Part 3: Rivaroxaban reduces myocardial fibrosis through PAR2 inhibition of TGF-β1/smads pathway in late stage of heart failure after myocardial infarction.Purpose: The exacerbation of fibrosis in the late stages of AMI reduces ventricular compliance,accelerating ventricular remodeling and the progression of HF.This part of the study aimed to explore whether rivaroxaban alleviates TGF-β1-mediated cardiomyocyte fibrosis response through PAR2 and improves ventricular remodeling in the early stage of HF after AMI.Methods: The in vivo experiment used the same rat model and administration method as in the second part,but the administration time was extended to 28 days to simulate the pathological process of late-stage HF after clinical AMI.Changes in cardiac function and cardiac structure were evaluated by transthoracic echocardiography and left heart catheterization.H&E staining and Masson staining were used to observe myocardial histology and fibrosis.Immunohistochemistry and Western blot techniques were used to detect the protein expression levels of fibrosis markers in ventricular tissue.In vitro experiments were conducted to establish a myocardial fibrosis model by stimulating primary fibroblasts(CFs)of neonatal mice with Ang II,and to explore the potential mechanism of rivaroxaban in improving fibrosis by overexpressing PAR2 in CFs.CCK8,immunofluorescence staining and Western blot technology were used to evaluate the effect of rivaroxaban on CFs proliferation and fibrosis-related pathways.Results:Rivaroxaban significantly improved the cardiac function in the late stage(28 days)of HF rats after AMI,as shown by the improvement of cardiac function and hemodynamic indicators,including increased LVEF and LVFS as well as hemodynamic parameters LVESP and ± dp/dtmax.Western blot results showed that rivaroxaban significantly inhibited the expression of fibrosis-related proteins(CollagenⅠ,CollagenⅢ,α-SMA)induced by Ang II in late-stage HF rats after AMI.Rivaroxaban inhibits the expression of PAR2,TGF-β1 and their downstream Smad2/3 phosphorylated effector proteins in late-stage HF rats and Ang II-induced rats after AMI.In vitro experiments,overexpression of PAR2 in CFs reversed the effects of rivaroxaban on the above effects.Fibrosis markers and inhibition of the TGF-β1/smads pathway.Conclusion: In the late stage of HF after AMI,rivaroxaban inhibits the expression of PAR2 protein and the TGF-β1/smads signaling pathway,thereby reducing the synthesis of CollagenⅠ and CollagenⅢ proteins and inhibiting the differentiation of CFs into myofibroblasts(shown by inhibiting α-SMA expression),thereby improving myocardial fibrosis and exerting an anti-ventricular remodeling effect. |
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