改善氧化应激微环境保护心肌细胞和骨髓间充质干细胞的降香新黄酮抗心肌梗死分子调控机制研究

Myocardial infarction（MI） received much attention around the world, with a mortality rate over 50%. Cardiomyocytes are terminally differentiated cells. MI is difficult to regenerate and repair. Nowadays there is no effective treatment to repair or reverse the necrotic myocardium. According to the literatures, BMSCs transplantation therapy for MI could improve cardiac function effectively. However, most MSCs of transplantation could not survive in hypoxia circumstance. Oxidative stress is the main factor that leads to the generation of free radicals and ischemic myocardial injury in vivo.Nrf2-ARE pathway plays a crucial role in anti-oxidative stress. Our previous studies demonstrated that new flavonoids latifoiln in Dalbergia odorifera has cardioprotective effect against myocardial hypoxia injury due to its antioxidant action via regulation the Nrf2-ARE pathway. Our studies also found that latifoiln could promote BMSCs proliferation mediated by ERK phosphorylation. These indicated that latifoiln could increase the activity of BMSCs. Therefore, this topic is based on the original work, we try to use modern pharmacology and molecular biology methods to investigate the effect of latifoiln on myocardial cells and BMSCs in oxidative stress circumstance and explore the relationship among them on the NrF2/ARE pathway. The project will clarify the molecular mechanism of latifolin in the treatment of myocardial infarction and provide a new way for clinical treatment of ischemic heart disease.

心肌梗死（MI）是全球备受关注的问题，死亡率高达50%。心肌细胞是终末细胞，目前治疗方法不能修复或逆转己坏死的心肌。文献报道，骨髓间充质干细胞(BMSCs)移植治疗 MI 能促进心功能恢复，但植入心肌后易受大量自由基的损害。氧化应激是导致体内自由基产生和缺血性心肌损伤的主要因素，而Nrf2-ARE是体内尤为重要的抗氧化应激通路。本课题组研究发现，中药降香中新黄酮latifolin对心肌缺血损伤具有显著保护作用，其机制与调控NrF2/ARE通路抗氧化应激有关；同时发现其可以通过激活ERK磷酸化促进BMSCs增殖，表明能增强BMSCs活性。因此，本课题拟在原有工作基础上，利用现代药理学与分子生物学手段，研究latifolin对氧化应激环境下心肌细胞及BMSCs的影响，并基于NrF2/ARE通路探索三者之间的关联性，阐明latifolin治疗心梗的分子调控机制，为临床治疗缺血性心脏病开辟新途径。

心肌梗死（MI）死亡率高达50%，目前治疗方法不能修复或逆转己坏死的心肌。文献报道，骨髓间充质干细胞(BMSCs)移植治疗 MI 能促进心功能恢复，但植入心肌后易受大量自由基的损害。本课题利用现代药理学与分子生物学手段，研究降香新黄酮latifolin对氧化应激环境下心肌细胞及BMSCs的保护作用以及对BMSCs促进受损心肌修复作用及机制，旨在为临床治疗缺血性心脏病开辟新途径。.研究显示latifolin能显著升高缺氧复氧损伤H9c2 细胞活力，降低细胞上清中 LDH 及细胞中ROS水平，升高胞浆中 Nrf2、HO-1 及胞核中 Nrf2 蛋白表达( P＜0. 05) ，说明latifolin 对 H9c2 细胞缺氧复氧所致损伤具有保护作用，其机制可能与激活 Nrf2/HO-1 通路有关。研究还发现，latifolin可降低缺血、缺氧和炎症环境中BMSCs细胞中TNF-α和IL-6水平，升高促炎因子IL-10水平，表明latifolin提高BMSCs活力，减少BMSCs凋亡。研究还显示，latifolin联合5Aza诱导BMSCs后，能显著增加早期转录因子GATA4、NKX2.5和MEF2C表达，分化后细胞有明显α-肌动蛋白和cTnI的表达，外形上细胞呈明显膨大凸起和连接，细胞内有明显的心房颗粒和肌丝，细胞质中有丰富的线粒体和粗面内质网，表明latifolin具有促进5Aza 诱导BMSCs分化成心肌细胞作用。Annexin V/PI法测定latifolin对BMSCs与H9c2共培养条件下H9c2细胞凋亡情况，结果表明latifolin可促进BMSCs对H9c2的保护。观察latifolin对大鼠左前降支结扎后移植P3代BMSCs对大鼠心肌梗死的保护作用。结果表明，大鼠血清中LDH和CK-MB含量显著降低，心功能显著提高， LVEF、LVFS和CO显著升高，梗死面积和胶原纤维化面积显著降低，梗死周边炎症细胞浸润和肌丝溶解情况明显改善， BMSCs生存率和心肌分化率显著提高，炎性巨噬细胞浸润降低。说明，latifolin能通过调控HIF-1α/NF-κB/β-catenin通路提高BMSCs移植生存率和心肌分化率的作用。.以上结果表明，latifolin对氧化应激环境下心肌细胞及BMSCs具有保护作用，还可以促进BMSCs向心肌细胞分化，促进BMSCs修复受损心肌。