VDR/Pin1轴矫正SIRT1/p66Shc失衡延缓糖尿病血管内皮衰老研究

High glucose promotes vascular endothelial senescence, participating in the pathogenesis of diabetic vasculopathy. Vitamin D improves diabetic vasculopathy, while the underlying mechanisms remain unclear. Recent studies revealed that dysregulated expression of two critical cellular senescence modulators ,i.e., downregulation of SIRT1 expression and upregulation of p66Shc expression, mediate the high glucose-induced, ROS-dependent endothelial senescence. Our preliminary study found that vitamin D, by activating vitamin D receptor (VDR), abolished the upregulation of prolyl imersorase1 (Pin1) expression (i.e. VDR/Pin1 axis) induced by high glucose and subsequently attenuated p66Shc-mediated mitochondrial-derived ROS generation. In addition, it has been documented that, Pin1 inhibitor could enhance SIRT1 expression in some conditions. Accordingly, we speculate that vitamin D may, by activating VDR/Pin1 axis, restore the dysregulated SIRT1 and p66Shc, and consequently prevent high glucose-induced endothelial senescence. Based on the above, we aim to elucidate sequentially at the cellular and animal levels the association of VDR/Pin1 axis with vascular endothelial senescence and dysregulation of SIRT1 and p66Shc , and reveal the potential mechanisms underlying the protective effect of vitamin D against diabetic vasculopathy. We also further compare the effects of different doses of vitamin D supplementation on endothelial senescence and vascular function in patients with type 2 diabetes and vitamin D deficiency. Our research may provide novel theoretical basis for use of vitamin D in the prevention and treatment of diabetic vasculopathy.

高糖诱导血管内皮衰老，参与糖尿病血管病变（DV）病理过程。维生素D（VitD）能够防治DV，然机制不清。研究发现，血管内皮衰老关键调控蛋白失衡即信息调节因子2相关酶类1（SIRT1）表达减少和p66Shc表达增加介导高糖诱导的ROS依赖性血管内皮衰老。我们前期研究发现，VitD通过激活VitD受体（VDR）抑制高糖诱导的脯氨酰异构酶1（Pin1）表达增加（即VDR/Pin1轴），减少p66Shc介导的线粒体途径ROS生成。结合最近研究显示Pin1能够下调SIRT1表达，我们推测，VDR/Pin1轴激活可能通过矫正SIRT1和p66Shc失衡，延缓高糖诱导的血管内皮衰老。本研究拟在细胞和动物水平深入探讨VitD/Pin1轴与血管内皮衰老、SIRT1和p66Shc之间的调控机制，并比较不同剂量VitD对糖尿病合并VitD缺乏患者血管内皮衰老和血管功能的影响，为VitD防治DV提供新的理论依据。

背景:心血管并发症被认为是糖尿病患者发病和致死的主要原因之一,而糖尿病血管病变和糖尿病心肌病是糖尿病患者心力衰竭再住院和死亡的重要病因，但其中的发病机制尚未明确。因此,阐明高糖环境下血管衰老和糖尿病心肌病的发生机制及影响因素对糖尿病心血管并发症的防治具有重要意义。.研究内容:本研究一方面在细胞和动物水平深入探讨维生素D对高糖所致血管内皮衰老的影响、VitD/Pin1轴和SIRT1/p66Shc失衡在其中的作用。另一方面在动物实验水平，用维生素D干预STZ诱导的糖尿病SD大鼠模型，探讨其是否通过影响Pin1/Ampk蛋白表达及线粒体内质网偶联（MAM）形成来改善糖尿病大鼠心脏的结构和功能；并在细胞实验水平探讨激活VDR或抑制Pin1是否通过拮抗糖尿病心肌线粒体途径的凋亡，改善线粒体功能进而改善DCM。.结果: 证实了维生素D能够通过抑制高糖诱导的Pin1表达增加，在不影响糖脂代谢的情况下，改善STZ诱导的糖尿病大鼠左心室的收缩与舒张功能障碍与胸主动脉内皮的舒张功能；证明了维生素D及胡桃醌可能通过矫正SIRT1/p66Shc失衡来抑制细胞氧化应激和周期阻滞，降低p21等衰老蛋白表达，从而延缓内皮细胞衰老；证实糖尿病大鼠心脏组织及高糖下NRCMS细胞Fundc1及Ip3r2蛋白表达明显增多，维生素D干预能抑制高糖诱导的Fundc1及Ip3r2异常升高，进而减少MAM的形成，且这一过程依赖于维生素D对Ampk的激活；证实了高糖作用下NRCMS细胞中促凋亡蛋白Bax增多，抗凋亡蛋白Bcl2减少，伴随线粒体Ca2+及线粒体ROS水平升高，线粒体膜电位下降，继而引起线粒体功能紊乱及心肌细胞凋亡，而维生素D治疗可以逆转Bax/Bcl2蛋白表达失衡，从而抑制糖尿病大鼠心肌细胞的凋亡。 .意义:本研究在细胞和动物水平探索了维生素D通过VitD/Pin1轴调控SIRT1/p66Shc失衡延缓血管内皮衰老及维生素D通过激活Ampk抑制Fundc1及Ip3r2异常升高减少MAM形成，抑制高糖所致心肌细胞凋亡，从而改善糖尿病心肌病的作用，有望为维生素D防治糖尿病心血管病变提供新的理论依据和实验证据。