Adropin内源性缺失及外源性干预对动脉粥样硬化的影响及机制研究

Atherosclerosis (AS) is one of the most widespread conditions that always threaten human health. Adropin (AD), a recently identified endogenous biologically active substance, is encoded by the energy homeostasis-associated (Enho) gene and associated with various risk factors for AS. In addition, AD play an important role in protecting endothelial function. Our previous study showed that serum adropin level was inversely associated with angiographic severity of coronary AS, and AD was expressed in aortas and myocardium of ApoE-/- mice. Therefore, we speculate that adropin may have the potential of anti-atherosclerotic effects. In this study, we generate Enho gene knock-out (KO) mice via the CRISPR-Cas system, and ApoE KO mice were administered adropin by i.p. injection. We aim to investigate the effects of adropin on energy and lipid metabolism and the process of AS, and to conform the effects of adropin on anti-atherosclerosis by biochemistry assay, histochemistry, western-blot and PCR. To elucidate the molecular mechanisms of adropin in anti-atherosclerosis, the blocking agents of signaling proteins will be used to observe the role of signal transduction pathways. AD may serve as a new therapeutic candidate for the prevention of AS.

动脉粥样硬化(AS)严重威胁人类健康。Adropin(AD)由能量动态平衡相关基因(Enho)编码，与AS的多种危险因素呈负相关，且具有血管内皮保护作用。我们前期研究显示，血清AD水平与患者冠状动脉粥样硬化严重程度呈负相关，AD表达在ApoE-/-小鼠主动脉及心肌组织。因此推测，AD可能具有抗动脉粥样硬化作用。本项目采用CRISPR-Cas 打靶系统构建Adropin缺失(Enho-/-)小鼠，同时予外源性Adropin干预高脂饮食的ApoE-/-小鼠，运用生物化学、组织化学、Western-blot、PCR等方法，观察Adropin内源性缺失及外源性干预对能量及脂质代谢、动脉粥样硬化病变进展的影响，使用信号蛋白阻断剂，探讨AD抗动脉粥样硬化的分子机制，将为抗动脉粥样硬化治疗提供新的靶点。

动脉粥样硬化(AS)严重威胁人类健康。Adropin(AD)与AS的多种危险因素呈负相关，且具有血管内皮保护作用。我们前期研究显示，血清AD水平与患者冠状动脉粥样硬化严重程度呈负相关，AD表达在ApoE-/-小鼠主动脉及心肌组织。因此推测，AD可能具有抗动脉粥样硬化作用。研究内容：本项目使用ApoE-/-小鼠，高脂饲养建立AS模型，分为ApoE-/-组和Adropin组：分别每天腹腔注射生理盐水和外源性Adropin。另取12只同窝对照野生型C57BL/6J小鼠作为对照组。取血检测血脂四项、IL-1β和IL-10的浓度；油红O染色分析主动脉根部AS斑块内脂质面积；HE染色测定胸腹主动脉血管内中膜厚度、内中膜厚度比值；免疫组化检测主动脉根部炎症因子CD68、TNF-α、MCP-1以及VCAM-1的表达；Western blot 检测主动脉CD68、MCP-1、VCAM-1；RISK通路中Akt蛋白、Hippo信号通路中yes-相关蛋白（YAP）及MAPK信号通路中氨基末端激酶（JNK）表达水平。重要结果：1、Adropin可降低血脂水平：与对照组比较，ApoE-/-组血浆中血脂四项表达水平明显升高，而Adropin可显著逆转上述改变。2、Adropin减轻动脉粥样硬化进展：对照组油红O、HE染色均未见明显动脉粥样硬化斑块形成，ApoE-/-组主动脉动脉粥样硬化斑块负荷严重，Adropin明显减轻粥样斑块负荷。3、Adropin通过减轻炎症反应抑制动脉硬化斑块进展：与对照组比较，ApoE-/-组IL-1β表达水平明显升高，IL-10表达水平明显降低，而Adropin可显著逆转上述改变。与对照组比较：ApoE-/-组见主动脉根部CD68、TNF-α、MCP-1及VCAM-1大量表达，Adropin干预明显降低上述炎症因子表达。4、Adropin通过上调YAK蛋白、下调JNK蛋白表达抑制动脉硬化斑块进展:与对照组相比，ApoE-/-组主动脉YAP表达降低，而JNK表达升高，Adropin干预可上调YAP、抑制JNK的表达，而对Akt表达没有影响。该研究首次明确外源性Adropin干预对AS形成过程的影响，探索其可能机制，为解析Adropin如何参与调控AS过程提供可靠的实验数据和理论基础，对Adropin将来应用于临床抗AS具有重要理论创新意义。