基于PI3K/Akt信号通路探讨微循环障碍相关microRNA在血瘀质非创伤性股骨头坏死中的调控机制及活血化瘀方调体干预研究

NONFH is a complex disease caused by multiple factors and regulated by multiple pathways. The study confirmed that the disease was associated with ischemia caused by local microcirculation disorder. And the dysfunction related to angiogenesis and platelet activation is one of the major factors leading to microcirculation disorder. The results of the previous project of NSFC showed that blood stasis constitution was NONFH and high incidence constitution in NONFH, and PI3K/Akt signaling pathway was the main enrichment pathway of NONFH of blood stasis constitution. At the same time, microcirculatory disorder related microRNA is an important regulatory factor, which is closely related to the occurrence of NONFH. So the project intends to take the PI3K/Akt signaling pathway as the breakthrough point, revealing the regulation mechanism of microRNA related to angiogenesis and platelet activation in NONFH of blood stasis constitution and the pathological mechanism of NONFH of blood stasis constitution at molecular level by the technology of RT-PCR and Western Blot, looking for valuable molecular biological markers. We will further study the effect of activating blood circulation to dissipate blood stasis prescription called for Longzhongsunshang Capsule on the expression of the signal pathway and analyze the intervention mechanism of it by constitution conditioning. It will provide new theoretical basis, ideas and targets for the prevention and treatment of NONFH by traditional Chinese medicine.

非创伤性股骨头坏死（NONFH）是多因素致病、多通路调控的复杂疾病。相关研究表明NONFH的核心病机与局部微循环障碍造成的缺血密切相关，而血管新生与血小板活化相关功能失常则是导致微循环障碍的主要因素之一。前期国家自然科学基金研究结果显示血瘀质为NONFH高发体质之一，PI3K/Akt信号通路为血瘀质NONFH主要富集通路。而微循环障碍相关microRNA是重要的调控因子，与NONFH的发生密切相关。故本项目拟以PI3K/Akt信号通路为切入点，利用RT-PCR及Western Blot等技术，通过揭示血管新生与血小板活化相关microRNA在血瘀质NONFH中的调控机制以阐明血瘀质NONFH发病机制，寻找有诊断价值的分子生物学标记物；并深入分析临床疗效确切的活血化瘀经验方——陇中损伤散对血瘀质NONFH调体干预机制，为NONFH的中医药防治提供新的理论依据、思路和治疗靶点。

目的：通过NONFH的流行病学调查，明确其高发体质类型及高发因素，基于PI3K/AKT信号通路探讨血管新生及血小板活化相关microRNA在血瘀质非创伤性股骨头坏死的调控机制，探讨部分不同体质NONFH患者血液指标差异，明确不同体质患者血液指标差异及应用活血化瘀方后血液指标差异变化。.方法：收集2015年1月至2021年12月在甘肃省中医院骨科就诊的NONFH患者共1286例，建立NONFH数据库，并对疾病发病人群、高发体质类型等进行统计学分析。采集患者及健康志愿者血液标本，提取血浆标本，采用RT-PCR法检测其血浆miRNA相对表达量，筛选差异表达的miRNA。应用实时PCR及wb等方法对差异表达的miRNA进行验证。构建兔SONFH模型，通过病理及实验室指标检测观察活血化瘀方对SONFH的治疗效果及miR-21、PTEN、VEGF、P2Y12等的影响。.结果：NONFH患者流行病学调查研究发现，患者体质为血瘀质286例、平和质196例、气虚质180例、痰湿质155例； 对患者骨组织进行HE、免疫组化、wb及pcr实验，对血瘀质 NONFH 差异性血清标志物进行检测，检测股骨头组织差异标志物，t-Pa 和 VEGF 是血瘀质 NONFH 的特异性指标；血瘀质NONFH的发生与 has-miR-486-5p 下调后降低了 t-Pa 基因 3’UTR 的能力，使 t-Pa 在血瘀质 NONFH的血清中升高。通过对患者血液指标PCR及wb方法检测得出血瘀质NONFH 患者外周血中：VEGF基因高表达，且经过活血化瘀方干预后，呈现降低趋势，PI3K与VEGF呈正相关，提示PI3K可能为VEGF 的上游调控因子。通过动物及细胞发现活血化瘀方治疗股骨头坏死机制与血管新生相关，通过上调血清miR-21的表达，激活PI3K/Akt/VEGF通路信号通络，促进内皮细胞增值，从而使局部血管新生。.结论：进一步明确了NONFH的高发体质类型，发现血瘀质是AIONFH的高发中医体质，完善了NONFH病例数据库，获得了血瘀质NONFH差异表达miRNA的特异性表达，及血管新生及血小板活化相关microRNA在血瘀质NONFH中的调控机制及血瘀质NONFH发病机制。