血府逐瘀汤对高血压心肌纤维化TGF-β1/Smads信号通路调控机制的研究

Hypertension is one of the most common worldwide diseases afflicting humans. Excessive myocardial fibrosis impairs cardiac function in hypertensive hearts. Tranforming growth factor-β1 (TGF-β1) plays a causal role in myocardial fibrosis and diastolic dysfunciton through fibroblast activation in pressure-overloaded hearts. Members of the transforming growth factor-β1 family control growth, differentiation and apoptosis of cells, and have important functions during embryonic development. A number of studies have demonstrated that it is effective in protecting the cardiovascular systerm from hypertensive injuries by inhibiting TGF-β1 signaling pathway. According to our fomer research, XueFuZhuYu Decoction, a well-known traditional Chinese medicine for treating cardiovascular diseases, can decrease level of TGF-β1 of blood and myocaridal in spontaneous hypertension rat. We designed this study to investigate whether XFZYD inhibit myocardial fibrosis by TGF-β1 signal pathway and explore its possible of mechanism of myocardial protection. Anti- TGF-β neutralizing antibodies are very effective tools tto investigate the roles of TGF-β1 in the fibrosid by inhibiting TGF-β1 activity in the SHR. Real -time RT-PCR was used to detect the mRNA expressions of Smad2、Smad3 and Smad7. Western blot were performed to determine bioactivities of Smads. Immunohistochemical analysis was used fo detect cardiac hypertrophy. Using Luciferase assay and siRNA interference also were used to check the possibly specific target in TGF-β1/Smads signal pathway.

TGF-β1/Smads信号传导是介导高血压心肌纤维化(CFs)的重要通路。前期临床与实验研究：血府逐瘀汤有良好的抗高血压CFs作用，能显著降低SHR心肌和血清中TGF-β1的表达水平，提示血府逐瘀汤抗高血压CFs的机制与TGF-β１/Smad通路有关。因此本项目从TGF-β1/Smads信号通路入手，采用特异性抗TGF-β抗体阻断此信号通路，运用免疫组化、Western blot和RT-PCR方法，研究血府逐瘀汤对SHR心肌结构、纤维化指标、心肌细胞凋亡率的变化和细胞信号分子Smad2、Smad3、Smad7 mRNA表达及蛋白含量的影响，明确血府逐瘀汤抗心肌纤维化是否直接作用在TGF-β1/Smads信号通路上；并采用荧光素酶报告基因实验进行验证。另选取Smad2、Smad3、Smad7、TGF-β受体等关键靶点，运用siRNA干扰技术，进一步明确血府逐瘀汤的具体作用靶点。

血府逐瘀汤是治疗心血管疾病的传统方剂，临床应用已有二百多年。然而，血府逐瘀汤对心肌纤维化的作用和机制还不明确。本项目旨在研究血府逐瘀汤对心肌纤维化的作用并阐明其潜在机制。本项目首先建立了血府逐瘀汤的中药指纹图谱，以确保药物质量的稳定性和可控性。从组织病理学、血流动力学、脏器功能、脏器指数、心脏超微结构等多个角度，观察了药物对高血压心肌纤维化的作用。在体外实验中，通过血府逐瘀汤预处理后再给予TGF-β1共同处理心肌成纤维细胞的方法研究血府逐瘀汤对体外心肌纤维化模型的作用。之后分别运用CCK-8法检测血府逐瘀汤对心肌成纤维细胞增殖的影响；qRT-PCR和Western Blot法分别检测细胞内ColⅠ、ColⅢ、α-SMA以及Smad2、Smad3、Smad7等mRNA和蛋白的表达影响；生化试剂盒检测细胞氧化应激相关因子水平的变化，包括SOD、ROS、MDA、GSH等。结果如下：①组织病理学、血流动力学、脏器功能、脏器指数、心脏超微结构等多个方面均证明了血府逐瘀汤治疗高血压心肌纤维化的有效性与安全性；②与空白对照组比较，TGF-β1组能够显著诱导细胞增殖；而使用血府逐瘀汤预处理细胞的增殖被明显抑制；③血府逐瘀汤组能够在mRNA和蛋白水平上降低TGF-β1诱导的ColⅠ、α-SMA、Smad2、Smad3的mRNA表达水平并上调Smad7的mRNA表达；④血府逐瘀汤组能够上调TGF-β1诱导的SOD、GSH水平，下调ROS、MDA的水平。综上，血府逐瘀汤能够抑制TGF-β1诱导的心肌成纤维细胞增殖，降低氧化应激水平，具有明确的体外抗心肌纤维化作用。血府逐瘀汤的以上作用可能与下调心肌成纤维细胞TGF-β1/Smad通路上Smad2、 Smad3，上调Smad7的mRNA和蛋白表达相关。本研究初步揭示了血府逐瘀汤抗高血压MF的网络作用机制，为其在抗高血压MF的临床应用中提供了重要的科学依据。