奈米/次微米枸杞對於氧化壓力引發的糖尿病視網膜病變之保護作用

Abstract

氧化壓力(Oxidative stress)目前被認為是造成許多眼球疾病的主因，其所產生的自由基可以破壞視網膜神經節細胞、色素上皮細胞以及眼球血管內皮細胞等，造成諸如青光眼、白內障、老年性黃斑部病變、葡萄膜炎、甚至是糖尿病視網膜病變；另外，氧化壓力還可引發下游許多炎性基因的活化，導致體內發炎因子濃度上升，對細胞組織形成進一步破壞，因此，如何降低氧化壓力所產生的自由基，以減少其對眼球內細胞及組織的傷害，是目前熱門的研究話題。 糖尿病視網膜病變，是目前西方國家工作年齡族群造成失明的主因，高血糖如何導致微血管病變，主要就跟氧化壓力，以及其在下游引發的發炎反應有密切相關。 「枸杞子」(Lycium barbarum fruits)是一種天然的植物果實，其作為藥用植物，在東亞地區已有相當久的歷史，在傳統中藥裡，更宣稱有「保肝明目」的功效。研究發現，枸杞中不僅含有豐富的玉米黃素，還有許多其他活性成分，例如枸杞多醣體 (Lycium barbarum polysaccharide)等，具有抗老化、抗氧化及神經保護等功能，對於其他器官的抗氧化功能(皮膚、肝臟、肌肉等)，在動物實驗上都已獲得證實；但是對於眼睛疾病的抗氧化功能，卻尚無完整的研究。其內含豐富的葉黃素及玉米黃素，是目前已知的一種抗氧化劑，它們可抑制自由基活性，避免細胞的DNA、蛋白質或脂質產生過氧化，影響正常的生理功能；而目前研究也發現，食用具有抗氧化功能的葉黃素或玉米黃素，能夠有效地防止氧化壓力對於眼球的傷害，對於老年性黃斑部病變具有預防和治療效果。 傳統枸杞製備方式，主要使用加水萃取的方法，本實驗使用新的奈米技術(Nanotechnology)，使用介質研磨方式，得出粒徑小於100奈米，我們稱為奈米/次微米的枸杞懸浮液，實驗此等製備方式是否能增強枸杞子的生體可用率。 本研究採用取自奈米製程之研磨枸杞懸浮液及傳統製程的細碎枸杞懸浮液，並建立糖尿病動物實驗模式，探討枸杞在眼睛，對於糖尿病引發之氧化壓力、續發的發炎反應及最終的糖尿病視網膜病變是否有保護作用，並比較奈米化前後是否能提高物質的生體可用率，實驗結果將可提供以奈米製程萃取枸杞的臨床應用，並作為推廣保健食品的基礎。 我們利用腹腔內注射STZ（Streptozotocin），在SD大鼠身上成功誘發糖尿病，建立一穩定且可用的糖尿病動物實驗模式；結果發現，餵食枸杞60天後，可有效降低糖尿病鼠的尿糖、血液中總膽固醇、三酸甘油酯及自由基含量，利用ELISA、Western blot及RT-PCR等方式分析眼內的房水液和視網膜，也可見各標的發炎因子的表現下降，包括ICAM-1、MCP-1、RANTES 及IL-8等，發炎相關酵素iNOS下降、抗氧化酵素GR活性增加等，另外，oxidative stress responder：HO-1, Prx-1及TRX表現在餵食枸杞後也有下降的情形，代表氧化壓力的下降，最後，視網膜組織的免疫化學染色方法，可見脂質、蛋白質、DNA的過氧化產物，也在食用枸杞後明顯下降，可證實枸杞的抗氧化、抗發炎反應。奈米化前後的枸杞效果，在不同的檢測因子，其結果不完全一致，但是奈米化枸杞在某些方面，的確有優於一般細碎枸杞的保護作用，不過這部分可能須更進一步的實驗以證實其效果。 在糖尿病動物實驗模式中，發現枸杞可降低眼內的氧化壓力及發炎反應，對糖尿病視網膜病變可能具有保護作用，因此，在傳統降血糖藥物之外，其有可能作為另一種針對糖尿病視網膜病變的輔助或替代療法。

Diabetes mellitus (DM) is an important disease not only because of its high prevalence but also the disabling systemic complications. In eyes, DM can cause microvascular complication, called diabetic retinopathy (DR). It is a major cause of vision loss in young adults in the developing world and therefore a critical public health issue. Multiple biochemical abnormalities have been documented to be responsible for the pathogenesis of diabetic retinopathy. However, no single event predominates. Among the various pathways and effector molecules, oxidative stress plays a crucial role. Hyperglycemia in DM patients can induce ROS (Reactive oxygen species) production, which stimulates downstream inflammatory mediators and finally causes retinal capillary cells apoptosis. These inflammation-related chemokines and cell adhesion molecules are proved to be related to DR. Lycium barbarum (LB) fruits, a kind of medicinal plant used in East Asia for over 2300 years, have been studied to posses anti-aging, antioxidant and neuroprotective effects. The active biological activity of LB fruits comes from LBP (Lycium barbarum polysaccharide), zeaxanthin and other small molecules. Some studied showed its antioxidant capacity in various animal models with oxidative stress induced in skin, muscle and liver. However, its protective effect on eyes or retinal tissues has not been fully elucidated. Therefore, in our study, we investigated if LB fruits had protective effects on DR via antioxidation and anti-inflammation properties. Besides, we also applied nano-technique to prepare LB fruits and assumed this would increase its bioavailability. In this study, we successfully established a diabetic animal model by intraperitoneal injection of STZ. The result proved that treatment with LB fruits for 60 days could reduce systemic total cholesterol, triglyceride and free radicals as well as urine sugar. In eyes, free radicals and some inflammatory markers including ICAM-1, MCP-1, RANTES, IL-8 and iNOS expressions in aqueous humor and retinas also decreased after treatment of LB fruits. Expression of three oxidative stress responders：HO-1, Prx-1 and TRX also attenuated, reflecting suppressed oxidative stress. In histological changes, retinal sections showed less staining for acrolein, nitrotyrosin and 8-OHdG after LB fruits treatment, indicating inhibited oxidative damages on lipid, protein and DNA. Milled LB fruits had better protection over blended ones in some aspects, bot not all of the tested markers. So the superior effects of LB fruits after nanotechnology -processing may need further investigations. Our study concluded that LB fruits had antioxidation and anti-inflammation effects in eyes, in diabetic animal models. The milled LB fruits may have enhanced protection over blended ones. Therefore, LB fruits may serve as an alternative or supplementary treatment in diabetic retinopathy.