A Study On The Changes Of Collagenic Metabolism And Comparison Of Antifibrotic Effects Of Oxymatrine And Captopril In Chronic Viral Myocarditis

Backgroud and objectivesAccording to the Dallas criteria,the chronic viral myocarditis (CVMC) is characterized by accumulation of connective tissue in heart muscle(fibrosis). CVMC is a frequent precursor to dilated cardiomyopathy in men. In general, fibrosis is characterized by substantial resident fibroblasts proliferation with subsequent deposition of the extracellular matrix (ECM) components like collagen, fibronectin or tenascin. But collagen is the most abundant protein in the mammals and the major component of the myocardial ECM. Myocardial fibrosis mainly results from the excessive accumulation of collagen. So it is very important to inhibit myocardial fibrosis in CVMC.A number of hormones, cytokines, and growth factors have been identified as important regulators of fibrosis. Matrix metalloproteinases (MMPs), the major regulators of collagen degradation, belong to a family of zinc-and calcium-dependent proteolytic enzymes. Generally synthesis and degradation of collagen, wound healing and tissue remodeling are maintained with the balance of MMPs and tissue inhibitors of metalloproteinases (TIMPs). A body of evidence suggests that the renin-angiotensin-aldosterone system (RAS) and transforming growth factor beta 1(TGF-β1) play essential roles in the progression of myocardial fibrosis. A distintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS-1) is a newly discovered metalloproteinase, originally cloned from a cancer cell line. It is characterized by an N-terminal metalloproteinase domain, a disintegrin-like domain and a carboxyl terminal region containing variable numbers of a repeat sequence with homology to thrombospondin-1. ADAMTS-1 binds the ECM through its C-terminal special regions and mediates cell-cell and cell-ECM interactions. Previous studies determined that ADAMTS-1 can cleave ECM components including cleave cartilage proteoglycans, aggrecan and versician. Lind et al. purified an insect derived recombinant human ADAMTS-1 protein and determined that ADAMTS-1 has novel gelatin (typeⅠcollagen) degrading activities. However, evidence for ADAMTS-1 function based on its pattern of expression is still very limited and has not been reported in vivo.TypeⅠcollagen is synthesized in the form of procollagen with additional sequences of amino acids at the N and C ends of its molecule. The carboxyterminal propeptide of typeⅠprocollagen (PICP) is released into the bloodstream during collagen typeⅠsynthesis. TypeⅢcollagen is synthesized in the form of its procollagen and the N-terminal propeptide of typeⅢprocollagen (PⅢNP) is released into the blood circulation during the conversion into collagen. So PICP and PⅢNP are considered degradation markers of typeⅠcollagen and typeⅢcollagen espectively.Carboxyterminal telopeptide of typeⅠcollagen (ICTP) is a degradation marker of typeⅠcollagen that is released in a 1:1 stoichiometric ratio with typeⅠcollagen molecules during collagen breakdown. So it is considered a degradation marker of typeⅠcollagen.Angiotensin converting enzyme inhibitors (ACEI) has been demonstrated to have protective effects on myocardial fibrosis. Its some side effects such as drop of blood pressure and persistent dry cough make some patients unendurable. Oxymatrine is one of the alkaloids extracted from Chinese herb Sophora japonica (Sophora flavescens Ait.). Current studies find oxymatrine has an activity of antihepatic fibrosis, with few side effects. There is to date no published studies about its effect on myocardial fibrosis.The aim of this study was to examine the changes of collagenic metabolism and compare the antifibrotic effects of oxymatrine and captopril in chronic viral myocarditis. Methods:1. CVB3 initiation of mouse CVMC model The mice were randomly divided into six groups:normal control group1 (group 1) (n=8), normal control group2 (group 2) (n=8), CVB3 CVMC groupl(group 3) (n=20), CVB3 CVMC group2 (group 4) (n=25), captopril therapy group (group 5) (n=25) and oxymatrine therapy group (group 6) (n=25). Specifically, mice in group 3, group 4, group 5 and group 6 were inoculated with CVB3 at days 0,14 and 28 using 0.20ml,0.25ml,0.30ml of CVB3; respectively. Mice in group 1 and group 2 received an equal-volume of normal saline without CVB3 at the 0,14, and 28 day time points.2. Echocardiographic analysis Mice in group 1 and group 3 were measured by Echocardiography at days 42 after inoculation. Mice were anesthetized with 3% chloralhydrate intrapritoneally (0.01～0.015ml/g). Transthoracic echocardiography was using PHILIPS 7500 (Phillips) equipped with 3.5 MHz linear transducer, parasternal long-axis and short-axis views of the left ventricle were obtained to measure heart rate (HR), left ventricular end-diastolic internal diameter (LVDD), left ventricular end-systolic internal diameter (LVSD), peak velocity of aorta (Vp) at least 3 consecutive cardiac cycles. The ejection fraction (EF) and fractional shortening (FS) were calculated respectively. Mice were killed after echocardiographic analysis. Peripheral blood was obtained by eye-ball excision, and heart was resected for the following examination. Following day 42, group 5 was treated with Captopril (100mg/kg) daily by gavage, group 6 was treated with oxymatrine (100mg/kg) daily by gavage for 28 days; concurrently group 2 and group 4 received daily equal-volume gastric perfusion of normal saline. Mice were anesthetized and measured by Echocardiography at days 70. Mice were killed after echocardiographic analysis. Peripheral blood was obtained by eye-ball excision, and heart was resected for the following examination to examine the changes of collagenic metabolism.3.To count the ratio of heart weight to body weight(HW/BW)4. Pathology examination by HE staining5.Morphometric analysis of collagen with picrosirius red staining 6. Calculation the collagen volume fraction (CVF) with image analysis software.7.The levels of serum PICP and PIIINP were detected quantitatively by ELISA method.8.The protein abundence for myocardial AngⅡ, ADAMTS-1, TGF-β1,MMP-1 and TIMP-1 were observed by immunohistochemical stain.9. Immunofluorescent Staining for myocardial ADAMTS-1 protein express10. The expressions of ADAMTS-1,TGF-β1, MMP-1 and TIMP-1 protein in myocardial tissue were characterized by Western blot analysis.11. Determination of the mRNA expression of ADAMTS-1, typeⅠcollagen,typeⅢcollagen and carboxyterminal telopeptide of typeⅠcollagen (ICTP) by RT-PCR method.12.To count the ratio of typeⅠcollagen to typeⅢcollagenResults:1.Two CVMC groups had significant increase in HW/BW compared to their normal control groups, but HW/BW of captopril therapy group and oxymatrine therapy group were markedly decreased compared with CVMC group2. HW/BW in two therapy groups had no difference.2. Compared with their normal control groups, the left ventricular end-diastolic internal diameters(LVDD), left ventricular end-systolic internal diameters(LVSD) and heart rates(HR) were significantly increased in two CVMC groups (P＜0.05, P<0.01, P<0.05, respectively), ejection fractions(EF), fractional shortenings(FS) and peak velocity of aorta(Vp) were all significantly decreased in the two CVMC groups. But LVDD, LVSD and HR were markedly decreased, and EF, FS, Vp were all significantly increased in captopril therapy group and oxymatrine therapy group compared with CVMC group2, while two therapy groups had no difference.3. Closely arranged muscle fibers and a few stromal fibroblasts were seen in normal control groups while two CVMC groups had more loosely arranged muscle fibers, hyperplasia of stromal fibroblasts and a minimal inflammatory cell infiltration. Pathological change of myocardium in captopril therapy group and oxymatrine therapy group were both obviously improved compared to CVMC group2. 4. In control hearts, thin collagen fibrils were found between closely arranged muscle fibers. Hearts from two CVMC groups mice had prominent, dense collagenous septa interspersed between more loosely arranged muscle bundles while collagen proliferation was markedly decreased in captopril therapy group and oxymatrine therapy group compared with CVMC group2. Two CVMC groups animals had significant increase in CVF compared to their normal control groups, while CVF in captopril therapy group and oxymatrine therapy group were markedly decreased compared with CVMC group2. But CVF in two therapy groups had no difference.5.TypeⅠcollagen, typeⅢcollagen levels and the ratio of TypeⅠcollagen to typeⅢcollagen in two CVMC groups were significantly lower than those in their normal control groups,while they were significantly increased in captopril therapy group and oxymatrine therapy group compared with CVMC group2. But typeⅠcollagen, typeⅢcollagen levels and the ratio of TypeⅠcollagen to typeⅢcollagen in two therapy groups had no difference.6.The levels of serum PICP and PⅢNP in two CVMC groups were significantly higher than those in their normal control groups,while they were significantly decreased in captopril therapy group and oxymatrine therapy group compared with CVMC group2. The ICTP level in two CVMC groups were significantly lower than those in their normal control groups,while they were significantly increased in captopril therapy group and oxymatrine therapy group compared with CVMC group2. But PICP, PⅢNP ICTP and levels in two therapy groups had no difference.7.The protein abundence for myocardial AngⅡand TGF-β1 were significantly increased in two CVMC groups compared to their normal control groups, and they were positively correlated with CVF level. The protein expression of AngⅡand TGF-β1 were significantly decreased in captopril therapy group compared to CVMC group2, while The AngⅡprotein expression in oxymatrine therapy group had no change, only TGF-β1 were significantly decreased compared to CVMC group2, and there were not different in TGF-β1 between captopril therapy group and oxymatrine therapy group.8.The protein abundence for myocardial MMP-1 was significantly decreased in two CVMC groups compared to their normal control groups, and it was negatively correlated with CVF level. The protein expression of TIMP-1 was markedly increased in two CVMC groups compared to their normal control groups, and it was positively correlated with CVF level. The protein expression of MMP-1 was significantly increased while TIMP-1 was obviously decreased in captopril therapy group and oxymatrine therapy group compared to CVMC group2. MMP-1 was positively correlated with ICTP level. The protein expression of MMP-1 and TIMP-1 in two therapy groups had no difference.9. ADAMTS-1 protein expression were significantly decreased two CVMC groups compared to their normal control groups, and it was negatively correlated with CVF level. The protein expression of ADAMTS-1 was significantly increased in captopril therapy group and oxymatrine therapy group compared to CVMC group2., and it was positively correlated with ICTP level. ADAMTS-1 protein expression in two therapy groups had no difference.10. AngⅡexpression was positively correlated with TGF-β1 and TIMP-1 levels, while it was positively negatively with MMP-1 and ADAMTS-1 levels in CVMC group2. TGF-β1 expression was positively correlated with TIMP-1 level, while it was also positively negatively with MMP-1 and ADAMTS-1 levels in CVMC group2. MMP-1 and ADAMTS-1 protein expressions were both negatively correlated with typeⅠcollagen and typeⅢcollagen levels, while TIMP-1 expression was positively correlated with typeⅠcollagen and typeⅢcollagen levels in CVMC group2. ADAMTS-1 protein expressions had no correlation with MMP-1 and TIMP-1.Conclusions:1.CVMC is characterized by accumulation of connective tissue in heart muscle. Myocardial fibrosis mainly results from the excessive accumulation of typeⅠcollagen and typeⅢcollagen and lack of proportion of typeⅠcollagen to typeⅢcollagen.2.The development of myocardial fibrosis is controlled by a regulatory network involving AngⅡand TGF-β1. AngⅡupregulates TGF-β1 expression in cardiac myocytes and fibroblasts and TGF-β1 acts downstream of AngⅡand promotes myocyte growth and fibrosis in the heart. They promote myocardial fibrosis by upregulating TIMP-1 expression and doownregulating MMP-1 and ADAMTS-1 expression.3. MMP-1 inhibits myocardial fibrosis via acceleration of typeⅠcollagen and typeⅢcollagen degradation.4. ADAMTS-1 also inhibits myocardial fibrosis via acceleration of typeⅠcollagen and typeⅢcollagen degradation, but ADAMTS-1 is another passway which is independent of MMP-1/TIMP-1 way.5.Captopril inhibits myocardial fibrosis via downregulation of AngⅡand TGF-β1 expression which can upregulating MMP-1 and ADAMTS-1 expression and doownregulating TIMP-1 expression.6.Oxymatrine has an antifibrotic effect equivalent to captopril in CVMC.7.The mechanism of oxymatrine antifibrotic effects might be related with the downregulation of TGF-β1 expression which can upregulating MMP-1 and ADAMTS-1 expression and doownregulating TIMP-1 expression.8.ADAMTS-1 is regulated by TGF-β1. AngⅡwhich acts upstream of TGF-β1 regulates ADAMTS-1 expression indirectly.Innovations and meanings:1.This study firstly demonstrated that ADAMTS-1 inhibited myocardial fibrosis via acceleration of typeⅠcollagen and typeⅢcollagen degradation, and ADAMTS-1 was another passway which was independent of MMP-1/TIMP-1 way. ADAMTS-1 was regulated by TGF-β1. AngⅡwhich acts upstream of TGF-β1 regulateed ADAMTS-1 expression indirectly.2.This study demonstrated that oxymatrine had an antifibrotic effect in CVMC in vivo for the first time, and the mechanism of oxymatrine antifibrotic effects might be related with the downregulation of TGF-β1 expression which can upregulating MMP-1 and ADAMTS-1 expression and doownregulating TIMP-1 expression.3.This study indicated oxymatrine could a new and potentially significant therapeutic target for the treatment of myocardial fibrosis. It might be provide a new thought for the clinical treatment of myocardial fibrosis and another choice for patients who were unendurable the side effects of ACEI.