基于miR-451调控TLR4/NF-κB/HIF-1α环路探讨真武汤预防CRS肾纤维化的机制研究

The inflammatory response and chronic hypoxia interact with each other to promote renal fibrosis in cardiorenal syndrome (CRS). TLR4/NF-κB/HIF-1α loop is activated in the state of inflammatory response and hypoxia in chronic heart failure (CHF). Recent studies have shown that miR-451 plays an important role in this loop regulation. Previously, we adopted with microRNA high-throughput sequencing technology and found that the expression of miR-451 in peripheral blood of patients with CRS Yang deficiency was significantly lower than those of CHF patients, and the levels of TLR4 and NF-κB p65 were increased. And Zhenwu Decoction can improve the heart and kidney function of patients with Yang deficiency and reduce renal fibrosis. Therefore, we hypothesis that Zhenwu Decoction could inhibit the inflammatory response and prevent CRS renal fibrosis by promote high expression of miR-451. Combine with the in vivo and in vitro experiments, using cell transfection, qPCR and Western blot technology, this study will elucidate the relationships between miR-451 expression, TLR4/NF-κB/HIF-1α activity, inflammatory response and renal fibrosis. And reveal the protective effect of Zhenwu Decoction on renal injury in CRS, and clarify the relationship between miR-451, TLR4/NF-κB/HIF-1α and renal fibrosis. Expand the new understanding of the mechanism of renal fibrosis in CRS and the Chinese medicine treatment.

炎性反应和慢性缺氧相互作用，共同促进心肾综合征（CRS）肾纤维化。TLR4/NF-κB/HIF-1α环路在慢性心衰（CHF）时显著激活，新近发现miR-451对该环路具有重要调控作用。课题组前期发现CRS阳虚证患者外周血miR-451低表达，TLR4和NF-κB p65水平上升。真武汤可显著改善CRS患者心肾功能，减轻肾纤维化。因此我们提出真武汤通过促进miR-451高表达，抑制炎性反应，预防CRS肾纤维化。本课题拟结合体内、体外实验，分析miR-451、TLR4/NF-κB/HIF-1α、炎性因子、肾脏纤维化标志分子的变化，从表观遗传学角度阐述真武汤对CRS肾损伤的保护作用，建立miR-451调控TLR4/NF-κB/HIF-1α激活防止肾纤维化的功能联系，拓展对CRS肾纤维化发生发展机制以及中医药治疗新认识。

炎性反应和慢性缺氧相互作用，共同促进心肾综合征（CRS）肾纤维化。TLR4/NF-κB/HIF-1α环路在慢性心衰（CHF）时显著激活，新近发现miR-451对该环路具有重要调控作用。课题组前期发现CRS阳虚证患者外周血miR-451低表达，TLR4和NF-κB p65水平上升。真武汤可显著改善CRS患者心肾功能，减轻肾纤维化。因此本课题结合体内、体外实验，分析miR-451、TLR4/NF-κB/HIF-1α、炎性因子、肾脏纤维化标志分子的变化，从表观遗传学角度阐述真武汤对CRS肾损伤的保护作用，建立miR-451调控TLR4/NF-κB/HIF-1α激活防止肾纤维化的功能联系，拓展对CRS肾纤维化发生发展机制以及中医药治疗新认识。研究结果显示：（1）动物实验方面，真武汤可以明显改善患者的心肾功能（LVIDD、LVIDS、CO、EF、NT-proBNP、Scr、BUN、24小时尿蛋白）；组织病理染色发现，真武汤可以改善肾脏结构，减轻肾间质纤维化的程度及炎症细胞的浸润；通过Elisa、Western blot、RT-PCR、免疫组化和免疫荧光等方法检测发现，真武汤可以上调 miR-451 表达，抑制TLR4/NF-κB/HIF-1α环路介导的炎症反应及肾纤维化。（2）细胞实验方面：TNF-α联合缺氧条件下，大鼠近端肾小管上皮细胞（NRK52E）细胞凋亡率显著增加，炎症因子及纤维化标志物表达水平明显升高，真武汤含药血清干预可以抑制上述反应；利用细胞转染方法，体外实验揭示了真武汤上调 miR-451 表达，抑制HIF-1α表达，减轻CRS肾纤维化，双荧光素酶实验发现HIF-1α是miR-451的靶蛋白。研究结果已发表、录用英文及中文核心期刊论文7篇，在投SCI论文1篇。研究期间培养博士研究生1名。本研究有助于加深对2型CRS发生机制的认识，真武汤保护 CRS 肾纤维化的作用做出新的阐述，为真武汤在此领域的拓展应用提供新的思路。