AGEs/NOX-1 介导的内皮微颗粒释放在糖尿病血管病变中的作用及机制

Vascular complications associated with endothelial dysfunction significantly influence the clinical prognosis of diabetes. Present studies indicate that endothelial microparticles (EMP) is not only one of endothelial injury markers, but also could aggravate endothelial dysfunction. Advanced glycation end products (AGEs), as one of the important injury factors involved in diabetes vascular injury, its impact on EMP releasing is unclear. In vitro cell culture, we used AGEs to simulate endothelial cells and found that not only the level of NADPH oxidase-1(NOX-1)and reactive oxygen species increased, but also EMP releasing elevated; furthermore, we observed that circulating level of EMP had a positive relation with the extent of vascular injury in diabetes. According to these findings, we supposed that AGEs/NOX-1 could mediate EMP releasing and further deterioratethe function of vascular function. In this study, we plan to use different dosages of AGEs to stimulate endothelial cell and diabetic rats, and investigate the effect of AGEs in EMP releasing. Then endothelia cells and diabetes rats with differential expression of NOX-1 are used to explore the regulating effect of NOX-1 in EMP releasing. Finally, we try to investigate the direct impact of EMP on the function of endothelial cells and aortic ring of diabetes rats, and clarify the probable mechanism. These findings could provide great support for early prevention and treatment of diabetic vascular injury.

内皮损伤导致的血管病变严重影响糖尿病患者的预后。目前研究提示，内皮微颗粒（EMP）不仅是血管内皮损伤的标志物，还可能进一步加重血管损伤。糖基化终末产物（AGEs）作为糖尿病血管损伤的重要因子，是否参与介导EMP释放，加重血管内皮损伤，尚不明确。课题组前期实验发现，AGEs体外刺激血管内皮细胞，不仅提高NADPH氧化酶1（NOX-1）表达和氧自由基生成，而且增加EMP的释放；同时发现，糖尿病患者外周血EMP水平与血管病变严重度密切相关。故推测：AGEs可能通过NOX-1途径介导EMP释放，导致内皮损伤，进而加重血管病变。本课题拟用不同浓度AGEs干预糖尿病大鼠及内皮细胞，观察AGEs促进EMP释放的效应；差异性表达NOX-1，探索其在AGEs诱导EMP释放中的调控作用；同时，应用EMP干预糖尿病大鼠离体主动脉和内皮细胞，观察其对内皮的损伤作用及其机制。为临床防治糖尿病血管并发症提供新的思路。

动脉粥样硬化是糖尿病大血管病变的基本病理改变。内皮损伤是动脉粥样硬化起始环节。内皮细胞膜受损及部分细胞膜脱落，进而形成外泌的膜性小囊泡，即内皮微颗粒（EMP）。内皮微颗粒不仅是血管损伤的生物学标记，其本身也参与血管损伤。同时糖基化终末产物（AGEs）在糖尿病血管并发症的发生发展中起着十分重要的作用。据此，本课题组体外人脐静脉内皮细胞培养，通过不同浓度糖基化终末产物对人脐静脉内皮细胞进行干预，观察内皮微颗粒的释放水平、内皮细胞糖基化终末产物的受体（RAGE）表达、NADPH氧化酶(NOX)，以及氧化应激产物(ROS)的变化。运用抗RAGE抗体、NOX阻断剂、ROS清除剂等干预措施，阐明AGEs诱导内皮微颗粒释放机制。在此基础上进一步探讨AGEs诱导分泌的内皮微颗粒对内皮细胞功能影响。我们初步实验结果显示AGEs诱导内皮细胞分泌内皮微颗粒，并且成剂量浓度依赖，与干预时间相关性不大。AGEs在诱导内皮微颗粒释放的同时，RAGE表达水平亦增高，予抗RAGE抗体拮抗能减弱AGEs诱导内皮微颗粒释放的能力；AGEs干预内皮细胞，伴随EMP水平增高，内皮细胞NOX、ROS水平亦增高，进一步予NOX抑制剂及ROS清除剂预处理内皮细胞，能明显减少AGEs诱导内皮微颗粒释放。高浓度AGEs诱导内皮细胞分泌的内皮微颗粒，在体外干预内皮细胞，观察到内皮微颗粒促进内皮细胞ROS产生，同时减少eNOS水平。以上研究结果提示AGEs诱导内皮微颗粒释放，其具体机制可能是AGEs与RAGEs相结合，激活下游NOX，诱导氧化应激，促进内皮微颗粒释放。内皮损伤产生的EMP可进一步参与血管内皮细胞的损伤，加重血管病变。这为早期防治糖尿病并发动脉粥样硬化提供了新的靶点。