基于R-Ras/Rac1通路多环节串扰募集周细胞探讨益气活血方药心脑血脉宁抑制斑块内出血的机制

Atherosclerosis (AS) with intraplaque hemorrhage (IPH) easily induces acute cardiovascular events. The main cause of IPH is pathological angiogenesis which appears pericytes absence in plaque, thus it is a new perspective that promotes new vessels to recruit pericytes for suppressing IPH. Qi deficiency and blood stasis are throughout AS, and preliminary study (No:81102699) has proved that Xinnao Xuemaining which supples Qi and activates Blood can facilitate pericytes recruitment, while the in-depth mechanisms remain to be elucidated. Recently researches discover that the signaling networks formed by R-Ras/Rac1 signaling and multi-link crosstalks are critical to regulate cells migration, therefore we propose a hypothesis that Xinnao Xuemaining promote pericytes migration through interventing the signaling networks on the basis of clinical and previous effects definitely. The study uses apoE - / - mousse for IPH model, new vessels in aortic ring and co-culture model of endothelial cells and pericytes as objects, and apply Phosphorylation protein chips conbined with RNAi technology to evaluate the correlation between signaling networks of R-Ras/Rac1 and IPH. From the point of Chinese Medicine Wholism and signaling networks, the study discuss the molecular mechanism that Xinnao Xuemaining raises pericytes to inhibit IPH, and has important significance to enrich the connotation of prescription which supples Qi and activates Blood The signaling proteins regulated in the study promise to be important target of inhibiting IPH in clinical.

动脉粥样硬化（AS）斑块内出血（IPH）易引起急性心脑血管事件。斑块内出现周细胞缺如的“病理性血管新生”是导致IPH的主因，故促新生血管募集周细胞是抑制IPH的新视角。气虚血瘀贯穿AS始终，前期（No：81102699）证实益气活血方药心脑血脉宁可促周细胞募集，然其深入机制尚待阐明。近期发现R-Ras、Rac1通路多环节串扰形成的信号网络调控细胞迁移，故在临床和前期疗效确切的基础上，提出心脑血脉宁干预上述信号网络促周细胞迁移的假说。拟以apoE-/-小鼠IPH模型、动脉环新生血管模型、内皮细胞/周细胞共培养模型为研究对象，以磷酸化蛋白芯片与RNAi 相结合，评价R-Ras、Rac1信号网络与IPH的相关性。从中医整体观角度、借助信号网络研究策略，探讨心脑血脉宁募集周细胞，抑制IPH的分子机制，对丰富益气活血方药的内涵具有重要意义。本研究所涉及的信号蛋白有望成为临床抑制IPH的重要靶点。

动脉粥样硬化斑块内出血是导致斑块破裂的重要因素。抑制斑块内病理性血管新生是稳定易损斑块的有效途径。心脑血脉宁为天津中医药物研究院的院内制剂（本质为补阳还五汤加减方颗粒剂），多年来用于临床治疗心血管疾病，本研究对其进行深入研究。本研究分为两部分：（一）采用6-8周龄ApoE-/-小鼠高脂喂养24周复制斑块内出血模型，给药12周之后发现补阳还五汤可明显改善AS模型小鼠血脂四项，减小斑块面积。此外，斑块内含铁血黄素沉积及斑块内出血情况减少，小鼠颈动脉PDGFRβ表达明显上调。提示补阳还五汤可促进斑块内新生血管成熟度，减少斑块内出血。新生血管动脉环实验及周细胞募集实验结果显示补阳还五汤促进斑块内新生血管成熟，减少斑块内出血的作用机制，与促进内皮细胞分泌bFGF、PDGFβ，上调周细胞PDGFRβ、Rac1、RhoA的表达有关，且ras/rac1通路为补阳还五汤促进内皮细胞募集周细胞，提高新生血管成熟进而减少斑块内出血的关键靶点。（二）炎症贯穿AS发展过程，肠道菌群紊乱导致LPS入血是引起炎症的关键因素。且越来越多的研究证明年龄在AS发展中起着不可或缺的作用，因此我们考察了青年及老年鼠AS的病变程度及其与肠道菌群紊乱的关系。采用高脂喂养６-８周龄ApoE-/-小鼠10周复制青年鼠AS模型（第一部分中6-8周龄ApoE-/-小鼠高脂喂养24周为老年鼠模型），给药10周，取青年鼠及老龄鼠肠道菌群进行测序，结果显示同年龄阶段模型组有益菌减少，有害菌增加；老龄对照与青龄对照相比有益菌减少；老龄模型与青龄模型相比，有益菌减少，有害菌增加。此外，高脂饮食和老龄均能降低肠道粪便中SCFAs及LPS水平、升高回肠末端紧密连接蛋白表达含量，并降低血清及主动脉组织中中炎症因子表达情况。因此，同样高脂条件下，老龄ApoE-/-小鼠表现为更为严重的AS病变，其机制与老龄个体脾胃痿瘁，脾失健运致肠道菌群失调，肠黏膜屏障受损，LPS入血，进而引发机体慢性低度炎症反应有关。