| Myocardial fibrosis(MF)is a common pathological characteristic in the late stage of many cardiovascular diseases,such as myocardial infarction(MI),coronary heart disease and cardiomyopathy.How to prevent and treat MF is a tough but hot field in the medical research.Cardiac fibrosis after myocardial infraction(CFMI)is one of the most popular clinical types of MF.The critical pathomechanism of CFMI is that the major effector cells of CFMI,cardiac fibroblasts(CFs),would undergo changes in biological behaviors such as proliferation,phenotypic differentiation,migration and secretion after induction of oxidative stress,inflammatory and other pro-fibrotic factors,which as a result cause the massive deposition of extracellular matrix(ECM),reducing the cardiac systolic function and electrical signal transduction,leading to heart failure,arrhythmia and poor prognosis of MI patients.The AMPK-mediated Integrin ?1 signaling pathways may play an important role in CFs involved excessive deposition of ECM.CFMI may belong to categories of "chest numbness" or "lump" according to theories of traditional Chinese medicine(TCM),which is closely related to the pathogenesis of blood stasis.Our previous study found that Chinese herbal medicine of activating blood circulation could regulate AMPK-mediated metabolic pathways and optimize myocardial energy metabolism,at the same time,interfere with ECM proteins like collagen ?,collagen ?,osteopontin and tenascin,then inhibit ventricular remodeling and improve cardiac dysfunction after MI.However,the current study on expression profiles of ECM in CFMI is limited to individual proteins,and the key mechanisms of TCM of activating blood circulation on the treatment of CFMI are still unclear.Hirudo is a classic Chinese medicine of activating blood circulation and removing blood stasis.In the view of pharmacology,Hirudin,the most important and active constituents in Hirudo,has distinguished effects of anti-coagulant,anti-thrombotic,anti-platelet and anti-MF in cardiovascular disease.Recently,there are evidences demonstrating the anti-MF and reduction of myocardial infarction area effects of bivarudine(a derivative of Hirudo)and patent medicine containing Hirudo.But the research on the anti-MF efficacy and mechanism of Hirudin itself is lacking.So Our study is designed by using the label-free sub-proteomics technology to research the composition characteristics of ECM.which are the main pathological products of CFMI.After that,the rat model of acute MI and Ang II-induced neonatal CFs model were established,respectively.Expression levels of AMPK mediated Integrin ?1 and its downstream FAK/PI3K/Akt TGF-?1/Smad2/3 signaling pathways were observed to elucidate the mechanisms of Hirudin in regulating key ECM deposition during CFMI repair process.This study would be the scientific evidence of TCM superiority in the prevention,treatment and also improvement of the prognosis in patients with CFMI1 Literature reviewThe pathogenesis mechanisms,clinical diagnosis and intervention treatments of CFMI were expounded,focused on ECM reconstruction as the core pathological process.The potential functions of TCM active ingredients in the treatment of CFMI were highlighted.The Hirudo basic sources,records in ancient Chinese books,usage in Chinese patent medicines,as well as preparations for clinical application,and pharmacological mechanisms of Hirudin in treating cardiovascular diseases were reviewed as comprehensive as possible.The literature review here is to build the theoretical foundation for the following experimental research,which would focus on the intervention of Hirudin from Hirudo in CFMI treatment and prevention2 Experiment researchPart 1 Expression profiles and bio-informatics analysis of ECM proteins in fibrotic myocardium after CFMIObjective:To analyze the components of ECM proteins and the interaction mechanisms within these differential proteins in fibrotic myocardium of CFMI rats model by label-free sub-proteomicsMethods:CFMI rats model was established by ligation of the left anterior descending coronary artery,then randomly divided into three groups:Sham(Sham),CFMI after 2 weeks(Model 2W)and CFMI after 4 weeks(Model 4W),with 10 rats in each group.Changes in ECM differential proteins after CFMI were analyzed using lable-free sub-proteomics.GO analysis was used to describe the characteristics of cellular component,molecular functions and biological processes of ECM proteins KEGG analysis was used to find the potential underline pathways in regulating expressions of these ECM differential proteins.Finally,the screened out ECM differential proteins were verified by Western Blot(WB).Results:(1)A total of 243 ECM differential proteins were identified.Compared with Sham.Osteoglycin was up-regulated and Nidogen-1 was down-regulated in Model 2W;Lumican and Collagen type VI alpha 2 chain(Collagen 6A2)were up-regulated while Nidogen-1 was down-regulated in Model 4W.Compared with Model 2W,Nidogen-1 and Talin-1 were down-regulated in Model 4W(P<0.05).Bio-informatics analysis showed that the cellular component of ECM differential proteins were significantly enriched in extracellular regions,extracellular matrix and other cell components.Molecular functions such as receptor binding,metal ion binding and Integrin binding were enriched.Biological processes such as immune regulation,protein transportation,membrane organization,intercellular signal transduction and cell adhesion were enriched.KEGG analysis showed that focal adhesion kinase(FAK).phosphatidylinositol-3-hydroxykinase(PI3K)/protein kinase B(Akt)and interaction between Integrin and ECM receptor pathways were significantly regulated.(2)WB results showed that expressions of Collagen 6A2,Lumican and Talin-1 were up-regulated in Model 2W group when compared with Sham group(P<0.05);Collagen 6A2 and Lumican were significantly increased and Nidogen-1 was significantly decreased in Model 4W group when compared with Sham group(P<0.05).When compared with Model 2W group,the expressions of Nidogen-1,Lumican and Talin-1 were down-regulated in Model 4W group(P<0.05)The results involving Collagen 6A2 and Lumican were up-regulated while Nidogen-1 was down-regulated in Model 4W group when comparing Sham group,Nidogen-1 and Talin-1 were down-regulated in Model 4W group when comparing Model 2W group,were coincident between label-free sub-proteomics and WB detection.Conclusion:The ECM differential proteins including Collagen 6A2,Nidogen-1,Lumican.Talin-1 and Osteoglycin might be the possible targets of CFMI.The mechanisms of regulation in deposition of these ECM differential proteins may relate to FAK,PI3K/Akt and Integrin signaling pathways.Part 2 Investigation on the effects and mechanisms of Hirudin treatment in ECM protein expressions in CFMI ratsObjective:To observe the effects and mechanisms of Hirudin intervention on cardiac function and myocardial pathology via regulation of the differential ECM proteins deposition in CFMI rats.Methods:The left anterior descending coronary artery of Wistar rats was ligated to establish the CFMI model.Rats were randomly divided into four groups:Sham group(Sham,normal saline),Model group(Model,normal saline),Valsartan group(Valsartan.144 mg·kg-1)and Hirudin group(Hirudin.270 mg·kg-1).20 rats in each group were treated once a day for four weeks.Echocardiography parameters including left ventricular diameter(LVD),ventricular septal thickness(IVST),left ventricular posterior wall thickness(LVPWT),end diastolic/systolic volume(EDV/ESV)and left ventricular ejection fraction(LVEF)in both diastolic phase and systolic phase were detected.The serum levels of peripheral markers involving myocardial injury(CK-MB,cTnT),heart failure(ANP,BNP)and oxidative stress(Ang ?,MDA,SOD)were measured.TTC,HE,Masson and Sirius red staining were used to observe the morphology of infarction and fibrotic lesions.Immunohistochemical staining and WB detection were used to observe the deposition and expression of ECM differential proteins.The expression of AMPK,Integrin ?1,FAK,PI3K p110?,PI3K p110?,Akt,TGF-?1 and Smad2/3 in fibrotic signaling pathways were measured by WB.Results:(1)Compared with Sham group,IVST,LVPWT and LVEF were decreased,while LVD,EDV and ESV were increased in Model group(P<0.05),indicating a satisfying CFMI model was established.Compared with Model group,IVSTs.LVPWT and LVEF were increased,while LVD.EDV and ESV were decreased in Hirudin group(P<0.05).(2)Compared with Model group,the serum levels of CK-MB,cTnT,ANP,BNP,Ang ? and MDA were decreased,while SOD was increased significantly in Valsartan group and Hirudin group(P<0.05).(3)Compared with Model group,heart index,infarction area and collagen volume fraction were significantly reduced in Valsartan group and Hirudin group;peripheral collagen volume fraction was significantly decreased in Hirudin group(P<0.05).Compared with Model group,Collagen 6A2 and Lumican expression were decreased,while Nidogen-1 expression was increased in Valsartan group and Hirudin group(P<0.05).(4)Compared with Model group,protein levels of AMPK,FAK,PI3K p110? and Akt were up-regulated,while Integrin ?1,TGF-?1 and Smad2/3 were significantly down-regulated in Valsartan group and Hirudin group(P<0.05).PI3K p1110? protein expression showed no significant difference among Model group,Valsartan group and Hirudin group(P>0.05).Conclusion:Hirudin can relieve left ventricle dilation,improve cardiac function,reduce myocardial injury,alleviate heart index,decrease infarction area and interstitial collagen deposition,regulate deposition of different ECM proteins including Collagen 6A2,Lumican and Nidogen-1,so as to delay the progression of CFMI in rat model.The mechanism is related to the Hirudin regulation of AMPK/Integrin ?1/FAK signaling and PI3K/Akt,TGF-?1/Smad2/3 pathways in CFMI rats.Part 3 Investigation on the effects and mechanisms of Hirudin in regulating ECM protein expressions in Ang ?-induced NRCFsObjective:To observe the effects and mechanisms of Hirudin on cellular biological behaviors,including morphology,phenotypic differentiation,migration and secretion of ECM proteins in Ang ? induced neonatal rat cardiac fibroblasts(NRCFs).Methods:The NRCFs were separated,extracted,cultivated and lastly identified via vimentin(+)/vWF(-)immunofluorescence staining.Different concentrations(10-9 mol·L-1,10-8 mol·L-1,10-7 mol·L-1,10-6 mol·L-1)and different times(6 h,12 h,24 h,48 h)of Ang II were given to NRCFs,then the vitality of cell proliferation in NRCFs were detected by CCK-8 to screen the appropriate concentration and time for Ang II induction of fibrotic model in vitro.NRCFs were randomly divided into four groups:Control group(Control),Model group(Ang ?),Valsartan group(Valsartan)and Hirudin group(Hirudin).After 30 min of pre-intervention with corresponding treatment,Ang II was employed to induce fibrotic NRCFs.Flow cytometry was used to observe cell cycles of NRCFs.Immunofluorescence staining of a-SMA was measured to quantify the phenotype transformation.Transwell assay and scratch wound assay were conducted to assess migration ability.The contents of hydroxyproline(HYP)and staining of ECM differential proteins were recruited to observe the intracellular ECM deposition.The protein expression levels of AMPK?1/?2,Integrin ?1,FAK,TGF-?1,Smad2/3 and Nidogen-1 were detected by WB.Results:(1)After 24 h culture of NRCFs.the ratio of NRCFs with vimentin(+)/vWF(-)was(98.09±1.03)%.Considering the OD values of CCK-8 assay,Ang ?10-6 mol·L-1 for 24 h was selected as the ideal stimulation to induce fibrotic changes in NRCFs.(2)Compared with Ang ? group,the percentage of cells in quiescent G0G1 phase was significantly increased,cells in proliferative S phase was significantly decreased in Valsartan group and Hirudin group;the number of cells with positive immunofluorescence staining of ?-SMA and migrating through the Transwell chamber were significantly reduced in Valsartan group and Hirudin group;the scratch width was significantly extended in Valsartan group and Hirudin group(all P<0.05).(3)Compared with Ang II group,the contents of HYP in supernate was reduced in Valsartan group and Hirudin group;the fluorescence intensity of Lumican and Collagen 6A2 were decreased in Valsartan group and Hirudin group,while Nidogen-1 and Talin-1 were increased in Hirudin group(P<0.05).(4)Compared with Ang ? group,the protein levels of AMPK?1/?2 and FAK were up-regulated,while Integrin ?1 was down-regulated in Valsartan group and Hirudin group;the expressions of TGF-?1 and Smad2/3 were decreased in Valsartan group(P<0.05).Compared with Ang II group,expressions of TGF-?1,Smad2/3 in Hirudin group,Nidogen-1 in Valsartan group and Hirudin group showed no difference(P>0.05).Conclusion:Hirudin can inhibit Ang ?-induced pro-fibrotic changes in NRCFs,involving cell proliferation,phenotypic differentiation,cell migration and secretion of ECM differential proteins.The mechanism of optimized NRCFs abilities in dealing with Ang ?-induced fibrogenesis is related to the regulation of AMPK/Integrin(31/FAK signaling pathways. |
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