丹参酮Ⅱ_A抑制主动脉瓣成肌纤维细胞向成骨细胞样表型转化的机制  

Mechanism of tanshinone Ⅱ_A in inhibiting transformation of aortic valvular myofibroblast to osteoblast-like phenotype

作　　者：沈迎念[1] 胡伟林[1] 陈正平[1] 蔡笠[1] 李永胜[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院急诊科/综合科,湖北武汉430030

出　　处：《中国中药杂志》2015年第18期3636-3643,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81070190);湖北省自然科学基金项目(2014CFB962)

摘　　要：心脏瓣膜钙化是与多种病因有关、由心脏瓣膜细胞主动调节的病理生理过程。该研究以体外培养的猪主动脉瓣成肌纤维细胞作为研究对象,用与心脏瓣膜钙化有关的病理因素肿瘤坏死因子α(TNF-α)50μg·L-1处理细胞,将50 mg·L-1丹参酮ⅡA磺酸钠(TSN)与TNF-α共同孵育细胞72 h(3 d),120 h(5 d)。采用Western blotting技术和Real-time PCR技术分别检测细胞中的平滑肌α肌动蛋白(α-SMA)、骨样表型相关的骨形成蛋白2(BMP2)、碱性磷酸酶(ALP)和Wnt/β-catenin信号通路上的关键效应蛋白GSK-3β,β-catenin基因表达的变化。研究发现,TNF-α能使成肌纤维细胞α-SMA的表达明显增加而成为有转化活性的细胞;骨相关蛋白BMP2,ALP的蛋白和mRNA在对照组和TSN组几乎没有表达,而在TNF-α组表达显著增加(P<0.01),呈现出成骨细胞样表型;Wnt/β-catenin信号通路中的上游调节蛋白GSK-3β的蛋白及mRNA表达在TNF-α组显著下调(P<0.01),关键效应蛋白β-catenin的蛋白表达显著增加,但mRNA与对照组、TSN组比较没有明显差异;TSN对TNF-α的这种病理性影响可以起到一定的抑制作用(P<0.05),并且存在时间依赖性效应(P<0.05)。炎性因子TNF-α可能通过激活心脏瓣膜成肌纤维细胞中的Wnt/β-catenin信号通路,促使心脏瓣膜成肌纤维细胞向成骨细胞样表型转化,进而导致心脏瓣膜的钙化。丹参酮ⅡA能够通过影响GSK-3β蛋白的活性,调控Wnt/β-catenin通路的信号传导,对由炎症所致的瓣膜钙化性疾病起到防治作用。Aortic valve calcification（ AVC） is a pathological process correlated with multiple disease causes and actively regulated by cardiac valve cells. In this study,porcine aortic valve myofibroblasts cultured in vitro were treated with 50 μg·L^- 1of pathological factor tumor necrosis factor α（ TNF-α）. Tanshinone ⅡA（ TSN） with the concentration of 50 mg·L^- 1and TNF-α were combined in incubating cells for 72 h（ 3 d） and 120 h（ 5 d）. The Western blotting and Real-time PCR were adopted to detect the changes in smooth muscle α actin（ α-SMA）,bone morphogenetic protein 2（ BMP2）,alkaline phosphatase（ ALP） in cells,and expressions of key effect proteins GSK-3β and β-catenin on Wnt / β-catenin signal pathway. According to the findings,TNF-α can significantly increase the expression of myofibroblasts α-SMA and add the transformation activity to them,with nearly no expression of BMP2,ALP and mRNA in the control group and the TSN group but significant increase in their expressions in the TNF-α group（ P〈0. 01）,which showed osteoblast-like phenotype. Moreover,TNF-α down-regulated the expression of up-streaming regulator GSK-3β and mRNA expression（ P〈0. 01）,notably increased the expression of key effect protein β-catenin,but with no significant difference in mRNA with the control group and the TSN group. The result demonstrated that TSN showed a certain inhibitory effect on TNF-α＇s pathological impact（ P〈0. 05） in a time-dependent manner. Inflammatory factor TNF-α may promote the transformation of aortic valvular myofibroblasts to osteoblast-like phenotype by activating Wnt / β-catenin signal pathway in aortic valvular myofibroblasts,so as to cause AVC.Tanshinone ⅡAcan have a preventive effect in AVC by activating GSK-3β proteins and regulating signal transduction of Wnt / β-catenin signal pathway.

关 键 词：丹参酮ⅡA TNF-α 瓣膜钙化 成肌纤维细胞 Wnt/β-catenin信号通路 GSK-3Β 

分 类 号：R285[医药卫生—中药学]