| Background:Myocardial fibrosis is a cardiac dysfunction disease,which is characterized bythe features such as the cell over-proliferation,deposition of extracellular matrix,accumulation of fibrillar collagens and the increased myocardial hardness.Additionally,the fibrosis develops from the erivascular area to the intermyocardium with a diffuse distribution pattern is also the main character.Traditionally,myocardial fibrosis occurs as a result of hypertension,ischemic injury,valvular,or heart disease,and left ventricular diastolic dysfunction is the major cause fibrosis,which finally resulted in heart failure.It has reported that the dysfunction of renin-angiotensin-aldosterone system(RAAS)is involved in the mechanism of myocardial fibrosis;in addition,inflammation reaction,oxidative stress,endothelial dysfunction,the anomalies of intracellular calcium concentration,and the abnormal of cytokines are all mediating myocardial fibrosis.However,due to the complex pathogenic mechanism,there is still no effective therapy method.Thrombin,a serine protease,is converted from soluble fibrinogen into fibrin in coagulation reactions.In addition to mediate the normal coagulation process of the body,thrombin is also acted as an important activator of platelets,and regulates the activity of platelet and inflammation response.Generally,thrombin works to promote inflammation and fibrosis,and it was activated in several diseases,such as tissue fibrosis,angiogenesis and thrombus.Thus,to regulate the activity of thrombin is important in various diseases.Functionally,thrombin cleaves an N-terminal domain to activate protease-activated receptors(PARs)on the cell surface to relay signaling cascades,such as activates the endothelial cells and immune cells for different biological responses.Protease activated receptor-1(PAR-1)mediates various physiology processes,such as tumor growth,cell apoptosis,inflammation response,tumor invasion and angiogenesis,as well as the toxic thrombin signaling pathway.It has been shown that PAR-1 is highly expressed in cardiac fibroblasts in adult rats and is involved in regulating myofibroblast transformation activation and the increasing in collagen(? and ?)synthesis.Studies indicate that thrombin could combine with PAR-1 and further activates a PAR-1-dependent pathway,and thus participateing various cellular responses.The Gq protein is an important protein in various signaling pathways,and plays an important role in signal transduction of cell transmembrane.In addition,Gq protein can couple various receptors to promote the activation of Gq,and thus activates the Protein kinase C in biological process.PKC plays an important role in fibrosis development in fibroblasts as well as in the thrombin induced myocardial fibrosis.New anticoagulant is a kind of targeted drugs;it suppresses the thrombosis formation by directly reducing the activity of thrombin.The drugs were considered as the important therapeutic drugs for myocardial fibrosis due to the goal-oriented trait,and few harm on the other normal tissues and organs.The mechanism of new anticoagulant is that the drugs suppressed the two target factor Xa and IIa in coagulation cascade.The coagulation factor Xa is the intersection of exogenous and endogenous coagulation pathways;it could activate prothrombin by formulating phospholipid complex on the surface of phospholipid membrane,and thus formulating thrombin-antithrombin complex.Apixaban is the direct inhibitor of Xa;it could prevent the transformation of thrombin form prothrombin by combining the active site of Xa,and further plays an important role in blood anticoagulant mechanism.While whether Apixaban mediates the progress of myocardial fibrosis by targeting thrombin is still unclear.In our present study,the ischemia induced myocardial fibrosis mouse models were received treatments of Apixaban with different concentration,meanwhile,Benazepil treated mice were acted as positive control,the myocardial fibrosis level,blood pressure,myocardium index,collagen accumulation,thrombin activity and PAR-1 expression were detected;secondly,the myocardial fibroblasts model treated by thrombin with different concentration were used to explore the protectiveness of Apixiban on the collagen deposition of myocardial fibroblasts.Finally,the inhibitor of Gq and PKC were used to verify the protective mechanism of Apixaban on myocardial fibroblasts in vitro,in addition,in vivo experiment was also performed to further verify the protective mechanism of Apixiban on myocardial fibrosis.The aim of this study was to clarify the protective effectiveness of Apixiban on myocardial fibrosis;the study provided experimental basis and theoretical guidance for the clinical application of Apixiban.Objectives:To investigate the protective effect of Apixaban on myocardial function of myocardial fibrosis in mice and its regulation on collagen deposition and thrombin activation.Then we investigate the molecular mechanism of the protective effects of Apixaban on ischemia-induced myocardial fibrosis in vitro,and verify the effects of Gq/PKC singlaing pathway on collagen deposition and ischemia-induced myocardial fibrosis.Methods:(1)?A mouse model of myocardial fibrosis was established and treated with different concentration of Apixaban,the mice treated with Benazepil were considered as control;?The levels of myocardial fibrosis were detected using immunohistochemical;?The left ventricular systolic pressure(LVSP)and left ventricular end-diastolic pressure(LVEDP)were detected using Automatic biochemical analyzer;?The mice weight,Left Ventricular and Right Ventricular were weighted to calculate the Left Ventricular Weight Index(LVWI)and Right Ventricular Weight Index(RVWI);? Real-time PCR and Western blot were used to detect the expression of Col1a1,Col1a3 and PAR-1;?ELISA was performed to detect the activity of thrombin.(2)?The primary passage of myocardial fibroblasts were isolated from the mice,the thrombin with different concentrations were used to treat the fibroblasts;?Construction of Gqknockdown in mycaridial fibroblasts;? Immunofluorescence was performed to detect the levels of Collal and Colla3 in myocardial fibroblasts;?Real-time PCR and Western blot were carried out to detect the expression of Col1a1 and Col1a3;?Western blot was performed to detect the activation of protein kinase C(PKC)signaling pathway.(3)?Western blot was performed to detect the protein expression of Col1a1,Col1a3,and PKC signaling activation;?Mice were divided into 5 groups,including sham,myocardial ischemia(MI),MI+Apixaban,MI+Api+GF and MI+Api+GPA;?The myocardial fibrosis was detected using immunohistochemisty;?The left ventricular systolic pressure(LVSP)and left ventricular end-diastolic pressure(LVEDP)were detected using Automatic biochemical analyzer;?The mice weight,Left Ventricular and Right Ventricular were weighted to calculate the Left Ventricular Weight Index(LVWI)and Right Ventricular Weight Index(RVWI);? Real-time PCR and Western blot were used to detect the expression of Col1a1,Co1a3 and PAR-1.Results:(1)The fibrosis level was significantly increased in myocardial fibrosis mice compared with sham group,Apixaban treatments significantly alleviated fibrosis,and high level of Apixaban showed same effect with Benazepil;The level of LVSP was significantly decreased,while LVEDP was increased in myocardial fibrosis mice compared with sham group,Apixaban treatments significantly ameliorates the blood pressure,and high level of Apixaban showed same effect with Benazepil;The levels of LVWI and RVWI were increased in myocardial fibrosis mice compared with sham group,Apixaban treatments significantly ameliorates the myocardial index,and high level of Apixaban showed same effect with Benazepil;The expression of Collal and Colla3 was increased in myocardial fibrosis mice compared with sham group,Apixaban treatments significantly decreased it,and high level of Apixaban showed same effect with Benazepil;The activation of thrombin was increased in myocardial fibrosis mice compared with sham group,Apixaban treatments significantly decreased it,and high level of Apixaban showed same effect with Benazepil;Apixaban did not change the level of PAR-1.(2)The Collagen depositon were increased in myocardial fibroblasts after treated with thrombin;The expressions of Collal and Colla3 were increased afterthrombin treatment in a concentration-dependent manner;The PKC signaling was activated after thrombin treatment in a concentration-dependent manner;Gqknockdown significantly decreased the levels of Collal,Colla3 and PKC activation induced by thrombi in mycardium fibroblasts.(3)GPA2A or GF109203X significantly reversed the overexpressed Collal,Colla3 and p-PKCa thatinduced by thrombin in myocardial fibroblasts;Apixaban significantly decreased myocardial fibrosis in mice,which was inhibited by GPA2A or GF109203X.Apixaban significantly promoted the level of LVSP,but decreased LVEDP in mice with myocardial fibrosis,which were inhibited by GPA2A or GF109203X.Apixaban significantly decreased the levels of LVWI and RVWI in mice with myocardial fibrosis,which were inhibited by GPA2A or GF109203X.Conclusion:(1)Apixaban could significantly ameliorate ischemia-induced myocardial fibrosis.(2)Thrombin promoted collagen deposition in myocardial fibroblasts by promoting the activation of PKC and Gq.(3)Gq/PKC signaling pathway mediated the mechanism of Apixaban on myocardial fibrosis. |
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