基于Wnt通路研究补肾活血复方对心力衰竭大鼠心肌肥厚及纤维化的影响

Effect of Compound Bushen Huoxue Decoction(补肾活i血复方)on Myocardial Hypertrophy and Fibrosis in Rats with Heart Failure Based on Wnt Signal Pathway

目的探讨补肾活血复方能否通过抑制Wnt通路改善心力衰竭大鼠心肌肥厚及纤维化。方法将45只SD大鼠随机分为4组,即假手术组、模型组、补肾活血复方组及赖诺普利组,按照实验分组采用结扎左冠状动脉前降支配合力竭式游泳造成慢性心力衰竭模型,在结扎冠状动脉前后进行超声心电图检测,判断手术结扎成功后进行力竭式游泳,并行超声心动图检查,当左室射血分数(LVEF)<45%为造模成功。模型建立完成后采用补肾活血复方或赖诺普利或0.9%氯化钠溶液持续灌胃4周。治疗结束后,称取体质量(BM)、全心质量(HM)和左心室质量(LVM),计算全心质量指数(HWI)和左心室质量指数(LVMI),判断心肌肥厚程度;ELISA检测大鼠中脑钠肽(BNP)、N末端B型脑钠肽前体(NT-proBNP)、心肌肌钙蛋白(cTnI)、Wnt家族成员3a(Wnt3a)含量;马松染色检测心肌纤维化情况,并测定心肌间质胶原容积分数(CVF)、心肌细胞横截面积(CSA)、血管周围胶原面积(PVCA)变化;Western blot检测心肌组织中Ⅰ型胶原(Col I)、Ⅲ型胶原(ColⅢ)、α平滑肌肌动蛋白(α-SMA)、蓬乱蛋白1(Dvl1)、磷酸化糖原合成酶激酶-3β(p-GSK3β)、β-连环蛋白(β-catenin)表达;Real-time PCR检测心肌组织中基质金属蛋白酶2(MMP2)、基质金属蛋白酶9(MMP9)mRNA表达。结果与结扎前比,结扎后大鼠心电图显示T波倒置或低平,ST段较术前明显抬高(绝对值≥0.1 mV)。与假手术组相比,模型组、赖诺普利组及补肾活血复方组的LVMI,血清中BNP、NT-proBNP、cTnI、Wnt3a、CSA、CVF及PVCA,心肌组织中Col I、ColⅢ、α-SMA、Dvl1、p-GSK3β、β-catenin蛋白表达及MMP2、MMP9 mRNA表达均上调,模型组与赖诺普利组HWI升高,差异有统计学意义(P<0.05),补肾活血复方组HWI升高,差异无统计学意义。与模型组相比,赖诺普利组及补肾活血复方组的HWI、LVMI,血清中BNP、NT-proBNP、cTnI、Wnt3a、CSA、CVF及PVCA,心肌组织中Col I、ColⅢ、α-SMA、Dvl1、p-GSK3β、β-catenin蛋白表达及MMP2、MMP9 mRNA表达均降低,差异有统计学意义(P<0.01),赖诺普利组与补肾活血复方组比差异无统计学意义。马松染色检测结果显示,假手术组心肌细胞呈红色,结构完整,心肌纤维排列完整,心肌间质可见少量胶原蓝染;模型组心肌细胞数量变少,心肌间质排列松散且无规则,出现大量胶原纤维的增生,呈波状;与模型组相比,赖诺普利及补肾活血复方组心肌细胞排列相对整齐,心肌间质内胶原纤维增生减少。结论补肾活血复方通过抑制Wnt通路改善心力衰竭大鼠心肌肥厚及纤维化。

Objective To explore whether Compound Bushen Huoxue Decoction(补肾活血复方)can improve myocardial hypertrophy and fibrosis in rats with chronic heart failure by inhibiting Wnt pathway.Methods 45 rats were randomly divided into 4 groups:sham-operation group,model group,Compound Bushen Huoxue Decoction group and lisinopril group.According to the experimental group,the left anterior descending coronary artery was ligated and exhaustive swimming was used to create the chronic heart failure model.Before and after the ligation of coronary artery,the echocardiography showed that the left ventricular ejection fraction(LVEF)<45%was successful.After the model was established,Compound Bushen Huoxue Decoction group or lisinopril or normal saline was used for 4 weeks.After the treatment,the body mass,whole heart mass and left ventricular mass were weighed,and the whole heart mass index HWI and left ventricular mass index LVMI were calculated to judge the degree of myocardial hypertrophy.The levels of BNP,NT-proBNP,cTnl and Wnt3a in ELISA rats were detected.Masson staining was used to detect myocardial fibrosis,and the changes of myocardial interstitial collagen volume fraction(CVF),myocardial cell cross-sectional area(CSA)and perivascular collagen area(PVCA)were measured.The expression of Col I,Col II,α-SMA,Dvll,p-GSK3βandβ-catenin in myocardial tissue was detected by Western blot.The expression of MMP2 and MMP9 mRNA in myocardial tissue was detected by Real-time PCR.Results Compared with before ligation,the ECG of rats after ligation showed T wave inversion or low level,