n-3多不饱和脂肪酸对糖尿病冠状动脉平滑肌细胞BK通道的激活作用及其机制研究

Diabetes mellitus accelerates the progress of coronary heart disease and promotes the clinical manifestations of myocardial ischemia in advance. Previous clinical studies have demonstrated that n-3 polyunsaturated fatty acids (n-3PUFA) have the potential dilated effects on diabetic coronary arteries, however, the mechanisms are not well understood. In the study of our previous national natural grant entiled "The effects and molecular mechanisms of TRPC1-BK complex on diabetic coronary function (No.81070157)" supported by Chinese National Natural Science Foundation in 2010, we have found that the large conductance calcium activated potassium channels (BK channels) are widely distributed in coronary smooth muscle cells and play an important role in adjusting coronary relaxation, however, the BK channel ?-subunit expression and current density were decreased in diabetic coronary smooth muscle cells, leading to the coronary contractions. In the meanwhile, we found that n-3PUFA could activate BK channels in normal coronary smooth muscle cells. In this study, we try to investigate the effects of n-3PUFA on BK channels of diabetic coronary smooth muscle cells. On the setting of our previous study results, we put forward the hypothesis that n-3PUFA can increase the expression of BK channel ?-subunit and the effects of n-3PUFA on BK channels in diabetic coronary arteries are performed by cytochrome P450 epoxygenase metablites and increased intracellular calcium.To test this hypothesis, we will investigate the activation mechanisms of n-3PUFA on diabetic coronary BK channels from the levels of ion channel, cell, tissue and animal by patch clamp technique, molecular biology technique, intracelluar calcium concentration assay and coronary tention measurement in this study. The purpose of this study is to elucidate the electrophysiologic mechanisms of n-3PUFA on BK channel activation in diabetic smooth muscle cells. The study will be performed from the new viewpoint of cellular electrophysiology, which may form the basic foundation of the dilated effects of n-3PUFA on diabetic coronary arteries and may provide a new strategy for preventing and retarding the progress of coronary heart disease in patients with diabetes mellitus.

糖尿病加速冠心病进展，并使心肌缺血临床表现前移。已有临床研究表明n-3多不饱和脂肪酸(n-3PUFA)对糖尿病冠状动脉(冠脉)具有扩张作用，但其机制不明。申请者在主持的前一个国家自然科学基金课题研究中发现糖尿病时冠脉大电导钙激活钾通道(BK通道)?-亚单位表达减少，BK通道电流密度降低，而n-3PUFA可激活BK通道。据此提出假说：n-3PUFA可增加糖尿病冠脉BK通道?-亚单位表达，并通过细胞色素P450环氧化酶代谢产物和增加细胞内钙离子浓度两种途径激活BK通道。为验证这一假说，我们拟通过建立糖尿病动物模型，采用膜片钳和分子生物学等实验技术，从离子通道、细胞、组织以及动物整体水平等多层次探讨n-3PUFA对糖尿病冠脉BK通道的激活机制。本项目将从细胞电生理这个新视点进行研究，为揭示n-3PUFA对糖尿病冠脉扩张作用机制奠定基础，研究结果可能为预防和延缓糖尿病患者冠心病的发病提供新思路。

糖尿病加速冠心病进展，并使心肌缺血临床表现前移。已有临床研究表明n-3多不饱和脂肪酸(n-3PUFA)对糖尿病冠状动脉(冠脉)具有扩张作用，但其机制不明。申请者在主持的前一个国家自然科学基金课题研究中发现糖尿病时冠脉大电导钙激活钾通道(BK通道)β1-亚单位表达减少，BK通道电流密度降低，而n-3PUFA可激活BK通道。据此提出假说：n-3PUFA可增加糖尿病冠脉BK通道β1-亚单位表达，并通过细胞色素P450环氧化酶代谢产物和增加细胞内钙离子浓度两种途径激活BK通道。为验证这一假说，我们从离子通道、细胞、组织以及动物整体水平等多层次探讨n-3PUFA对糖尿病冠脉BK通道激活作用及其机制。研究内容包括：① n-3PUFA对糖尿病冠脉BK通道亚单位表达的影响；② n-3PUFA对糖尿病冠脉平滑肌细胞钙离子浓度和钙离子通道表达的影响；③ n-3PUFA对糖尿病冠脉平滑肌细胞BK通道的激活作用；④ n-3PUFA对糖尿病冠脉平滑肌细胞BK通道的体内作用影响。主要研究结果如下：① n-3PUFA灌胃增加糖尿病大鼠冠脉BK通道β1-亚单位表达；② n-3PUFA灌胃降低了糖尿病大鼠冠脉平滑肌细胞钙离子浓度和钙离子通道表达；③ n-3PUFA灌胃增加糖尿病大鼠冠脉对BK通道特异性抑制剂IBTX的收缩反应；④ n-3PUFA之一的二十二碳六烯酸（DHA）对正常及糖尿病大鼠BK通道具有激活作用。低浓度DHA可通过细胞色素P450代谢酶途径激活BK通道；高浓度DHA则对BK通道具有直接激活作用；而不同浓度DHA均可以通过磷脂酶C-三磷酸肌醇信号通路升高细胞内钙离子浓度并激活BK通道；⑤ n-3PUFA之一二十碳五烯酸（EPA）对正常及糖尿病大鼠BK通道具有激活作用。EPA可通过细胞色素P450代谢酶途径激活BK通道；⑥ n-3PUFA对糖尿病冠脉平滑肌细胞BK通道的体内激活作用可被细胞色素 P450环氧化酶抑制剂SKF525A部分阻断，表现为SKF525A阻滞了n-3PUFA引起的糖尿病冠脉平滑肌细胞BK通道表达的增加、BK通道电流的激活、细胞内钙离子浓度的降低以及冠脉对IBTX收缩反应的增加。本研究已发表核心期刊以上论文30篇，其中SCI论文4篇，中华级论文14篇。本研究初步阐明了n-3PUFA对糖尿病冠脉的扩张作用及机制，研究结果可能为预防和延缓糖尿病患者冠心病的发病提供新思路。