GHRH信号通路介导炎症细胞活化在糖尿病视网膜病变中的作用及机制研究

An important hallmark of diabetic retinopathy (DR) is breakdown of the blood-retinal barrier. In recent years, much attention has been paid to the contribution of inflammatory cells in this process, but it remains unclear how the activation of the local immune cells recruit the white blood cells to the retina. The finding in our previous studies showed that GHRH receptor (GHRH-R) was obviously expressed in the infiltrating macrophages and neutrophils of the iris. Blocking GHRH-GH-IGF1 axis exerted inflammatory alleviation in the anterior segment of the rat eyes. We therefore hypothesize that GHRH secreted from the activated retinal microglia and upon binding to GHRH-R on the immune cells involves in the pathogenesis of DR through mediating leukocyte chemotaxis which in turn leads to dysfunction of the vascular endothelium. By investigation of the samples taken from the patients with proliferative DR, culturing retinal microglial cells and establishment of experimental DR model using the techniques such as immunohistochemistry, the present study is conducted to elucidate the mechanism of advanced glycationend products (AGEs)–induced GHRH signaling activation in the occurrence and development of DR in term of its action on up-regulating GHRH-R expression on the immune cells and promoting their migration toward to the retina. Also, taking advantage of GHRH antagonist to block GHRH-GH-IGF1 axis could further validate our hypothesis. The results of the present study reveal a novel mechanism underlying GHRH-mediated immune cell activity in DR with a view to investigate the potentials of GHRH as targeted molecule for DR control.

血-视网膜屏障破坏是糖尿病视网膜病变（DR）发生的关键。炎症细胞在其中的作用备受关注，但视网膜组织免疫细胞活化如何募集炎症细胞参与DR发生的机制尚未明确。申请者前期研究发现GHRH受体（GHRH-R）显著表达于虹膜组织上移行中的巨噬细胞与中性粒细胞，且证实拮抗该受体能抑制GHRH-GH-IGF1通路，缓解眼前部炎症。据此提出，活化的视网膜小胶质细胞释放GHRH，通过GHRH-R介导炎症细胞趋化破坏血管内皮屏障，参与DR发生发展。本课题拟采集增殖DR患者标本、培养视网膜小胶质细胞及构建DR模型，采用免疫组化等技术阐明糖基化终末产物激活小胶质细胞GHRH信号通路，上调GHRH-R表达，募集炎症细胞参与DR的致病机理；并利用GHRH拮抗剂阻断该信号通路的优势，进一步验证上述假说。本研究将揭示GHRH信号通路介导炎症细胞活化在DR发病中的新机制，为深入研究GHRH作为潜在治疗DR的靶分子奠定基础。

血-视网膜屏障破坏是糖尿病视网膜病变发生的关键。炎症细胞在其中的作用备受关注，但视网膜组织免疫细胞活化如何募集炎症细胞参与糖尿病视网膜病变发生的机制尚未明确。申请者前期研究发现促生长激素释放激素受体（GHRH-R）显著表达于虹膜组织上移行中的巨噬细胞与中性粒细胞，且证实拮抗该受体能抑制GHRH-GH-IGF1通路，缓解眼前部炎症。据此提出，活化的视网膜小胶质细胞释放GHRH，通过GHRH-R介导炎症细胞趋化破坏血管内皮屏障，参与糖尿病视网膜病变发生发展。 .本项目成功采集增殖性糖尿病视网膜病变患者的视网膜前增殖膜、玻璃体液、房水及血清，培养人视网膜小胶质细胞及利用链脲佐菌素STZ构建大鼠糖尿病视网膜病变的模型。利用免疫组化等实验技术，首次发现增殖性糖尿病视网膜病变是一种纤维素性炎症反应，其视网膜前增殖膜由大量渗出的纤维蛋白原及中性粒细胞组成，揭示了“纤维素性炎症”在糖尿病视网膜病变发生中的新机制；进而发现，GHRH及GHRH-R高表达于增殖性糖尿病视网膜病变增殖膜的纤维细胞、血管内皮细胞及炎症细胞，GHRH及生长激素GH的浓度在玻璃体液中分别较对照组显著升高1.8倍和72.8倍（P<0.001），但IGF-1的浓度却无明显升高。因此，GHRH/GHRH-R可能独立于GH-IGF1信号通路，参与糖尿病视网膜病变的发生与发展；实验进一步证实，高糖诱导视网膜小胶质细胞可上调GHRH-R的表达，并释放炎症介质TNF-α与MCP-1，而GHRH拮抗剂可抑制高糖（20 μM）诱导视网膜小胶质细胞释放炎症因子；且在DR动物模型中，GHRH拮抗剂可抑制大鼠视网膜血管的血管渗漏及小胶质细胞的活化。.本课题共发表3篇SCI论文（第一作者，第一基金标注），分别发表在Am J Ophthalmol（IF=4.5， 2020）、 IOVS（IF=3.8，2018）及BMC Ophthalmol（IF=1.4，2018），部分数据正在撰写论文。纤维素性炎症的提出，为强化抗炎治疗在糖尿病视网膜病变的作用提供有力的证据；GHRH/GHRH-R参与增殖性糖尿病视网膜病变的病理过程及视网膜小胶质细胞的活化，为深入研究GHRH/GHRH-R作为治疗糖尿病视网膜病变的靶分子奠定基础。