Nrf2通过抑制Notch1信号通路改善内皮祖细胞功能治疗糖尿病下肢缺血的效应及机制研究

Abstract: In diabetes mellitus, the number of circulating endothelial progenitor cells (EPCs) is decreased and the angiogenic function of EPCs is attenuated, which is one of key factors leading to ischemic vascular complications in diabetes. The preliminary data showed that Nrf2 expression in diabetic EPCs is decreased, which coincided with impaired survival and tube formation abilities; furthermore, the expression of Notch1 is negatively associated with Nrf2; elevating the expression of Nrf2 or knockdown the expression of Notch1 could similarly improve the survival and angiogenic function of diabetic EPCs. Based on these preliminary data, we hypothesized that the decreased expression and function of Nrf2 in diabetic EPCs resulted in activating Notch1-mediated anti-angiogenic signaling, which leads to the angiogenic function impairment in diabetic EPCs. Inhibiting Notch1-mediated anti-angiogenic signaling pathway by elevating Nrf2 expression or activating Nrf2 signaling can effectively improve the angiogenic function of diabetic EPCs to promote diabetic ischemic vascular regeneration and blood flow recovery. The main objective of this project is to elucidate the relationship between Nrf2 and the function of diabetic EPCs; to investigate whether Nrf2 is a key target to improve the function of diabetic EPCs by regulating Nrf2 expression or Nrf2 specific agonist, and to clarify the underlying mechanism; translationally to evaluate the feasibility of Nrf2 agonist in the treatment of diabetic ischemic vascular disease.

外周循环中内皮祖细胞（endothelial progenitor cells, EPCs）数量减少和血管形成功能受损是导致糖尿病缺血性血管并发症的关键原因。预实验研究发现糖尿病小鼠来源的EPCs中Nrf2表达下调，并伴随存活和血管形成功能受损，且发现Nrf2与Notch1的表达呈负相关，上调Nrf2的表达或抑制Notch1的表达均能改善EPCs的存活和血管形成功能。据此，我们提出科学假设：提高Nrf2的表达或激活Nrf2信号从而抑制Notch1信号，能有效改善糖尿病EPCs的血管形成能力，促进糖尿病缺血性血管再生。本课题的主要目标是阐明Nrf2与糖尿病导致的EPCs数量减少和功能受损的关系，利用基因干预或Nrf2特异性激动剂考察Nrf2对糖尿病EPCs功能的改善作用；并阐明Nrf2改善糖尿病EPCs功能的分子机制；最后从转化医学的角度，评价Nrf2激动剂治疗糖尿病缺血性血管疾病的可行性。

外周循环中内皮祖细胞（endothelial progenitor cells, EPCs）数量减少和血管形成功能受损是导致糖尿病缺血性血管并发症的关键原因。项目组前期实验研究发现糖尿病病人和糖尿病小鼠来源的EPCs中Nrf2表达下调，并伴随存活和血管形成功能受损，但Nrf2对糖尿病EPCs血管形成功能的直接调控作用及其分子机制尚不清楚。本项目拟通过分别分离正常人和糖尿病人外周血中EPC，并利用Nrf2-shRNA和Nrf2过表达慢病毒载体分别调控正常人和糖尿病人EPC中Nrf2的表达，通过体外评价Nrf2的表达变化与EPC存活和血管形式功能的关系，结果发现Nrf2正向调控EPC的存活和血管形成功能；进一步体内考察Nrf2对小鼠EPC移植治疗糖尿病下肢缺血修复的作用，结果表明Nrf2改善糖尿病EPC功能，促进缺血下肢的血流恢复和血管新生。. 为阐明Nrf2改善糖尿病EPC血管新生功能的分子机制，基于糖尿病导致EPCs功能受损的关键因素是氧化应激损伤，而线粒体作为活性氧（ROS）生成的主要细胞器，同时线粒体的形态及功能受ROS水平的调节，因此，在本研究中重点聚焦Nrf2是否通过调控EPC线粒体形态及功能，从而改善EPC血管形成功能，并通过转录组学分析找到其下游的分子作用靶点，证实Nrf2是通过转录调控IDH2改善EPC线粒体形态和功能。. 最后，从临床转化医学的角度出发，通过考察Nrf2特异性激动剂莱菔硫环（Sulforaphane，SFN）对糖尿病小鼠下肢缺血的治疗效果，并评价SFN对db/db小鼠外周血中EPC数量的影响，并考察SFN对体外高糖条件下EPC存活及血管新生功能的影响。 结果发现：SFN能有效改善高糖条件下EPCs的存活和血管形成功能；同时体内实验发现SFN能有效促进糖尿病小鼠下肢缺血的血流恢复和血管新生。. 综上所述，该项目明确了Nrf2对糖尿病EPC血管新生功能的保护作用，并初步阐明其主要作用机制是通过转录激活IDH2而调控EPC线粒体形态及功能，从而改善糖尿病EPC血管新生功能，最后从转化医学的角度，利用Nrf2激动剂干预治疗，证实Nrf2作为改善糖尿病EPC功能的有效靶点，为糖尿病血管病变的治疗提供新的分子靶点。