血糖波动通过蛋白激酶C/核因子κB/肌环指蛋白1信号通路对冠状动脉平滑肌细胞BK通道的损伤作用及其调控机制

Diabetes is a disease characterized by hyperglycemia. There are two main kinds of hyperglycemia, namely constant hyperglycemia and intermittent hyperglycemia (glucose fluctuation). More and more studies in recent years have demonstrated that the occurrence and development of diabetic vascular complications is closely related to glucose fluctuation in addition to the levels of blood glucose. The more serious the glucose fluctuation is, the higher the incidence of chronic diabetic vascular complications becomes. Furthermore, glucose fluctuation is an independent risk factor and a predictor of adverse cardiovascular events in patients with diabetes. However, the underlying regulatory mechanisms remain elusive and may be associated with abnormal intracellular signaling pathways. ..In our completed national natural grant entitled “The effects and molecular mechanisms of TRPC1-BK complex on diabetic coronary function (No.81070157)” supported by Chinese National Natural Science Foundation from 2011 to 2013, we have found that large-conductance calcium-activated potassium channels (BK channels) are widely distributed in normal coronary arterial smooth muscle cells and play an important role in adjusting coronary artery relaxation, however, coronary artery tension is increased in diabetes, leading to coronary artery contractions. In the basis of the preliminary work of this study, we found that glucose fluctuation enhanced PKC activity, activated nuclear factor kappa B (NF-κB), increased the protein expression of muscle RING finger protein 1 (MuRF1), and decreased BK channel β1-subunit expression in cultured human coronary arterial smooth muscle cells. On the setting of these results, we put forward the hypothesis that glucose fluctuation reduces the expression of BK channel β1-subunit of diabetic coronary arterial smooth muscle cells via PKC/NF-κB/MuRF1 signaling pathway, consequently decreasing BK channel current density, reducing BK channel open probability, and increasing coronary artery tension, which may lead to the increase of adverse cardiovascular events. ..In order to test this hypothesis, we will investigate the injurious effects and mechanisms of glucose fluctuation on BK channels of diabetic coronary arterial smooth muscle cells from the levels of ion channel, cell, tissue and animal by patch clamp technique, molecular biology technique, and coronary artery tension measurement in this study. The purpose of this investigation is to elucidate the injurious mechanisms of glucose fluctuation on BK channels of coronary arterial smooth muscle cells via PKC/NF-κB/MuRF1 signaling pathway. The research of this work has not been reported at home and abroad at present. The study will explore the injurious mechanisms of glucose fluctuation on BK channels of coronary arterial smooth muscle cells. The results may suggest that PKC/NF-κB/MuRF1 signaling pathway is a potential therapeutic target and may provide a theoretical basis for further research of diabetes and its complications.

目前越来越多研究证实血糖波动比持续性高血糖更能促使糖尿病血管并发症的发生，但其机制不明。申请者在主持的前一个已结题的国家自然科学基金课题研究中发现糖尿病时冠状动脉平滑肌细胞BK通道β1-亚单位表达减少，在本项目前期工作研究中，申请者发现血糖波动可使冠状动脉平滑肌细胞PKC活性增强，NF-κB磷酸化水平升高，MuRF1蛋白表达增加，BK通道β1-亚单位表达减少。据此提出如下科学假说：血糖波动通过PKC/NF-κB/MuRF1信号通路降低冠状动脉平滑肌细胞BK通道β1-亚单位表达，从而使BK通道功能受损。为验证这一假说，申请者拟通过建立血糖波动模型，采用膜片钳、分子生物学和血管张力测定等实验技术，从离子通道、细胞、组织及动物整体水平多层次探讨血糖波动对BK通道的损伤作用及其调控机制，为揭示血糖波动对BK通道的损伤作用机制奠定基础，研究结果可能为糖尿病及其并发症的进一步研究提供理论依据。

越来越多研究证实血糖波动比持续性高血糖更能促使糖尿病血管并发症的发生，但其机制不明。申请者在主持的前一个已结题的国家自然科学基金课题研究中发现糖尿病时冠状动脉平滑肌细胞BK通道β1-亚单位表达减少，在本项目前期工作研究中，申请者发现血糖波动可使冠状动脉平滑肌细胞PKC活性增强，NF-κB磷酸化水平升高，MuRF1蛋白表达增加，BK通道β1-亚单位表达减少。据此提出如下科学假说：血糖波动通过PKC/NF-κB/MuRF1信号通路降低冠状动脉平滑肌细胞BK通道β1-亚单位表达，从而使BK通道功能受损。为验证这一假说，我们通过建立血糖波动模型，采用膜片钳、分子生物学和血管张力测定等实验技术，从离子通道、细胞、组织及动物整体水平多层次探讨血糖波动对BK通道的损伤作用及其调控机制。研究内容主要包括：（1）血糖波动对冠状动脉平滑肌细胞BK通道电流和表达的影响；（2）血糖波动对冠状动脉血管张力的影响；（3）血糖波动对冠状动脉平滑肌细胞 BK 通道的损伤作用机制。主要研究结果表明，血糖波动时冠状动脉平滑肌细胞PKC活性增加，激活的PKC使核因子NF-κB活化，导致NF-κB入核增多，诱导MuRF1基因转录，增加MuRF1蛋白表达，从而使BK通道β1-亚单位表达减少，冠状动脉张力增加，血管收缩加剧。本研究已发表论文16篇，其中SCI论文10篇，中华级论文6篇。本研究初步阐明了血糖波动对冠状动脉平滑肌细胞BK通道的损伤作用及其调控机制，研究结果可能为糖尿病及其并发症的进一步研究提供理论依据。