花色苷对血小板趋化因子调节动脉粥样硬化炎症反应的影响及机制研究

Atherosclerosis (AS) is a chronic inflammatory disease, and activated platelets has been recognized to play an essential function in the development of atherosclerosis. Platelets are the source of chemokines which are well known to be the consequence of pro-inflammatory mediators stored in platelets and released during atherosclerosis, efficiently attract leukocytes to the site of inflammation. Therefore, chemokines released by platelets play a crucial role in atherosclerosis via their strong potency to attract circulating inflammatory cells. In the applicant's lab, we have found that anthocyanins significantly inhibited inflammation, platelet activation and aggregation, decreased platelet deposition and prolonged the occlusion time in AS. However, the effects of anthocyanins on platelet-derived chemokines are poorly studied. Our primary data recently indicated that anthocyanins affect significantly the highly specific platelet-derived chemokines of patients with hyperlipidemia, such as CXCL7 and CCL5 both in vitro and in vivo. Therefore, the ApoE-/- mice fed with high fat diet will be used to observe the detailed markers of the different platelet-derived chemokine treated by anthocyanins in the present study, and explore whether anthocyanins could disrupt or decrease leukocyte recruitment to the site of inflamed endothelium via prohibiting the release of platelet-derived chemokines, blocking CXCL7 receptor CXCR1/R2 and activating its integrin Mac-1 to its platelet ligands as well, we also will study the effects and mechanisms of anthocyanins on platelet-derived chemokines in the different stages of AS as well as leukocyte recruitment, adhesion, migration to injured endothelial surfaces and leads to a reduction of atherosclerotic lesions in vivo. Collectively, the study will demonstrate for the first time the effects and mechanisms of anthocyanins on platelet-derived chemokines, the interactions of the different chemokines produced by platelets participate in leukocyte functions in vivo at the animal level. The highly specific platelet-derived chemokines may be a feasible approach or target for preventing and treating AS by anthocyanins.

动脉粥样硬化(AS)是慢性炎症过程，血小板在此过程中发挥重要作用，被激活后释放促炎趋化因子，招募白细胞等到达炎症部位促进斑块形成。我们以往研究表明花色苷有抑制血小板活化聚集及血栓和抗炎等效应，但其对血小板趋化因子的调控及机制缺乏研究。我们近来发现，花色苷在体内和体外均显著抑制高脂血症患者血小板趋化因子CXCL7、CCL5等的释放，故本研究拟利用高脂饲料喂饲的ApoE-/-小鼠，首先在体内明确花色苷对小鼠纯化血小板分泌的趋化因子的影响，重点探讨花色苷是否通过抑制CXCL7的受体整合素信号通路而调控白细胞的激活黏附迁移等功能及斑块形成，从而在明确花色苷对血小板趋化因子和AS炎症的体内调控效应。该研究将从动物水平明确花色苷通过调控血小板趋化因子及白细胞相互作用而抑制AS炎症的关键靶点及机制，为揭示花色苷防治AS提供科学依据。

本课题以血小板趋化因子在动脉粥样硬化中的作用为切入点，分别从体外和体内两方面来探讨了膳食花色苷对血小板趋化因子及受体的影响。首先，通过体外试验证实了花色苷可抑制健康人血小板的活化、聚集、粘附等过程；通过动物体内实验证明了花色苷对血小板颗粒物释放的抑制作用。高胆固醇血症人群膳食补充花色苷干预24周后，发现血浆中CCL5、CXCL7、CXCL5、CXCL8、CXCL12和CCL2等血小板趋化因子均有显著下降；且血浆中趋化因子的变化值与炎症因子IL-1β和血脂LDL-C的变化值呈正相关。随后开展了动物实验，在高脂饲料中分别添加200、400、800 mg/kg三个剂量的花色苷Cy-3-g，饲喂ApoE-/-小鼠16周，结果显示花色苷可显著抑制小鼠动脉斑块形成，并抑制血小板活化聚集及CXCL4、CCL2、CCL5、CXCL5、CXCL7、CXCL12等血小板趋化因子的水平；花色苷可显著抑制白细胞和血小板表面趋化因子受体CXCR4、CXCR7和CCR5的表达；花色苷还可使主动脉斑块内促炎巨噬细胞mRNA的表达减少，抗炎巨噬细胞mRNA的表达增多。为了阐明作用机制，进一步探讨了花色苷对血小板趋化因子介导的血小板与白细胞相互作用的影响，结果显示，花色苷可通过抑制单核细胞表面趋化因子受体CCR5及下游PLC-PKC信号通路，实现对CCL5/CCR5介导的单核细胞迁移的抑制作用。细胞实验明确了花色苷在“血小板趋化因子--靶细胞跨膜受体--胞内信号分子”这一信号传导通路中的作用靶标。以上体外和体内实验，证明了血小板趋化因子可作为营养干预防治动脉粥样硬化的关键靶点，为植物化学物花色苷防治心血管疾病提供了新的理论方向和实验依据。. 本课题共发表SCI论文3篇，其中1篇为JCR 1区文章，另一篇SCI论文正在撰写过程中；中文核心期刊1篇，收稿1篇，另1篇正在修稿。课题负责人获得了中华预防医学科技奖二等奖和广东省自然科学二等奖。课题团队多次参加国内外学术会议，发表会议论文摘要与壁报5篇。并保持与加拿大多伦多大学Heyu Ni教授团队和哈佛大学医学院的Wagner Denisa D教授定期的国际交流与合作。在该项目支助下，已培养硕士研究生1名，在读硕士研究生1名，本科生4名。