藍光發光二極體造成視網膜細胞傷害的保護策略-發散光譜調整與營養素補充

Blue Light Light-emitting Diode Induced Retinal Cell Damage and the Protective Strategies-Emitted Light Spectrum Modification and Nutrients Supplement

林昭文(Chao-Wen Lin)

指導教授 ： 楊長豪 ； 楊偉勛

國立臺灣大學/醫學院/臨床醫學研究所/博士(2021年)

https://doi.org/10.6342/NTU202100061

摘要

3C產品的使用在現代人的日常生活中已經是不可或缺的一部分，液晶顯示器(Liquid crystal displays, LCDs)作為這些3C產品的螢幕，利用發光二極體(Light-emitting diode, LED)當做背光模組，提供了高能量效率的光源。但是其所發出的光線相較於傳統光源帶有較高比例的短波長藍光，在我們要求提高影像品質的同時，也伴隨著光源高能量光線的發散，對我們的視網膜造成更嚴重的傷害，藍光並且會影響生理時鐘的規律性，成為健康的重大議題。傳統的藍光濾鏡和抗藍光鏡片因為會降低亮度而犧牲部分視覺品質，並非完美的解決方式。取而代之，我們藉由改變液晶顯示器LED背光模組的發散光譜，在維持亮度的情形下降低能量的發散，試圖減少對網膜細胞的傷害，這也是我們研究的第一個部分。
我們眼睛吸收光線主要是藉由感光細胞和帶有黑色素的視網膜色素上皮細胞，這兩種細胞也最容易受到光線照射的傷害，所以感光細胞和視網膜色素上皮細胞也是光線造成視網膜損傷很好的細胞研究模組。在我們研究的第一部分，選擇661W感光細胞做為我們的細胞模組。我們建立了一個顯示器入眼的能量指標(ocular energy exposure index, OEEI)，計算公式為顯示器可見光頻譜的總輻射率(Radiance)除以總亮度(Luminance)。暴露於較高發散能量的液晶顯示器照射後，細胞的活性(viability)顯著下降，細胞凋亡(apoptosis)的發生也顯著增加。這些細胞損傷的原因是透過含氧自由基(reactive oxygen species, ROS)的產生，增加氧化壓力並且影響粒線體的功能，其分子機轉牽扯到Nuclear factor-κB (NF-κB) pathway的活化以及和氧化壓力、發炎反應及細胞凋亡相關蛋白質的表現量增加，影響的程度和OEEI的強度有關聯性。我們的實驗結果證明了液晶顯示器照射對感光細胞的傷害程度和其能量的發散有密切關聯，如果可以使用較低能量發散的顯示器將可減少對視網膜的傷害，對人類的眼睛提供較多保護。
我們在第一部分實驗中已經證實了光線照射對視網膜傷害的機轉是透過含氧自由基和發炎反應的產生，增加氧化壓力並且導致網膜細胞凋亡。在我們研究的第二部分，將探討使用強的抗氧化劑甲殼素(chitosan oligosaccharides, COSs)和蝦紅素(astaxanthin)對藍光LED造成的視網膜細胞傷害可能的保護作用，並且試圖釐清其作用的機轉。
甲殼素是幾丁質的水解和去乙醯化(deacetylated)產物，富含於甲殼類動物的外骨骼和黴菌的細胞壁中，其具有抗腫瘤、抗菌、抗發炎、抗氧化和抗細胞凋亡的特性。在這個實驗中我們使用ARPE-19細胞作為我們的實驗模組，細胞先給予不同濃度的甲殼素後再接受2500 lx藍光LED照射。我們的實驗結果發現接受較長時間的光線照射細胞的凋亡顯著增加，而使用甲殼素可以顯著減少細胞凋亡的發生，並且其效果和甲殼素的濃度有關聯性。甲殼素同時可以抑制含氧自由基的產生和發炎反應及細胞凋亡相關蛋白質的表現，並且穩定粒線體膜電位及活化抗細胞凋亡蛋白Bcl-2。甲殼素藉由抑制NF-κB入核作用，繼而降低下游基因inducible nitric oxide synthase (iNOS)和monocyte chemoattractant protein-1 (MCP-1)的表現。我們的研究確立了甲殼素在藍光LED照射對視網膜色素上皮細胞傷害的保護作用及其背後機轉。
蝦紅素則是一種葉黃素類(xanthophyll)的營養素，富含於海鮮食物中，它是很強的含氧自由基清除劑和抗發炎物質。它的抗氧化能力來自於其與細胞膜之間的生化交互作用，蝦紅素的共軛雙鍵會將自由基箝制於細胞膜上進行清除，消除含氧自由基並且終止自由基連鎖反應。雖然蝦紅素並非人類視網膜的組成成分，但是它可以通過血液視網膜障壁(blood–retina barrier)在視網膜發揮其抗氧化作用。
我們的實驗是第一個證明蝦紅素對藍光LED照射造成網膜細胞損傷的保護作用的研究，使用661W細胞做為細胞光傷害的實驗模組，細胞先給予不同濃度的蝦紅素後再接受2000 lx藍光LED照射。我們的實驗結果發現蝦紅素確實可以抑制藍光LED造成的細胞凋亡和死亡，其保護作用強度和蝦紅素的濃度有關聯。蝦紅素抑制含氧自由基及氧化壓力代謝產物的產生，並且減少藍光照射造成的粒線體損傷，西方墨點法(western blot)的分析則驗證了其作用機轉是透過活化phosphoinositide 3-kinases (PI3K)/Akt pathway，進而促使Nuclear factor erythroid 2-related factor 2 (Nrf2)移入細胞核內，增加phase II抗氧化酵素Heme oxygenase-1 (HO-1)和NAD(P)H:quinone oxidoreductase-1 (NQO1)的表現，抗氧化酵素的活化與細胞凋亡相關蛋白的抑制最終發揮了保護作用減低藍光LED造成的661W細胞傷害。我們的研究確認了蝦紅素在藍光LED照射對感光細胞傷害的保護作用，並且了解了保護作用的機轉。總和而言，我們的第二部分研究結果顯示甲殼素和蝦紅素具有潛能做為保護眼睛減少藍光造成視網膜傷害的補充營養素。液晶顯示器發散光譜的調整以及抗氧化營養素的補充可以減少高能量藍光LED照射的網膜傷害，是值得將來持續探討研究的方向。

關鍵字

藍光 ； 發光二極體 ； 氧化壓力 ； 甲殼素 ； NF-κB ； 蝦紅素 ； Nrf2

並列摘要

Consumer electronics are indispensable in daily life in the modern era of computing. Liquid crystal displays (LCDs) are used as screens in consumer electronics. LCDs utilize light-emitting diode (LED) as the backlight modules. LED provides light source with high energy efficiency. However, it emits higher levels of short wavelength blue light than conventional light sources. The quest for increased image quality may be accompanied by higher energy emission of the light source, in turn resulting in more severe damage to the retina and enhancement of other blue light hazards such as circadian rhythm desynchronization. This represents a major societal health concern. Traditional blue light filters and blocking lenses may be poorly suited, as consumers may be unwilling to sacrifice visual quality. Alternatively, in the first part of our research, we adjusted the emitted light spectrum of LED backlight modules in LCDs and reduced the energy emission but maintained the luminance.
The main sites of light absorption are the photoreceptors and the melanin pigment in retinal pigment epithelial (RPE) cells, which can easily be injured by light exposure. Therefore, photoreceptors and RPE cells could be suitable cell models for light induced retinal cell damage. In the first part of our research, we chose 661W photoreceptor cell line as the model system. We established a formula of the ocular energy exposure index (OEEI), which could be used as the indicator of LCD energy emission. Cell viability decreased and apoptosis increased significantly after exposure to LCDs with higher emitted energy. Cell damage occurred through the production of reactive oxygen species (ROS), the induction of oxidative stress and mitochondrial dysfunction. The molecular mechanisms included activation of the Nuclear factor-κB (NF-κB) pathway and upregulation of the expression of proteins associated with oxidative stress, inflammation and apoptosis. The effect was correlated with OEEI intensity. We demonstrated that LCD-induced photoreceptor damage was correlated with LCD energy emission. LCDs with lower energy emission may serve as suitable screens to prevent light-induced retinal damage and protect consumers’ eye health.
We already proved that the mechanism of light-induced retinal injury was related to the generation of ROS and inflammatory reactions, which induced oxidative stress and cell apoptosis within the retina. In the second part of our research, we used strong antioxidants chitosan oligosaccharides (COSs) and astaxanthin to investigate the potential protective effects on blue light LED induced retinal cell injury and try to elucidate the mechanisms of action.
COSs are the deacetylated and hydrolyzed products of chitin, which is abundant in the exoskeleton of crustaceans and cell walls of fungi. COSs are known to exert various biological effects including anti-tumor, anti-bacterial, anti-inflammation, anti-oxidative, and anti-apoptotic activities. We used ARPE-19 cell as the model system. These cells were treated with various concentrations of COSs and then exposed to 2500 lx blue light LED. Our results confirmed that RPE cell apoptosis increased significantly with longer light exposure. Treatment with COSs significantly reduced apoptosis in a dose-dependent manner. These molecules also suppressed the production of ROS and the expression of inflammation- and apoptosis-related proteins. Moreover, COSs stabilized the mitochondrial membrane potential of cells and up-regulated the anti-apoptotic protein Bcl-2. These compounds also down-regulated the NF-κB pathway by decreasing its translocation to the nucleus and subsequently inhibiting the expression of downstream genes, such as inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1). Through our study, the protective effects of COSs on blue light LED-induced RPE cell damage, as well as the associated mechanisms, were demonstrated.
Astaxanthin, a xanthophyll that is abundantly available in seafood, is a potent free radical scavenger and anti-inflammatory agent. Its antioxidant properties are attributed to the physical and chemical interactions with cell membranes. The conjugated double bonds trap and scavenge radicals in the cell membrane, quench singlet oxygen, and terminate free radical chain reactions. Although astaxanthin is not a component of human retina, it could cross the blood–retina barrier and exerts anti-oxidative effects in human retina.
Our study was the first effort to investigate and demonstrate the protective effects of astaxanthin on retinal cells against damage mediated by blue light LED exposure. The photo-injury model was established by using 661W photoreceptor cell line. The cells were treated with various concentrations of astaxanthin and then exposed to 2000 lx blue light LED. Our results showed that pretreatment with astaxanthin inhibited blue light LED-induced cell apoptosis and prevented cell death. Moreover, the protective effect was concentration dependent. Astaxanthin suppressed the production of ROS and oxidative stress biomarkers and diminished mitochondrial damage induced by blue light exposure. Western blot analysis confirmed that astaxanthin activated the phosphoinositide 3-kinases (PI3K)/Akt pathway, induced the nuclear translocation of Nuclear factor erythroid 2-related factor 2 (Nrf2), and increased the expression of phase II antioxidant enzymes Heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 (NQO1). The expression of antioxidant enzymes and the suppression of apoptosis-related proteins eventually protected the 661W cells against blue light LED-induced cell damage. Thus, our results demonstrated that astaxanthin exerted a dose-dependent protective effect on photoreceptor cells against damage mediated by blue light LED exposure. Taken together, our study supported the idea that COSs and astaxanthin could be potentially used for the prevention of blue light-induced damage to the human eye.

並列關鍵字

blue light ； light-emitting diode (LED) ； oxidative stress ； chitosan oligosaccharides ； NF-κB ； astaxanthin ； Nrf2

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