【作者】 吴平；

【导师】 彭惠；

【作者基本信息】 重庆医科大学 ， 临床医学（专业学位）， 2025， 博士

【摘要】 目的:年龄相关性黄斑变性（Age-related macular degeneration,AMD）是一种黄斑区视网膜逐渐衰退的常见疾病,目前被广泛认为是50岁以上中老年人中心视力丧失的首要诱因。AMD的发展过程中,视网膜色素上皮（Retinalpigmentepithelium,RPE）细胞因氧化应激而受损起着至关重要的作用,这种损伤显著加速了AMD的进程。然而,关于RPE细胞退化的具体机制及潜在的预防手段,目前尚不完全清晰。值得注意的是,β淀粉样蛋白（amyloidβ-protein,Aβ）作为AMD视网膜玻璃膜疣的关键成分之一,能够激发视网膜细胞释放炎症因子和血管生成因子,且研究表明Aβ与铁死亡现象密切相关。因此,探究Aβ是否诱导RPE细胞发生铁死亡,可能为AMD的治疗开辟新的潜在途径。另一方面,褪黑素（Melatonin,MEL）通过作用于线粒体,对视网膜的氧化应激、炎症反应及细胞凋亡产生调节作用。深入研究褪黑素在AMD防治中的潜力及其作用机理,有望为AMD的治疗带来创新思路与策略。方法:将ARPE-19细胞在37℃、5%CO₂的孵箱中培养一定时间后,用Aβ1-40干预48小时后构建RPE细胞铁死亡模型,其后通过检测脂质过氧化（C11-BODIPY和MDA）、活性氧（ROS）水平、透射电子显微镜（TEM）观察、铁含量测定（Ferro Orange）、实时荧光定量PCR（q-PCR）、蛋白质印迹（Western Blotting）和免疫荧光等方法来评估铁死亡情况,此外,采用褪黑素进行预处理ARPE-19细胞来评估其抑制Aβ1-40诱导RPE细胞铁死亡的作用。通过玻璃体腔注射Aβ1-40构建Aβ1-40诱导的视网膜损伤小鼠模型,其后采用腹腔注射褪黑素进行干预实验,利用视网膜电图（ERG）、光学相干断层成像（OCT）以及苏木精-伊红（H&E）染色等技术,系统评估小鼠视网膜结构和功能的变化。采用网络药理学方法,探究褪黑素在AMD中对RPE细胞铁死亡保护作用的潜在分子机制。为阐明RPE细胞铁死亡的分子调控机制,本研究通过Western blotting和脂质过氧化水平检测,分析了PI3K/AKT/MDM2/P53信号通路及SLC7A11的表达变化。为进一步验证MDM2的功能,利用si RNA技术在体外构建MDM2敲低模型,评估其对RPE细胞铁死亡进程的影响。结果:Aβ1-40能够诱导RPE细胞发生铁死亡,而褪黑素干预可以有效缓解这一病理变化。在小鼠动物模型中,褪黑素可减轻Aβ1-40导致的视网膜组织损伤,促进视觉功能修复。基于网络药理学的分析显示,褪黑素对AMD的作用靶点与铁死亡调控网络存在显著交集,其中PI3K/AKT/MDM2/P53信号轴可能发挥关键作用。进一步的机制研究证实,该信号通路介导了褪黑素的细胞保护效应,而特异性抑制MDM2表达则会显著降低其对氧化损伤和铁死亡的调控能力。结论:我们的研究显示Aβ1-40诱导RPE细胞发生了铁死亡,同时褪黑素可通过PI3K/AKT/MDM2/P53途径抑制RPE细胞发生铁死亡,进而预防和减缓AMD的进展。更多还原

【Abstract】 Objective:Age-related macular degeneration（AMD）is a prevalent condition characterized by the gradual deterioration of the macula in the retina,currently recognized as the primary cause of central vision loss in individuals aged 50 and older.In the progression of AMD,damage to retinal pigment epithelium（RPE）cells due to oxidative stress plays a pivotal role,significantly accelerating the disease process.However,the specific mechanisms underlying RPE cell degeneration and potential preventive measures are not yet fully understood.Notably,amyloidβ-protein（Aβ）,a crucial component of drusen in AMD retinas,can stimulate the release of inflammatory and angiogenic factors by retinal cells.Studies have also indicated a close relationship between Aβand ferroptosis.Therefore,investigating whether Aβinduces ferroptosis in RPE cells may open up new potential avenues for AMD treatment.On the other hand,melatonin（MEL）exerts regulatory effects on oxidative stress,inflammatory responses,and apoptosis in the retina by acting on mitochondria.Further research into the potential and mechanisms of melatonin in the prevention and treatment of AMD could lead to innovative ideas and strategies for managing this condition.Methods:After culturing ARPE-19 cells in an incubator at 37°C with5%CO₂for a specific duration,we induced ferroptosis in RPE cells by treating them with Aβ1-40 for 48 hours.Subsequently,we evaluated the ferroptosis status through various methods,including detection of lipid peroxidation（using C11-BODIPY and MDA）,reactive oxygen species（ROS）levels,transmission electron microscopy（TEM）observation,iron content measurement（using Ferro Orange）,real-time fluorescent quantitative PCR（q-PCR）,Western blotting,and immunofluorescence.Additionally,melatonin was used to pretreat ARPE-19 cells to assess its inhibitory effect on Aβ1-40-induced ferroptosis in RPE cells.The Aβ1-40-induced retinal injury mouse model was established through intravitreal injection of Aβ1-40 oligomers,followed by intraperitoneal melatonin administration as the interventional treatment.Comprehensive evaluations of structural and functional alterations in murine retinas were conducted using electroretinography（ERG）,optical coherence tomography（OCT）,and hematoxylin-eosin（H&E）staining techniques.Network pharmacological methods were employed to explore the potential molecular mechanisms of melatonin’s protective effect on RPE cell ferroptosis in AMD.To elucidate the molecular regulatory mechanisms of RPE cell ferroptosis,this study analyzed changes in the PI3K/AKT/MDM2/P53 signaling pathway and SLC7A11 expression through Western blotting and lipid peroxidation level detection.To further validate the function of MDM2,an MDM2 knockdown model was constructed in vitro using si RNA technology to assess its impact on the ferroptosis process in RPE cells.Results:Aβ1-40 can induce ferroptosis in RPE cells,while melatonin intervention can effectively alleviate this pathological change.In mouse models,melatonin reduces retinal tissue damage caused by Aβ1-40 and promotes visual function recovery.According to network pharmacological analysis,melatonin’s targets for AMD significantly overlap with the ferroptosis regulatory network,with the PI3K/AKT/MDM2/P53 signaling axis potentially playing a pivotal role.Further mechanistic studies confirm that this signaling pathway mediates the cytoprotective effects of melatonin,and specific inhibition of MDM2 expression significantly reduces its regulatory capacity for oxidative damage and ferroptosis.Conclusion:Our study demonstrates that Aβ1-40 induces ferroptosis in RPE cells,while melatonin inhibits ferroptosis through the PI3K/AKT/MDM2/P53 pathway,thereby preventing and mitigating the progression of AMD.更多还原