基于Wnt/β-catenin靶点的益气活血方抗糖尿病视网膜病变作用机制研究

Diabetic retinopathy is one of the most common complications of diabetes with high incidence of blindness which seriously impact on human health. To study the modulation mechanism of "Yi-Qi-Huo-Xue fang" on diabetic retinopathy based on wnt/β-catenin signaling pathway, a primary diabetic retinopathy model on rats, a human retinal microvascular endothelial cell line (RMECs) and retinal ganglion cell line (RGC-5) would be built with high fat-high sucrose feeding, the expression of β-catenin in nucleus and cytoplasm would be observed by immunofluorescence staining and immunoprecipitation assays et al., the target gene of wnt/β-catenin signaling pathway would be verified. Moreover, the wnt/β-catenin signaling pathway would be stimulated by TWS119 (a GSK-3β inhibitor) both in vivo and in vitro. Later, the regulation of wnt/β-catenin signaling pathway on inflammation, vascular neogenesis and neuron apoptosis in model rats' retinas would be investigated. Based on the above built model in ras and cell lines treated with high fat-high sucrose and TWS119, the molecule mechanism of Yi-Qi-Huo-Xue fang" on diabetic retinopathy would be found. The chief achievement of this study would put forword a new medication target for retinal diseases and enrich the basic theory for their clinical uses.

糖尿病视网膜病变具有很高的致盲率，严重影响人类健康。为了探明糖尿病视网膜病变发生过程中Wnt/β-catenin信号通路的调控规律，确立益气活血方改善糖尿病视网膜病变的药理机制，本项目将建立大鼠糖尿病视网膜病变模型及体外培养的视网膜血管内皮细胞和视神经节细胞体系，采用荧光免疫染色、免疫沉淀等技术检测模型形成中Wnt/β-catenin信号通路相关蛋白如GSK-3β、β-catenin等分布及表达变化，并确立通路靶基因。采用工具药TWS119（GSK-3β抑制剂）构建体内外Wnt/β-catenin信号通路，研究信号通路调节大鼠视网膜炎症发生、新生血管形成和神经细胞进行性丢失的规律。基于上述构建的糖尿病视网膜病变模型和Wnt/β-catenin信号通路，探明益气活血方改善糖尿病视网膜病变的作用机制。本项目拟取得的研究成果能丰富糖尿病视网膜疾病发生的基本理论，为视网膜疾病新药开发提供靶点。

益气活血方是治疗视网膜病变的中药复方，本项目旨在探明该方治疗糖尿病视网膜病变的作用，并观察其是否通过调控Wnt/β-catenin通路产生视网膜神经节细胞（RGCs）保护作用。①首先采用HPLC法对益气活血方进行质量控制，测得复方中8个特征峰含量。②采用GK大鼠构建糖尿病视网膜病变（DR）模型，给予益气活血方12周，发现40周龄GK大鼠随机和空腹血糖升高、视盘水肿，视网膜血管迂曲肿胀扩张、RGCs显著丢失、视网膜神经节细胞层（RGCL）β-catenin和VEGF表达显著增加。益气活血方能显著降低随机和空腹血糖、有效改善大鼠视网膜水肿和血管扩张，显著降低RGCL层β-catenin和VEGF过表达，减缓RGCs丢失。NMDA诱导大鼠视网膜病变模型，给予益气活血方干预。NMDA显著增加β-catenin、COX-2、VEGF、Cleaved-caspase-3、-8和-9蛋白表达，导致RGCs凋亡。NMDA抑制剂MK801和β-catenin抑制剂DKK-1、COX-2抑制剂NS398均能缓解NMDA所致的β-catenin、COX-2表达增加和神经元丢失，益气活血方通过降低β-catenin、COX-2、VEGF、Cleaved-caspase-3、-8和-9蛋白表达，抑制RGCs凋亡。④丙酮醛（MGO）是DR形成过程中重要参与因子。采用MGO诱导视网膜神经节细胞（RGC-5）建立体外DR模型。抗氧化、抑制AGEs、抑制β-catenin通路能改善MGO对RGC-5的诱导损伤。益气活血方及其主要活性成分丹酚酸B能改善MGO对RGC-5的诱导损伤。⑤采用Wnt/β-catenin信号通路激活剂TWS119构建大鼠视网膜损伤模型。TWS119显著增加COX-2表达，并降低RGCs数目，说明β-catenin通路激活导致COX-2表达升高，导致神经元丢。NS398和IVB均能缓解RGCs丢失，说明COX-2和VEGF是β-catenin通路导致神经元损伤的关键蛋白，可以通过阻滞该通路、调控蛋白阻止神经元丢失。益气活血方通过减少TWS119诱导的β-catenin、VEGF和COX-2过度表达，减少神经元丢失。⑥综上，活血祛瘀方是通过调控Wnt/β-catenin信号通路产生视网膜神经节细胞保护作用，进而阻止DR的发生。本项目探明了益气活血方的临床作用及药理机制。