SIRT1介导的Resveratrol对糖尿病视网膜病变“代谢记忆”的作用及其机制研究

Oxidative stress, chronic inflammation and apoptosis plays an important role in the pathogenesis of diabetic retinopathy (DR). These changes have metabolic memory phenonmenon which means that good glycemic control, if reinstituted after a period of poor control, fails to halt the disease. Epigenetic modifications, especially the post-translational modifications of histone contributes to the metabolic memory. Class III histone deacetylase sirtuin 1 (SIRT1) is a multifunctional protein critically involved in stress responses, cellular metabolism, and aging through deacetylating a variety of substrates, including histones and transcription factors and coregulators, to regulate target gene expression both positively and negatively. SIRT1 may be an underlying mechanism for the metabolic memory of diabetic cells. Oxidative stress, chronic inflammation and apoptosis plays an important role in the pathogenesis of diabetic retinopathy (DR). These changes have metabolic memory phenonmenon which means that good glycemic control, if reinstituted after a period of poor control, fails to halt the disease. Epigenetic modifications, especially the post-translational modifications of histone contributes to the metabolic memory. Class III histone deacetylase sirtuin 1 (SIRT1) is a multifunctional protein critically involved in stress responses, cellular metabolism, and aging through deacetylating a variety of substrates, including histones and transcription factors and coregulators, to regulate target gene expression both positively and negatively. SIRT1 may be an underlying mechanism for the metabolic memory of diabetic cells. Resveratrol is an activator of SIRT1. It can attenuates renal hypertrophy and alleviates cardiac dysfunction in streptozotocin-induced diabetes. But its effect on DR, particularly its impact on the metabolic memory and the underlying molecular mechanisms have not yet been clearly elucidated. TXNIP has a critical role in inflammation and retinal injury in early stages of DR. It can regulate the expressions of NF-κ B, NLRP3 and cell adhesion molecules. It has been considered to be an important target to control the development of DR. Nrf2 is a master regulator of cellular antioxidant response and is a transcriptional factor that controls the expression of phase 2 genes like GCL and HO-1 This study is to investigate the effects and mechanisms of Resveratrol on diabetic retinopathy, focusing on the metabolic memory phenomenon.

氧化应激，慢性炎症及细胞凋亡在糖尿病视网膜病变（DR）的发病机制中发挥重要作用，这些改变具有代谢记忆现象。SIRT1调控的组蛋白乙酰化改变可能是糖尿病视网膜损伤代谢记忆产生的潜在机制。Resveratrol是SIRT1的激活剂，可改善糖尿病肾脏病变。但Resveratrol对糖尿病视网膜病变的保护研究较少，特别是其对代谢记忆的影响未清晰阐明。高血糖早期， TXNIP 表达升高，可活化 NF-κ B，激活 NLRP3，调控细胞粘附因子表达，被认为是控制DR发生发展的重要靶点。Nrf2是细胞内调节氧化应激反应的重要转录因子。本实验旨在探讨Resveratrol对糖尿病视网膜病变的作用，重点观察其能否阻止代谢记忆现象，并验证这一作用是否通过 SIRT1 介导的组蛋白乙酰化修饰，调控 TXNIP-NLRP3， NF-κ B 以及 Nrf2 通路，继而调节凋亡蛋白，炎症因子水平及抗氧化酶表达实现。

本研究旨在探索Resveratrol对糖尿病视网膜病变的保护作用，重点观察其能否阻止代谢记忆现象，并探讨这一作用的机制。第一部分利用人视网膜微血管内皮细胞进行研究，细胞分为低糖对照组、高糖对照组、高渗对照组、高糖转低糖组。对SIRT1，乙酰化 NF-κB，NLRP3的表达水平，细胞增殖活性及脂质氧化指标进行检测，结果显示，高糖条件下，SIRT1的表达明显降低，乙酰化 NF-κB，NLRP3的表达显著升高，细胞增殖活性降低，同时脂质氧化水平升高。且这些改变在高糖培养4天后转为低糖培养仍不会改变。显示高糖培养 4 天后转为低糖培养 4 天可建立稳定的高糖代谢记忆模型。随后利用该模型开展进一步研究，发现高糖条件下，炎症因子TNF-α、NLRP3、caspase 1、IL-1β、iNOS 的表达水平显著升高，且转为低糖条件后无法逆转，而Resveratrol可以提高SIRT1的表达，并抑制上述炎症因子的持续高表达。抗氧化因子Nrf2，HO-1在高糖条件下有所升高，Resveratrol可以进一步提高其表达，发挥抗氧化保护作用。为了深入探讨其作用机制，本课题利用SIRT1 抑制剂,AMPK抑制剂进行研究，结果显示，SIRT1 抑制剂 及AMPK抑制剂可部分阻断Resveratrol的保护作用。表明该药物的作用部分依赖于 SIRT1 及AMPK通路的激活。第二部分利用糖尿病大鼠模型开展研究，验证细胞实验的结论。综上，本研究显示，Resveratrol可以改善糖尿病所致视网膜代谢记忆损伤，且这一效应主要通过激活SIRT1，AMPK，抑制NF-κB、NLRP3通路及下游炎症因子，同时活化Nrf2通路，提高抗氧化酶的表达实现。Resveratrol是一种多酚类天然物质，目前所有研究均未报道其长期治疗有严重不良反应的发生，同时各类技术也被用来改进其生物利用度和稳定性， 因此， 该药物有很好的临床应用前景及临床实用价值。本研究力图为以后糖尿病视网膜保护新药的研发及药物的临床试验提供更新更多的理论与实验依据，为糖尿病视网膜病变的治疗开发新的途径。本项目资助发表文章4篇，项目经费共计21.6万元，支出193144.28元，结余22855.72元。剩余经费用于本项目研究后续支出。