黄芪生脉饮对大鼠放射性心脏损伤的防护作用及机制研究

Radiotherapy for thoracic and abdominal tumors affects heart and cause damage to the heart, which is known as radiation-induced heart damage (RIHD). The incidence of RIHD has been increasing and this damage becoming a popular topic for research. However, the pathogenesis of RIHD remains unclear and there are no effective treatments. The main pathological change of RIHD is fibrosis, TGF-β1 is the key. Our previous study used PCR-array to shown that the X-ray induced fibrosis effect and the high expression of TGF-β1 was related to the abnormal expression of Smads and metal matrix protease system. While, astragalus inhibiting radiation-induced fibrosis effect, and these molecular abnormality expression have been reversed at the same time. Thus, we believe that Smads and metal matrix system maybe adjust the TGF-β1 signal activation which involved RIHD fibrosis pathological process. . Traditional Chinese Medicine thought radial as "heat evil", for radiation-induced fibrosis, it was due to clogging of blood, damage of Qi and Yin, long illness into meridians and collaterals, and the stagnation of Qi and blood. Treatments should boost Qi and nourish Yin and benefit Qi for activating blood circulation based on "strengthening the body resistance and eliminating evil". So, on the basis of the previous research we choose HuangqiShengmaiYin (Astragalus membranaceus pulse-activating decoction) to intervene the RIHD rat model. Based on the cardiac fibrosis in rats caused by X-ray radiation, the objectives of this study is to: 1) Investigate the effect of HuangqiShengmaiYin on X-ray induced fibrosis damage, from the aspects of rat cardiac function, cardiac pathological morphology, myocardial enzymology, etc; 2) Applicate RT-PCR and Western blot analysis to confirme the differentially expression of Smad2, Smad3, Smad4, Smad7, MMP14, TIMP1, etc, which screened out by PCR chip in previous experiment, to further explore the mechanism of Smads and metal matrix system maybe adjust the TGF-β1 signal activation which involved RIHD fibrosis pathological process; 3) Clarify the pharmacological mechanism of HuangqiShengmaiYin, which in order to provide the basic theory support for its prevention and treatment of RIHD.

放射性心脏损伤（RIHD）是胸部肿瘤放疗常见副反应，但因防治机制不明确，缺乏有效干预措施。研究证实RIHD主要病理改变是纤维化，TGF-β1是其病理机制的关键。课题组前期细胞实验曾用PCR芯片技术观察到X线诱导TGF-β1高表达的促纤维化效应与Smads分子、金属基质蛋白酶系统的异常表达有关，黄芪抑制纤维化效应的同时这些分子异常表达被逆转。由此，我们认为，Smads分子和金属基质蛋白酶系统可能通过调节TGF-β1信号的活化参与RIHD的纤维化病理过程。本项目从“射线损伤人体正气阴血，致热积血淤，气阴两伤”出发，以“益气养阴，滋阴养血”为原则，用黄芪生脉饮干预RIHD大鼠模型，从大鼠心功能、心脏病理形态学、心肌酶学等方面探讨其药效，并应用分子生物学技术检测分析Smads分子和金属基质蛋白酶系统对TGF-β1信号活化的影响，探讨黄芪生脉饮干预RIHD的药理机制，为其应用于临床提供基础研究支持。

背景：放射性心脏损伤（RIHD）是胸部肿瘤放疗常见副反应，其病理分子机制不明，缺乏有效干预。中医认为射线是热毒，引起心气血不足是RIHD的主要病机，本研究从“益气养阴，滋阴养血”治则出发，探讨黄芪生脉饮（HSY）干预RIHD。.研究内容：（1）构建动物模型；（2）围绕TGF-β1探讨RIHD损伤机制；（3）观察HSY干预的药效机制。.重要结果及关键数据：（1）大鼠全身单次照射耐受剂量小于16Gy；心脏局部单次照射剂量至少是25Gy才能引起显著炎性—纤维化病损，是模型构建条件。模型组大鼠，心功能变差，病理损伤评分、CVF和血清心肌酶CK-MB、cTn增高；炎性因子p65、NF-κB (p50) 和TNF-α等照射后第1天开始出现高表达, 纤维化分子TGF-β1、Col1/3表达呈渐进性升高，第4周高表达达峰值，说明X线照射引起心脏炎性-纤维化损伤。（2）模型组HIF-1α、CTGF的表达呈渐进性升高的趋势，第4周达高峰值，Smad 2/3/4表达增高， Smad 7表达降低；模型组MMP14表达增高TIMP1表达降低。（3）HSY能改善大鼠心功能尤其是舒张功能，可剂量依赖的降低照射后心脏病理损伤评分和CVF。中、高剂量HSY下调X线引起的炎性和纤维化分子的高表达，Smad 2/3/4、MMP14的高表达被下调，同时逆转Smad7、TIMP1的表达抑制。.科学意义：（1）25Gy心脏局部单次照射可损伤大鼠心功能，构建稳定的RIHD模型；病理观察和CVF能动态反映RIHD模型大鼠早期炎性病理变化和随着时间渐进性的纤维化病理进程；炎性因子p65、NF-κB (p50)、TNF-α的早期持续高表达和纤维化因子TGF-β1、Col1/3的渐进性升高可评价RIHD炎性-纤维化动物模型的急性炎性损伤和纤维化迟发效应；（2）MMPs-TGF-β1、TGF-β1/Smads、NF-κB/HIF-1α/CTGF等通路参与RIHD的病理进程；（3）HSY有改善放射诱导的炎性-纤维化损伤效应，其药理作用涉及纤维化关键因子TGF-β1相关的上游金属基质蛋白酶系统（MMPs）及下游Smads信号分子的平衡调节，也和介导炎性反应向纤维化病理转变的NF-κB/HIF-1α/CTGF信号机制有关，可多途径发挥保心抗炎抗纤维化作用，为HSY临床干预放射性心脏损伤提供了一定的实验依据和理论依据。