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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。
Cardiac valve calcification is associated with a variety of causes, by the heart valve cells actively regulate the pathophysiological process. In this study, pig aortic myofibroblasts were cultured in vitro and treated with 50 μg · L-1 of tumor necrosis factor-α (TNF-α), a pathological factor associated with calcification of heart valves, and 50 mg · L -1 Tanshinone Ⅱ A sulfonate (TSN) incubated with TNF-α for 72 h (3 d) and 120 h (5 d). Western blotting and Real-time PCR were used to detect the expression of α-SMA, BMP2, ALP and Wnt / Changes of expression of GSK-3β and β-catenin in β-catenin signaling pathway. The results showed that TNF-α could significantly increase the expression of α-SMA in myofibroblasts and become transformed cells. The protein and mRNA of bone-related proteins BMP2 and ALP were almost not expressed in control group and TSN group, The expression of GSK-3β protein in Wnt / β-catenin signaling pathway was significantly down-regulated in TNF-α group (P <0.01), and the expression of GSK-3β in Wnt / 0.01). The protein expression of the key effector protein β-catenin was significantly increased, but there was no significant difference between the mRNA and the control group and the TSN group. The pathological effects of TSN on TNF-α could be inhibited (P <0.05 ), And there was a time-dependent effect (P <0.05). The inflammatory cytokines TNF-α may induce the cardiac valve myofibroblasts to transform into osteoblast-like phenotype by activating Wnt / β-catenin signaling pathway in cardiac valve myofibroblasts, leading to calcification of heart valves. Tanshinone IIA can regulate the signal transduction of Wnt / |Â-catenin pathway by affecting the activity of GSK-3|Â protein and play a preventive and therapeutic role in valve calcification caused by inflammation.