晚期糖基化终末产物损伤冠状动脉平滑肌细胞Kv及冠脉舒张功能的作用机制

Impairment of vasodilator function of coronary arterioles in diabetes mellitus plays a key role in the development of coronary artery disease. Our previous study proved that high glucose induced oxidative stress in rat small coronary arterioles (RSCAs), resulting in impairment of voltage-gated K+ (Kv) channels mediated vasodilation. However, the mechanisms remain unclear. It’s known that AGE interacting with RAGE can lead to inhibition of PPAR-γ pathway and activation of NF-κB pathway which are major mechanisms responsible for high glucose induced.production of reactive oxygen species. Based on these findings, we hypothesize that AGE/RAGE cause impairment to Kv mediated vasodilation through regulating PPAR-γ and NF-κB pathways. We will use diabetic rats in vivo, vascular loops and coronary vascular smooth muscle cells (CVSMCs) in vitro to investigate the role of AGE/RAGE pathway in the impairment of Kv channels in CVSMCs and vasodilation of coronary arterioles, and secondly we will explore the underlying mechanisms responsible for the effect of AGE/RAGE. Our study is of great significance on clarifying the mechanisms responsible for the impairment of vasodilation in coronary arterioles exposed to high glucose, which will help to provide new ideas for prevention of coronary dysfunction in patients with diabetes mellitus.

糖尿病导致冠状动脉舒张功能异常是冠心病的重要发生机制之一。我们前期研究证实高糖能引起氧化应激增加，导致Kv介导的冠状动脉舒张功能受损，但机制尚不明确。目前认为晚期糖基化终末产物（AGE）与其受体（RAGE）结合，可抑制PPARγ通路，并激活NF-κB通路，是高血糖状况下活性氧增加的主要途径。因此我们推测AGE/RAGE可能通过调控PPARγ及NF-κB通路导致Kv受损，进而使冠脉舒张功能受损，目前国内外尚无相关报道。本研究将通过糖尿病大鼠模型、血管环及体外细胞培养实验，探讨①AGE/RAGE通路对大鼠冠状动脉平滑肌细胞Kv通道及冠脉舒张功能的影响；②PPARγ通路和NF-κB通路在AGE/RAGE损伤冠状动脉平滑肌Kv通道功能中的作用。本课题结果将阐明高糖通过氧化应激导致冠脉舒张功能受损的重要分子机制，为糖尿病患者冠状动脉功能障碍提供新的治疗靶点。

背景：糖尿病引起的冠状动脉舒张功能受损在冠状动脉粥样硬化性心脏病的进程中起着重要作用。我们的前期研究证实大鼠冠状动脉平滑肌细胞（coronary artery smooth muscle cells，CSMCs）中过量产生的晚期糖基化终末产物（advanced glycation end products，AGEs）损伤电压门控的钾离子（voltage-gated potassium，Kv）通道功能，进而导致Kv通道介导的冠状动脉舒张功能减退。然而，其具体机制仍不明确。.主要研究内容：分离大鼠原代冠状动脉平滑肌细胞。CSMCs分别经100 μg/mL AGEs-牛血清白蛋白（AGEs-bovine serum albumin, AGE-BSA）、100 μg/mL非糖基化的牛血清白蛋白, 100 μM阿拉氯胺（alagebrium, ALA）, 50 μM吡咯烷二硫代甲酸（pyrrolidine dithiocarbamate，PDTC）和1 μM佛波醇12-豆蔻酸13-醋酸盐（phorbol 12-myristate 13-acetate，PMA）干预24小时。ALA是一种常用的AGEs裂解剂。PDTC和PMA分别用作NF-κB信号通路阻滞剂和激动剂。经过siRNA沉默AGER或RELA基因干预。采用膜片钳技术检测CSMCs的Kv通道电流及活性。ZDF大鼠是一种2型糖尿病模型，将ZL（对照鼠）和ZDF大鼠分组用于在体动物实验研究。应用微血管环技术检测了4-氨基吡啶阻断Kv通道后ZL与ZDF大鼠中毛喉素（forskolin）诱导的离体冠状动脉舒张反应。蛋白免疫印记和rt-PCR实验用来检测各种相关蛋白和mRNA表达水平。.重要结果：与ZL大鼠相比，ZDF大鼠血清AGEs浓度水平，冠状动脉组织中AGEs、RAGE表达以及NF-κB的活性均显著升高；然而，ZDF大鼠冠状动脉组织中Kv1.2/1.5表达则明显减少。给予氨基胍、ALA或PDTC干预以降低NF-κB活性，从而降低炎症反应与氧化应激水平，进而使CSMCs中减少的Kv1.2/1.5表达与受损的Kv通道介导的冠状动脉舒张功能得到明显改善。.科学意义：阐明糖尿病导致冠脉舒张功能受损的重要分子机制,为糖尿病患者冠状动脉功能障碍提供新的治疗靶点。