SIRT1/miR-182调控的细胞自噬对糖尿病角膜神经的保护机制

Diabetic neuropathy is the most prevalent complication of diabetes mellitus (DM). The sensory neurons are targeted and damaged by DM before significant motor neurons involvement. Management of this disease is complex and only a few approaches can unequivocally arrest progressive neuropathy beyond strict control of the glucose levels. Corneal innervation derives from the ophthalmic division of the trigeminal nerve. Diabetic keratopathy is considered to be a manifestation of peripheral neuropathy. In vivo corneal confocal microscopy represents a novel noninvasive way of diagnosing and defining the severity of diabetic neuropathy. .Sirtuin 1 (SIRT1) is a class III histone deacetylase that mediates the protective effects of neurons in neurodegenerative disorders, including Alzheimer’s and prion disease. However, the mechanism directly involved in neuroprotection of diabetic keratopathy is still poorly understood. Recent evidence has demonstrated that activating SIRT1 induces autophagy, and that activating autophagy protects neurons against neurodegenerative disorders by regulating mitochondrial homeostasis. In our preliminary experiments, we found that miRNA-182 was activated by SIRT1 and it can enhance neurite elongation in isolated trigeminal sensory neurons and overcome the detrimental corneal wound-healing effects of hyperglycemia by stimulating corneal nerve regeneration. Thus, we focused on the mechanism of the SIRT1/miR-182 mediated neuroprotective effect that was associated with regulating mitochondrial homeostasis via autophagy. These results suggest that SIRT1/miR-182 could be used as a potential neuroregenerative factor in diabetic sensory nerve injury.

角膜神经损害是糖尿病周围神经病变（DPN）的表现形式之一，而DPN与活性氧ROS介导的线粒体自噬缺陷有关。但糖尿病状态下TG神经元是否存在线粒体自噬缺陷且其在角膜神经损害中的作用鲜有研究。我们前期工作发现SIRT1通过激活三叉神经节（TG）细胞miR-182的表达，保护角膜神经免受高糖造成的损害，且SIRT1也可以减轻高糖诱导神经细胞的线粒体损伤。因此我们拟以角膜和TG作为整体，通过基因敲除鼠与ChIP-Seq等技术，探讨SIRT1/miR-182通路介导的线粒体自噬在糖尿病状态下对线粒体产生ROS和TG神经元存活的影响，该通路促进角膜神经修复的关键靶点以及调控角膜神经与上皮细胞相互作用的关键因子及通路，旨在阐明SIRT1对糖尿病角膜神经损害的保护机制，为糖尿病角膜病变的治疗提供新思路。

本研究选用2型糖尿病模型BKS-db/db小鼠，探讨了沉默信号调控因子1（SIRT1）对糖尿病角膜神经的保护作用。构建了过量表达SIRT1基因的腺病毒，使SIRT1在TG组织中过量表达，采用miRNA Array基因芯片技术，检测TG组织中受到SIRT1调控的一系列mRNAs。通过生信分析，探讨miR-182可能的靶基因，并在体外培养的TG神经细胞上进行验证。体外原代培养小鼠TG三叉神经节细胞，检测miR-182 agomir对细胞突触生长及存活情况的影响。建立小鼠角膜损伤的动物模型，结膜下注射miR-182的模拟物mimic或者抑制剂inhibitor，检测上皮细胞与神经修复相关基因与蛋白的表达，并观察角膜神经的变化。通过western blot以及定量PCR技术，结合免疫荧光共定位技术，发现SIRT1在2型糖尿病小鼠TG神经节的表达显著下降。选取其中随SIRT1表达升高，差异表达最显著的miR-182为研究对象。体外培养TG细胞，发现随SIRT1的表达显著升高，miR-182的表达也显著升高。为进一步验证这个结果，构建了过量表达SIRT1的腺病毒系统，发现随SIRT1的表达显著升高，miR-182的表达也显著升高。与正常培养三叉神经节细胞相比，miR-182 agomir处理后三叉神经节细胞突触长度显著增加。与对照组BKS-db/+小鼠相比，SIRT1在BKS-db/db小鼠的TG和角膜中表达显著下调。结膜下注射miR-182 agomir 后可以显著促进BKS-db/db小鼠角膜上皮损伤后的愈合，并显著促进角膜神经的修复，恢复角膜敏感度。