LL-37/CRAMP-FPR2介导的ERK通路调控Müller细胞活化促进糖尿病视网膜病变纤维血管膜形成的机制研究

Müller glial cells (MGCs) activated ERK pathway contributes to the formation of fibrovascular membrane (FVM) in diabetic retinopathy, but the governing mechanism of this pathway has not been reported. FPR2, an important chemokine receptor, binding to LL-37/CRAMP are involved in cell migration and proliferation. Our previous studies were the first time to discover that high glucose significantly upregulated FPR2 in MGCs, in associated with increased MGCs chemotaxis in response to the CRAMP. High glucose also enhanced the migration and proliferation of MGCs activated by CRAMP. In addition, high glucose increased the activation of ERK1/2 pathways in response to CRAMP. Based on this, we put forward the hypothesis that high glucose promotes inflammatory responses of MGCs by upregulating FPR2 binding to LL-37/CRAMP for chemotaxis and proliferation to exacerbate fibrovascular membrane. In this study, we detected the expression of LL-37 and FPR2 in vitreous humor and FVM from patients with PDR. Then we investigate the role of CRAMP-FPR2 in MGCs and the mechanism by which it regulates the activation of ERK pathway in the development of FVM by interfering with the expression of FPR2 and CRAMP in vivo and in vitro. This study is expected to provide a novel therapeutic target to control FVM, which has a profound theoretical and clinical significance.

Müller细胞(MGCs)介导的ERK通路调控糖尿病视网膜病变纤维血管膜(FVM)的形成，但其上游调控机制尚不清楚。FPR2是重要的趋化因子受体，能够与配体LL-37/CRAMP结合激活ERK通路,调控细胞增殖和趋化。课题组首次发现高糖能上调MGCs中FPR2表达，CRAMP刺激MGCs促进细胞增殖、移行并激活ERK通路。基于此提出假设：高糖环境中，MGCs上的FPR2通过识别结合LL-37/CRAMP而活化,激活 ERK通路促进MGCs增殖、迁移、炎症因子释放从而参与FVM形成。本研究在糖尿病视网膜病变患者玻璃体和增殖膜标本中检测LL-37、FPR2表达，并建立体外高糖刺激MGCs模型、STZ诱导糖尿病小鼠模型，通过体内外干预FPR2、CRAMP，探究LL-37/CRAMP和FPR2相互作用对ERK通路的调控及在FVM发生中的作用。该研究有望为FVM的治疗提供新的靶点，具有重要作用。

Müller细胞介导的ERK通路调控糖尿病视网膜病变纤维血管膜(FVM)的形成，但其上游调控机制尚不清楚。Fpr2是重要的趋化因子受体，能够与配体CRAMP结合激活ERK通路,调控细胞增殖和趋化。本项目利用C57BL/6J和Fpr2 KO小鼠提取视网膜Müller细胞，构建高糖细胞模型，发现高糖刺激Müller细胞上的Fpr2在mRNA水平和蛋白水平均有增加。细胞增殖实验发现高糖促进Müller细胞上Fpr2介导的细胞增殖；趋化实验显示高糖刺激Müller细胞对CRAMP的趋化反应增加，抑制Fpr2后趋化受到抑制。划痕实验显示高糖促进Müller细胞上Fpr2介导的细胞迁移。高糖还能促进Fpr2介导的Müller细胞上P38和ERK1/2的磷酸化表达。CRAMP能够促进高糖刺激下Müller细胞炎症因子的分泌。构建STZ诱导糖尿病小鼠模型，发现Fpr2、CRAMP在糖尿病小鼠视网膜中表达增加，视网膜胶质细胞增生。糖尿病小鼠视网膜中TNFα、IL-1β和IL-6的mRNA表达升高，而抑制Fpr2后细胞因子表达降低。与糖尿病WT小鼠相比，糖尿病Fpr2 KO小鼠视网膜中ERK1/2和P38 MAPKs磷酸化水平降低。视网膜铺片染色显示高糖促进小鼠视网膜新生血管生成，抑制Fpr2使视网膜中的无细胞毛细血管减少。氧诱导视网膜病(OIR)小鼠模型发现Fpr2 KO OIR小鼠视网膜中血管内皮细胞增殖减少，新生血管的形成和病理性血管无灌注区面积均减少，在OIR小鼠玻璃体内注射CRAMP后，新生血管形成和血管闭塞的面积均增加。PDR患者玻璃体液中VEGF的分泌表达显著高于对照组，增殖膜中发现有FPR2的表达。这些结果表明在视网膜中存在Fpr2-CRAMP相互作用轴，影响胶质细胞活化增生、炎症反应和新生血管生成调控FVM的形成。已发表SCI论文3篇，中文3篇，参加学术会议3次。