【作者】 陈启兰；

【导师】 龚一萍；

【作者基本信息】 浙江中医药大学 ， 中医临床基础， 2013， 博士

【摘要】 目的第一部分：观察复方黄芪养心合剂(compound astragalus mixture nourishing heart, CAMNH)对SD大鼠冠状动脉结扎术后再灌注心律失常(reperfusion arrhythmias, RA)及心肌缝隙连接蛋白43(connexin43,Cx43)分布的改变和表达的影响,并探讨RA与心肌Cx43的关系。第二部分：观察CAMNH对SD大鼠冠状动脉结扎术后心脏重构(cardiac remodeling, VR)超声心动图(ultrasonic cardiogram, UCG)的影响,心肌及血管周围胶原纤维化的影响,以及心肌Cx43分布的改变和表达的影响,并探讨胶原纤维化与心肌Cx43的关系。方法第一部分：应用CAMNH高剂量(CAMNHh)、中剂量(CAMNHm)、低剂量(CAMNH1)分别按28g·14ml-1·1kg-1·d-1、14g·7ml-1·1kg-1·d-1、7g·3.5ml·1kg-1·d-1剂量灌胃,琥珀酸美托洛尔缓释片(metoprolol succinate sustained-release tablets, MSSRT)(9.5mg·7ml-1·1kg-1·d-1)灌胃2周后,对SD大鼠行冠状动脉左前降支结扎30min后再灌注60min造成RA模型,记录心电图,进行室性心律失常(ventricular arrhythmias, VA)评分；免疫组织化学法(immunohistochemistry, IHC)观察心肌Cx43的分布,运用Image Pro Plus6.0图像分析软件计算Cx43的平均光密度(average optical density of Cx43, Cx43-AOD,%)。对VA评分与Cx43-AOD(%)进行相关性分析。第二部分：对SD大鼠行冠状动脉左前降支结扎术后观察10天。存活大鼠随机分组后应用CAMNHh、CAMNHm、CAMNH1及MSSRT灌胃8周。术后给药第4周和第8周分别进行UCG测定,并进行前后对比及变化幅度对比。计算全心室重与体重比(Vt/Wt,%)。使用改良Masson染色法观察大鼠心室重构,用Image Pro Plus6.0图像分析系统,计算心肌组织胶原容积分数(collagen volume fraction, CVF,%)和血管周围胶原面积(perivascular collagenvolume area, PVCA,%)。IHC法观察心肌Cx43分布的改变,免疫印迹法(western blot, WB)检测心肌Cx43的表达,应用Quantity One1-D Analysis Software分析计算心肌Cx43比值(the ratio of Cx43, Cx43-T,%)。对CVF(%)、 PCVA(%)与心肌Cx43-T(%)进行相关性分析。结果第一部分：VA评分(分)CAMNH1组与MSSRT组显著低于I/R组及CAMNHh组(P<0.01)。梗死区和缺血区多位于心肌内膜下,非缺血区多位于心肌中层。心肌内膜下缺血区域Cx43-AOD(%)MSSRT组、CAMNHm组与CAMNH1组均高于I/R组(P<0.05)。VA评分与缺血区心肌Cx43-AOD(%)呈负相关(P<0.05)。第二部分：术后第6周UCG:LVEF1(%)及FS1(%) CAMNHm组、CAMNH1组较SO组明显降低(P<0.01)；较MSSRT组降低(P<0.05)；与VR组相近(P>0.05)。术后第10周UCG:LVDs1(mm)MSSRT组、CAMNHm组较VR组明显减小(P<0.01)；CAMNH1组较VR组减小(P<0.05)。LVEF2(%)与FS2(%)：CAMNHm组、CAMNH1组、MSSRT组明显高于VR组(P<0.01)。前后对比：LVDs(mm):VR组增大(P<0.05); LVEF(%)及FS(%)在VR组及MSSRT组进一步降低(P<0.01)。变化幅度对比：ΔLVDs(mm):MSSRT组、CAMNHh组、CAMNHm组、CAMNH1组增大幅度均较VR明显减小(P<0.01)；ΔLVEF(%)和△FS(%)：CAMNHh组、CAMNHm组、CAMNH1组及MSSRT组降低幅度较VR组明显减小(P<0.01)。Vt/Wt (%): MSSRT组小于VR组(P<0.05); CAMNHm组、CAMNH1组明显小于VR组(P<0.01)。CVF(%)与PVCA (%):CAMNHm组、CAMNH1组及MSSRT组与VR组相比明显降低(P<0.01);CAMNHh组与VR组相比降低(P<0.05)。心肌Cx43非梗死区Cx43表达与SO组比较有所减少。心肌Cx43-T(%)CAMNH1组、MSSRT组较VR组明显增加(P <0.01)。CAMNHh组、CAMNHm组较VR组增加(P<0.05)。CVF(%)、PCVA(%)与心肌Cx43-T(%)呈显著负相关(P<0.01)。结论第一部分：CAMNH1有抗RA作用,与MSSRT相近。CAMNHm、CAMNH1可促进再灌注心肌缺血区Cx43的表达,其作用与MSSRT相近。CAMNH1抗心律失常作用可能与其促进再灌注心肌缺血区Cx43的表达有关。第二部分：CAMNHm、CAMNH1能够持续改善VR LVDs(mm)的增大以及LVEF(%)、 FS(%)的降低,减轻Vt/Wt(%),其作用与MSSRT相当。CAMNHh、CAMNHm、CAMNH1能降低VR CVF(%)、PVCA(%),改善VR非梗死区心肌Cx43的表达减少,与MSSRT相当,其作用无剂量依赖性。CAMNH抗VR作用可能与其改善非梗死区心肌Cx43的表达减少有关。更多还原

【Abstract】 Objective To study the effects of Compound Astragalus Mixture Nourishing Heart (CAMNH) on reperfusion arrhythmia(RA) and myocardial connexin43(Cx43) in male sprague dawley(SD) rats of ischemia/reperfusion injury(I/R) after coronary artery ligation, and to explore the relationship between RA and myocardial Cx43in the first part of this study.To study the effects of CAMNH on ultrasonic cardiogram(UCG), myocardial and perivascular collagen fibration, and myocardial Cx43in SD rats of cardiac remodeling(VR) after coronary artery ligation, and to explore the relationship between myocardial and perivascular collagen fibration and myocardial Cx43in the second part of this study.MethodIn the first part, the following method were adopted. SD rats were were randomly divided into6groups, as high-dose group of CAMNH(CAMNHh),medial-dose group of CAMNH(CAMNHm), low-dose group of CAMNH(CAMNH1), MSSRT group, I/R group and sham-operated(SO) group. Then the rats were treated for14days with CAMNHh(28g·1kg-1·d-1), CAMNHm(14g·1kg-1·d-1), CAMNH1(7g·1kg-1·d-1) or Metoprolol Succinate Sustained-Release Tablets(MSSRT,9.5mg·1kg-1·d-1) before acute myocardial infarction(AMI). AMI was induced by ligation of Left Anterior Descending(LAD) coronary artery for30mins, followed by reperfusion of60mins. The ventricular arrhythmia(VA) score.The distribution and expression of myocardial Cx43were observed with immunehistochemistry(IHC). Average optical density of myocardial Cx43(myocardial Cx43-AOD) were measured with Image Pro Plus6.0. The correlation between VA score and myocardial Cx43-AOD in the ischemic regions was analyzed.In the second part, the following method were adopted. Watched10days after ligation of LAD coronary artery, the survived rats were randomly divided into5groups, as CAMNHh group, CAMNHm group, CAMNH1group, MSSRT group and VR group. The other group was SO group. Then the rats were treated for8weeks with CAMNHh(28g·1kg-1·d-1), CAMNHm (14g·1kg-1·d-1),CAMNH1(7g·1kg-1·d-1) or MSSRT(9.5mg·1kg-1·d-1). On the end of the sixth week and the tenth week after ligation of LAD coronary artery, diameter of left ventricle at end-systole(LVDs, mm), left ventricular eject fractions(LVEF,%) and fraction shortening (FS,%) were measured with UCG. Then to compare the change between the sixth week and the tenth week after ligation, and to compare the amplitude of variation among groups. At the end of the experiment, the ratio of weight(Wt,g) and ventricle weight(Vt,g), Vt/Wt(%) was calculated. The VR of rats were observed with upgrading Masson, then collagen volume fraction(CVF,%) and perivascular collagenvolume area(PVCA,%) were measured with Image Pro Plus6.0. The distribution of myocardial Cx43were observed with IHC, and the ratio of myocardial Cx43(myocardial Cx43-T,%) were detected with western blot(WB). The relative content of myocardial Cx43was measured by Quantity One1-D Analysis Software. CVF(%) and PVCA(%) were used to analyze the correlation with myocardial Cx43-T(%) in the non infarct regions.ResultIn the first part, the following results were obtained. The VA score of CAMNH1group and MSSRT group was less than I/R group. Most of the infarction regions and ischemic regions were located in subendocardial myocardium, and the non ischemic regions in midmyocardium. CAMNHm group, CAMNH1and MSSRT group vs I/R group, the change of the distribution of myocardial Cx43was modified, and myocardial Cx43-AOD(%) in ischemic region were inVReased(P<0.05). There were negative correlation between the VA score and myocardial Cx43-AOD(%) in ischemic region(P<0.05).In the second part, the following results were obtained. On the end of the sixth week after ligation, the following results were gotten with UCG. CAMNHm group and CAMNH1group vs MSSRT group, LVEF (%) and FS(%) deVReased (P<0.05). CAMNHm group and CAMNH1group vs VR group, LVEF (%) and FS(%) didn’t deVRease (P>0.05). Then on the end of the tenth week, LVDs(mm), LVEF (%) and FS(%) were obtained. VR group vs MSSRT group, VR group vs CAMNHm group, LVDs(mm) significantly inVReased (P<0.01). VR group vs CAMNH1group, LVDs(mm) inVReased (P<0.05). VR group vs MSSRT group, CAMNHm group, and CAMNH1group, LVEF (%) and FS(%) significantly reduced (P<0.01). When comparing the change between the sixth week and the tenth week after ligation, it was found that LVDs(mm) inVReased in VR group(P<0.05), LVEF (%) and FS(%) significantly reduced in VR group and MSSRT group (P<0.01). After comparing VR group with the other groups, the inVRescent amplitudes of LVDs(Δ LVDs, mm) significantly inVReased(P<0.01), and the reduced amplitude of LVEF(Δ LVEF,%) and FS(ΔFS,%) significantly inVReased(P<0.01).CAMNHm group and CAMNH1group vs VR group, Vt/Wt(%) significantly deVReased(P<0.01). MSSRT group vs VR group, Vt/Wt(%) deVReased(P<0.05). MSSRT group, CAMNHm group and CAMNH1group vs VR group, CVF(%) and PVCA(%) significantly deVReased(P<0.01). The expression of myocardial Cx43in non infarction region slightly reduced. VR group vs MSSRT group and CAMNH1group, myocardial Cx43-T(%) in non infarction region significantly deVReased(P<0.01), and VR group vs CAMNHh group, VR group vs CAMNHm group, deVReased(P<0.05). CVF(%) and PCVA(%)were negatively correlated significantly with myocardial Cx43-T(%) in non ischemic region(P<0.01).ConclusionIn the first part, the following conclusions are reached. CAMNH1induce VA score, the effects are similar to MSSRT. Most of the infarction region and ischemic region locate in subendocardial myocardium, and the non ischemic region in midmyocardium. CAMNHm and CAMNH1can inVReases the expression of myocardial Cx43in ischemic region, and the effects is near to MSSRT. There were negative correlation between the VA score and myocardial Cx43-AOD(%) in ischemic region.In the second part, the following conclusions can be gotten. CAMNHm and CAMNH1can persistently ameliorate the acVRetion of LVDs(mm) and the reduction of LVEF(%) and FS(%), and the effects is near to MSSRT. CAMNHm and CAMNH1can reduce Vt/Wt(%), and the effects is near to MSSRT. CAMNHh, CAMNHm and CAMNH1can reduce CVF(%), PVCA(%) and myocardial Cx43-T(%)in non infarction region. The effects are near to MSSRT and no dose-dependent. CVF(%) and PCVA(%) are negatively correlated significantly with myocardial Cx43-T(%) in non ischemic region.更多还原