AMPK/mTOR介导的自噬在糖尿病早期视网膜Müller细胞反应性活化与神经节细胞损伤中的作用

Diabetic retinopathy is not only a microvascular disease, but a retinal neurodegenerative disorder as well. The pathogenesis that Müller cells reactive gliosis and ganglion cell apoptosisIt, take part in the progression of DR. Researchers found that autophagy defects or abnormalities associated pathways can lead to the activation of glial cell reactivity and neuronal apoptosis, and AMPK-mTOR signal pathway is an important regulatory elements involved in regulation of autophagy. Our previous work found that the expression of autophagy related protein LC3B, beclin1 decreased in early stage of DR, which suggesting that autophagy defects participate in Müller cell gliosis and ganglion cell apoptosis, and the activation of AMPK-mTOR signal pathway possibly repaire the retinal neuronal apoptosis at early stage of DR. In this research, we intends to establish DR model induced by high-fat diet and injection of STZ, and high glucose induced RGC and Müller cell models to investigate autophagy participates in Müller cell reactive gliosis and retinal ganglion cell apoptosis. This study aims to elucidate the of role of AMPK / mTOR signaling pathway involved in regulation the mechanism of autophagy of retinal ganglion cells and Müller cells, and provide a new target for early retinal neuroprotection in DR .

DR不仅是一种微血管病变，也是一种眼的“神经退行性疾病”，其中Müller细胞反应性胶质化及RGC细胞凋亡是DR发病与进展的重要环节。研究表明自噬相关通路的缺陷或异常参与早期DR形成的重要环节，而AMPK-mTOR信号通路是参与调控自噬现象的重要调节元件。本课题组前期工作发现糖尿病小鼠视网膜组织中自噬相关蛋白LC3B、beclin1表达降低，推测自噬缺陷可能参与DR早期视网膜神经退行性改变的重要原因，AMPK-mTOR信号通路激活可能抑制Müller细胞反应性胶质化及修复RGC细胞损伤。本课题拟建立高脂饮食联合小剂量STZ注射诱导C57小鼠2型糖尿病模型及高糖诱导Müller细胞及RGC细胞损伤模型，探讨自噬在Müller细胞反应性活化及RGC细胞损伤中的关键作用，以及AMPK/mTOR信号通路参与调控细胞自噬的机制，为DR早期神经修复及保护提供了新靶点。

AMPK是研究糖代谢相关疾病的核心，本研究拟探讨自噬及凋亡在糖尿病早期视网膜神经退行性病变中的作用，并进一步探讨AMPK参与视网膜神经退行性病变的相关机制。1.本研究建立了高脂饮食联合STZ构建2型糖尿病小鼠模型以及体内实验建立高糖诱导Müller细胞及661W细胞损伤模型，TUNEL法、免疫组化法及Westernblot等方法检测高糖组凋亡水平升高(Bcl-2降低，Bax增加，caspase-3增加等)，而自噬水平降低（Beclin-1降低、LC3-II/LC3-I降低、Ulk-1降低）。结果提示细胞凋亡和自噬异常参与早期DR形成。2.本研究体内及体外实验中，通过TUNEL法、免疫组化法及Westernblot法等发现HG组p-AMPK表达水平降低，而p-mTOR表达水平增加，AMPK/mTOR 信号通路被抑制，AMPK的激活能抑制 mTOR 活性，提示自噬相关AMPK/mTOR信号通路在DR中可能发挥潜在调节作用。3. 体内实验中通过加入AMPK抑制剂或激活剂及Berberine预处理不同视网膜细胞。结果表明加入AMPK激动剂AICAR或小檗碱可以增强AMPK磷酸化水平和下调mTOR磷酸化水平，激活AMPK/mTOR 信号通路，诱导细胞自噬形成，减少细胞凋亡，从而逆转糖尿病视网膜退行性损伤。结论：AMPK参与调节视网膜神经细胞自噬与凋亡，对糖尿病早期视网膜神经退行性病变起到一定保护作用，能够为DR的治疗提供潜的靶点。