【摘要】 目的：心肌纤维化是多种心血管疾病进展过程中常见的病理改变，其主要特征为心肌间质中细胞外基质(extracellular matrix,ECM)过度沉积，并伴随心脏结构和功能异常。微RNA(microRNA,miRNA)在心肌纤维化相关调控过程中发挥重要作用。已有研究提示miR-34a参与调控心肌纤维化相关病理过程，但其在体内条件下对心肌纤维化及心功能的影响，以及其与微管相关肿瘤抑制因子1(mitochondrial tumor suppressor 1,MTUS1)之间的关系尚不明确。本研究旨在通过体内试验探讨miR-34a靶向MTUS1对大鼠心肌纤维化及心功能改变的影响。方法：选取健康成年雄性SD大鼠，采用异丙肾上腺素(isoproterenol,ISO)皮下注射建立心肌纤维化模型(模型组)，给药剂量为10 mg/(kg·d)，连续14 d，对照组给予等体积生理盐水。采用经胸超声心动图(transthoracic echocardiography,TTE)检测心脏结构和功能变化。取左心室组织制备石蜡切片，采用苏木精-伊红(hematoxylin-eosin,HE)染色观察心肌组织结构改变，Masson染色评估胶原沉积情况。采用实时聚合酶链反应(real-time PCR)和蛋白质印迹法检测miR-34a、MTUS1及纤维化相关蛋白α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)和I型胶原(collagen I)的表达水平。利用生物信息学方法预测miR-34a与MTUS1的潜在结合位点，并通过双荧光素酶报告基因实验验证二者之间的靶向关系。进一步采用腺相关病毒(adeno-associated virus,AAV)在体内下调miR-34a及MTUS1的表达，评估其对心功能及心肌纤维化的影响。结果：ISO处理后，大鼠心肌组织出现明显纤维化相关病理改变。HE染色显示心肌细胞排列紊乱、间质增宽，Masson染色显示胶原纤维沉积增加。TTE检测结果显示，模型组左心室射血分数较对照组下降(P<0.05)。与对照组相比，模型组心肌组织中miR-34a表达上调，MTUS1表达下调，α-SMA和collagen I蛋白质表达水平升高(均P<0.05)。双荧光素酶报告基因实验结果表明，miR-34a可直接与MTUS1 3’非翻译区结合并抑制其表达。AAV介导下调miR-34a表达后，大鼠心肌纤维化程度减轻，胶原沉积减少，心功能指标较模型组改善；在同时下调MTUS1表达的条件下，上述改善作用发生相应变化。结论：在ISO诱导的大鼠心肌纤维化模型中，miR-34a可通过靶向MTUS1参与调控心肌纤维化相关病理过程及心功能改变。体内AAV干预结果提示miR-34a-MTUS1调控轴与心肌纤维化的发展相关。更多还原

【Abstract】 Objective: Cardiac fibrosis is a common pathological feature during the progression of various cardiovascular diseases and is characterized by excessive deposition of extracellular matrix(ECM) in the myocardial interstitium, accompanied by alterations in cardiac structure and function. MicroRNAs(miRNAs) play important roles in the regulation of cardiac fibrosis. Previous studies have suggested that miR-34a is involved in fibrotic processes; however, its in vivo effects on cardiac fibrosis and cardiac function, as well as its relationship with mitochondrial tumor suppressor 1(MTUS1), remain unclear. This study aimed to investigate the effects of miR-34a targeting MTUS1 on cardiac fibrosis and cardiac function in rats through in vivo intervention.Methods: Healthy adult male Sprague-Dawley(SD) rats were used to establish a myocardial fibrosis model by subcutaneous injection of isoproterenol(ISO) at a dose of 10 mg/(kg·d) for 14 consecutive days(model group), while the control group received equal volumes of normal saline. Transthoracic echocardiography(TTE) was performed to evaluate cardiac structural and functional changes. Left ventricular tissues were collected and paraffin sections were prepared. Hematoxylin-eosin(HE) staining was used to observe histological changes in myocardial tissue, and Masson staining was used to assess collagen deposition. Real-time polymerase chain reaction(real-time PCR) and Western blotting were performed to detect the expression levels of miR-34a, MTUS1, and fibrosis-related proteins including α-smooth muscle actin(α-SMA) and collagen I. Bioinformatics analysis was used to predict potential binding sites between miR-34a and MTUS1. A dual-luciferase reporter assay was performed to verify the targeting relationship between miR-34a and MTUS1. Furthermore, adeno-associated virus(AAV) was used in vivo to downregulate the expression of miR-34a and MTUS1, and the effects on cardiac function and myocardial fibrosis were evaluated.Results: ISO-treated rats exhibited pronounced myocardial fibrotic changes. HE staining revealed disorganized myocardial fibers and widened interstitial spaces, and Masson staining demonstrated increased collagen deposition. TTE analysis showed that left ventricular ejection fraction was reduced in the model group compared with the control group(P<0.05). Compared with control rats, ISO-treated rats showed increased miR-34a expression and decreased MTUS1 expression in myocardial tissues, accompanied by elevated protein levels of α-SMA and collagen I(all P<0.05). Dual-luciferase reporter assays demonstrated that miR-34a directly bound to the 3’ untranslated region of MTUS1 and suppressed its expression. AAV-mediated downregulation of miR-34a attenuated myocardial fibrosis, reduced collagen deposition, and improved cardiac functional parameters compared with the model group; these effects were correspondingly altered when MTUS1 expression was concurrently modulated.Conclusion: In an ISO-induced rat model of cardiac fibrosis, miR-34a participates in the regulation of myocardial fibrosis and cardiac functional alterations by targeting MTUS1. The in vivo AAV intervention results suggest that the miR-34a-MTUS1 regulatory axis is associated with the progression of cardiac fibrosis.更多还原