生长抑素sst5受体在慢性高眼压视网膜神经节细胞损伤保护中的作用及机制研究

Glaucoma is one of the leading causes for irreversible blindness, and the retinal ganglion cell (RGC) apoptosis is the fundamental pathology of glaucoma. However, the mechanisms underlying RGC death are not clearly understood. They are vital scientific questions to study the mechanisms of RGC injury in glaucoma and to explore the neuroprotective strategies for delaying RGC apoptosis. Previous studies have shown that somatostatin (SRIF) exerts neuroprotection effects in the central nervous system through inhibiting Ca2+ influx and reducing oxidative stress damage, and so on. In retina, SRIF reduces the ischemia-induced cell injury and the AMPA-induced excitotoxicity by activating different subtypes of SRIF receptors (sst1-5). However, the effects of SRIF and its receptors on RGC injury in glaucoma are still largely unknown, which are urgently needed to be studied. Our preliminary results obtained from the acutely isolated rat RGCs showed that activating sst5 receptors could regulate the T-type Ca2+ currents and outward K+ currents, suggesting that sst5 receptors may play an important role in RGC injury. In the present proposal, we aim to investigate the effects and mechanisms of SRIF and its sst5 receptors on RGC apoptosis in a chronic intraocular hypertension model by using patch-clamp electrophysiology, immunohistochemistry, calcium imaging, Western blot and molecular biology techniques. The experiments will be done in retinal slices, acutely isolated RGCs, whole animal models,etc. Our prospective research achievements would provide a scientific basis for illuminating the mechanisms of RGC injury and for effctively prevention and cure of glaucoma.

青光眼是重要的不可逆致盲性眼病，视网膜神经节细胞(RGC)凋亡是其病变的根本，但机制仍未完全阐明。青光眼RGC损伤机制的研究和探寻阻止RGC凋亡的策略是该领域的重要科学问题。生长抑素(SRIF)在中枢神经系统通过抑制Ca2+内流和降低氧化应激损伤等发挥神经保护作用。在视网膜，通过激活不同的受体亚型(sst1-5)，SRIF降低缺血导致的细胞损伤和AMPA引起的神经兴奋毒性作用。但SRIF及其受体在青光眼RGC损伤中的作用还是亟待研究的科学问题。我们在急性分离的大鼠RGC上的预实验结果显示，激活sst5受体可调控RGC的T-型Ca2+通道和外向K+电流，提示sst5受体可能在RGC损伤中有重要作用。本课题拟利用电生理、免疫组化、钙成像、Western blot和分子生物学等多种技术，研究SRIF及其sst5受体在慢性高眼压RGC凋亡中的作用及机制，为有效地防治青光眼提供理论参考依据。

视网膜神经节细胞(retinal ganglion cell,RGC)的凋亡是青光眼视神经损伤的最终病理结局。生长抑素(SRIF)通过作用于不同的受体亚型(sst1-5)对视网膜细胞损伤具有神经保护作用，但sst5受体激活对青光眼RGC损伤的保护作用及其机制不明。我们发现慢性高眼压大鼠腹腔注射sst5受体激动剂L-817,818可显著减少RGC中TUNEL阳性细胞数目，增加RGC存活。分光光度法测定视网膜组织中线粒体呼吸链复合物(MRCC)的活性以及过氧化氢(H2O2)和脂质过氧化物丙二醛(MDA)的含量，发现L-817,818可明显逆转高眼压导致的MRCCIII活性降低以及H2O2和MDA水平升高。qPCR和Wes全自动蛋白质表达定量分析结果表明高眼压中Bax基因及蛋白表达均上调，Bcl2表达均下调，而L-817,818可显著逆转这种表达变化。此外，L-817,818还可下调高眼压中Caspase-3和Caspase-9蛋白表达升高。在急性分离的大鼠RGC上，利用全细胞膜片钳技术发现L-817,818分别通过胞内NO/cGMP/PKG信号通路和PLC/PKC信号通路压抑T-型Ca2+通道和外向K+通道电流。这些结果表明，sst5受体激活通过改善线粒体功能，减少氧化应激反应，调控离子通道，对高眼压导致的RGC凋亡起到抑制作用。