Notch3信号通路介导MCT诱导的PAH大鼠肺血管ECM的重塑以及分子机制

摘要

Objective To investigate the molecular mechanisms of Notch3 signaling pathway-mediated pulmonary vascular extracellular matrix (ECM) remodeling in monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) rats.Methods Totally 32 male 4-week-old SD rats were randomly divided into 3 groups:control group(n =10),MCT-induced PAH model group(MCT,n =12),PAH model + DAPT group(MCT + DAPT,n =10).The PAH model was induced by intraperitoneal injection with MCT(60 mg/kg body weight) at day 1.DAPT(10 mg/kg) was administered by intraperitoneal injection once a day after administrated with MCT at day 1 and continuously treated for 28 d.Rats in each group were anesthetized at day 28 to measure the right ventricle systolic pressure (RVSP) and index of right ventricular hypertrophy(RVH),ECM in pulmonary vessel was localized by Masson staining,and the activity of MMP-2/9 from the lung tissue lysates was assessed by gelatin zymography.The contents of Notch3,NICD3,tissue inhibitor of matrix metalloproteinase-1 (TIMP-1)protein were detected by Western blot.Results The levels of Notch3 and its cleavage domain NICD3 protein in lung tissue in MCT group were significantly higher than that in control group.Compared with control group,the deposition of pulmonary vascular ECM increased in MCT group,the activity of MMP-2/9 was enhanced in lung tissue,and TIMP1 protein level from lung tissue lysates was increased.The levels of Notch3 and its cleavage domain NICD3 protein in lung tissue in MCT + DAPT group were significantly lower than those in MCT group(P ＜0.05),and the activity of MMP-2/9 and the level of TIMP1 protein were also lower(P ＜ 0.05),accompanied with decreased deposition ECM of pulmonary vascular wall.Conclusion Notch3 signal may promote the pulmonary vascular ECM remodeling through increasing the activity of MMP-2/9 and the expression of TIMP1.%目的 探讨Notch3信号通路介导野百合碱(monocrotaline,MCT)诱导的肺动脉高压(pulmonary arterial hypertension,PAH)大鼠肺血管细胞外基质(extracellular matrix,ECM)重塑以及分子机制. 方法 32只4周龄SD雄性大鼠被随机分为3组,对照组(Con组)10只;MCT诱导的PAH模型组(MCT组)12只,一次性腹腔注射2％ MCT 60 mg/kg;3,5-二氟苯乙酰-L-丙氨酰-S-苯基甘氨酸t-丁酯(N-[N-(3,5-difluorophena-cetyl-L-alanyl)]-S-phenylglycine t-butyl ester,DAPT)处理的PAH模型组(MCT+ DAPT组)10只,一次性腹腔注射2％ MCT 60 mg/kg后立即给予腹腔注射Notch抑制剂DAPT 20 mg/kg,1次/d,连续治疗28 d.第28天麻醉各组大鼠,测量右心室收缩压和右心室肥大指数,Masson染色观察肺血管ECM的分布,明胶酶谱法检测肺组织基质金属蛋白酶-2/9(matrix metalloproteinases-2/9,MMP-2/9)的活性.Western blot方法检测肺组织中Notch3、NICD3,以及TIMP-1蛋白质水平. 结果 在MCT诱导的PAH模型组大鼠的肺组织中,Notch3以及其裂解片段NICD3蛋白水平较对照组明显升高(P＜0.05),伴有肺血管ECM过量沉积,MMP-2/9活性较对照组明显增加(P＜0.05),TIMP1蛋白水平亦较对照组增加(P＜0.05);在MCT+ DAPT组大鼠的肺组织中,Notch3和NICD3蛋白水平较MCT组明显下降(P＜0.05),MMP-2/9活性亦较MCT组降低(P＜0.05),TIMP1蛋白水平亦较MCT组下降(P＜0.05),伴有肺血管壁ECM较MCT组明显减少. 结论Notch3信号通路可能通过上调MMP-2/9活性和TIMP1蛋白的表达,介导野百合碱诱导的大鼠肺血管壁ECM的重塑.