HIF-1/BNIP3/Beclin1激活线粒体自噬促进光感受器-RPE细胞功能重建的机制研究

Our previous studies demonstrated that Beclin1 was induced and autophagy was activated in the retina after photoreceptor separation from the sub-RPE layer that leads to photoreceptor cell apoptosis. The mechanism of photoreceptor apoptosis after separation from the RPE is complex and still unclear. Low cellular oxygen levels act through either AMPK/mTORC activity, or through hypoxia-inducible-factor 1 (HIF-1). HIF-1 has been characterized as a central regulator of hypoxia-induced autophagy of mitochondria (termed mitophagy), thereby down-regulating oxidative phosphorylation during the adaption of primary cells to hypoxia. Hypoxia-induced HIF-1-mediated autophagy via BNIP3 is an important survival mechanism for cells. BNIP3, a member of the BH3-only subfamily of Bcl-2 family proteins, may promote survival either by preventing ATP depletion or by eliminating damaged mitochondria. BNIP3 can also negatively regulate the mTORC1 activation pathway through binding of the small GTPase Rheb to relieve anoxia stress-induced cell death. Thus we proposed that BNIP3 might play a neuroprotective role in the hypoxic stress by HIF-1/BNIP3/Beclin1 pathway after photoreceptor separation from RPE cell in a retinal detachment model. This project will investigate how the photoreceptor changes when BNIP3 gene is silenced or over-expressed in low cellular oxygen conditions. Using siRNA technology and genetic recombination technology in vivo, in vitro and 3D cell culture, we will study the HIF-1/BNIP3/Beclin1 pathway, to clarify the mitophagy mechanism. We expect to clarify the role of BNIP3 in the hypoxia after retinal detachment in order to provide new understanding of the molecular mechanisms of photoreceptor stress response, to better protect the photoreceptors and to provide new ideas for clinical rehabilitation and reconstruction of visual function after the photoreceptor-RPE separation.

课题组前期研究发现，光感受器与视网膜色素上皮细胞（RPE）分离后视网膜内Beclin1上调、自噬激活，进而导致光感受器细胞凋亡，但其作用尚不明确，分子机制待探讨。低氧应激通过诱导HIF-1上调BNIP3表达，进而促进Beclin1活化引发线粒体自噬，维持内环境氧自稳态。由此推测，线粒体自噬可能是光感受器-REP复合体分离后细胞内源性保护机制之一，HIF-1/BNIP3/Beclin1信号途径在低氧环境下起到重要调控作用。为证实以上假设，本项目以视网膜脱离为动物模型，采用基因转染调节光感受器细胞BNIP3水平，通过体内外实验探讨低氧应激对光感受器细胞存活的影响，阐明分子机制；体外构建三维细胞培养系统，研究BNIP3对光感受器-RPE细胞复合体在低氧应激后功能重建过程中的调控及其信号通路，为发现新的信号分子，干预有效信号途径和发现视功能保护新靶点提供理论依据。

视网膜脱离（RD）是严重影响患者视觉功能的致盲性眼病。课题组前期研究发现，RD后光感受器细胞发生死亡是视功能损伤的重要因素。因而明确RD后光感受器细胞死亡的调控机制是进一步保护视功能的理论基础。在本课题研究工作中，课题组首先采用大鼠制作实验性视网膜脱离动物模型，发现脱离的视网膜组织中HIF-1α和BNIP3表达均显著增加。课题组进一步采用PHI球旁注射的方法稳定视网膜组织中HIF-1α的表达，通过Western blot、免疫荧光染色、透射电镜观察等方法发现PHI干预可增强RD后视网膜组织中HIF-1α/BNIP3介导的线粒体自噬，同时减少视网膜中氧化应激产物ROS的生成，并且减少RD后光感受器细胞的凋亡的发生，在一定程度上保护视网膜，初步阐明了HIF-1α/BNIP3介导的线粒体自噬途径参与RD后光感受器细胞损伤修复的作用及机制。本课题的完成丰富了RD后光感受器细胞损伤/修复相关理论，为寻找干预RD后光感受器细胞损伤新的治疗靶点、进而更好地保护视功能提供了理论依据。