动脉粥样硬化干预新靶点--TRPC6对巨噬细胞功能的调控

Atherosclerosis (AS) is the common pathological basis of many cardiovascular and cerebrovascular diseases including coronary heart disease and stroke, which have long been the leading causes of death worldwide. Our previous studies found that macrophages in atheroma expressed higher level of transient receptor potential canonical channel 6 (TRPC6) in apolipoprotein E-deficient (Apoe-/-) mice fed with high-fat diet, a well-established animal model of AS, compared to the control mice. Trpc6 and Apoe double knockout mice，we generated for the first time，developed reduced atherosclerosis lesion than the model mice. Furthermore, the inflammatory factors induced by oxLDL and receptor-operated calcium entry (ROCE) evoked by OAD (a DAG analogue) were markedly reduced in Trpc6-deficient macrophages, companying with the dramatic decrease of calcineurin (CaN) and NFκB activation. These preliminary data hint that TRPC6 might play a pivotal role in atherogenesis via regulating inflammatory response of macrophages. In this study, Trpc6 and Apoe double knockout mice and bone marrow chimera mouse will be used to determine the roles of TRPC6 in the pathological process of AS on cell, tissue and whole animal levels with dual wavelength ion imaging technique and multiple molecular biology techniques. The potential molecule mechanisms will be further explored that TRPC6 is involved in macrophage inflammatory response through Ca2+-CaN-NFκB signaling pathway, and regulates key pathological processes such as foam cell formation, polarization, proliferation and apoptosis. The current study will provide an expanding vision for the pathological mechanisms of AS and the experimental basis to confirm TRPC6 as a new target for prevention and treatment of AS.

以动脉粥样硬化（AS）为病理基础的冠心病和脑卒中等心脑血管疾病已成为人类死亡的主因。我们前期发现：Apoe敲除的AS模型小鼠巨噬细胞瞬时受体电位通道6(TRPC6)表达显著升高；首次构建的Trpc6和Apoe双基因敲除小鼠较模型小鼠AS损伤显著减轻；Trpc6敲除使巨噬细胞炎症因子与受体操纵性钙流入减少，钙调磷酸酶（CaN）和NFκB的活性显著降低。提示：TRPC6可能是调控巨噬细胞炎症反应，参与AS形成的关键分子。本研究拟应用Trpc6和Apoe双敲除和骨髓嵌合体小鼠，采用双波长离子影像和分子生物学等技术，从细胞、组织和整体水平明确TRPC6在AS中的作用，阐释其通过Ca2+-CaN-NFκB通路影响巨噬细胞炎症反应，进而调控泡沫化、极化和增殖与凋亡等关键病理环节的分子机制。研究结果将是现有AS病理机制及调控通路的重要拓展，将为寻找新的AS干预靶点提供实验依据，具有潜在的应用和转化价值。

以动脉粥样硬化（AS）为病理基础的心脑血管疾病已成为人类死亡的主因，深入研究AS发病机制，寻找有效的治疗方法，倍显重要。本项目通过建立高脂饲料诱导的Trpc6-/-ApoE-/-基因双敲除和骨髓嵌合体小鼠AS模型，采用基因和药理干预的研究策略，应用小动物超声影像、免疫荧光染色、双波长离子影像、基因组测序等组织病理、细胞生物和分子生物学领域的技术方法，从分子、细胞、组织和整体水平系统阐释了TRPC6在AS形成中的作用，明确了TRPC6对其调控的机制：（1）Trpc6敲除可显著抑制AS模型小鼠主动脉斑块的形成、提高斑块稳定性、降低血流阻力和血清中炎症因子水平，对AS发生发展产生显著的抑制作用。（2）TRPC6在巨噬细胞内的表达特异性地升高，骨髓特异性Trpc6基因缺失可显著抑制AS模型小鼠的血管壁增厚、主动脉斑块的形成、全身炎症反应和血流阻力，提高斑块稳定性，故TRPC6可通过调控巨噬细胞功能进而促进AS的发生发展。（3）Trpc6基因敲除可显著降低oxLDL诱导的巨噬细胞泡沫化、脂质摄取、细胞凋亡、促炎因子的表达、M1和MOX型极化，同时明显增强M2型极化。（4）TRPC6可通过Ca2+-CaN-NFAT-NFκB通路促进oxLDL诱导的巨噬细胞炎症反应，重要性地调控AS的发生发展。（5）TRPC6可通过调控Ca2+-CALPAIN1-ABCA1通路和上调MARCO蛋白的表达，分别抑制oxLDL诱导的巨噬对胆固醇的外排、促进胆固醇的摄取，影响AS的发展进程。综合上述结果证明TRPC6是AS病理发展过程中调控巨噬细胞多种功能的关键蛋白；提示TRPC6可能成为AS诊断和治疗的新靶点。本课题研究结果拓展了AS发生发展过程中巨噬细胞功能变化的分子机制，发掘了AS有效诊断和治疗的新靶点，为AS的临床治疗、诊断和新药研发奠定了坚实的实验基础。