新穎化合物Aza-PBHA在離體視網膜色素上皮細胞及
活體 C57BL/6 小鼠以高血糖-缺氧環境誘發糖尿病視
網膜病變的作用機制之探討

由於科技的高速發展與人類壽命的延長，加上飲食習慣及生活型態改變，使得慢性疾病盛行率逐年增加，而糖尿病 (Diabetes mellitus, DM)即為其一。根據統計，糖尿病視網膜病變 (Diabetic retinopathy, DR)在先進國家當中是造成工作年齡內成年人失明的最主要原因。DR 形成的主要的原因，是由於血液中長期慢性的血糖濃度過高，引發生理變化及有害物質累積，使眼球內的微血管產生病變，而其組織也因而受到牽連並損害。病程上，DR 可以分為兩個階段：前期的非血管增生性(Non-proliferative)及後期的血關增生性 (Proliferative)。在 DR 前期，可以觀察到由於血管與細胞間隙損傷所導致的出血 (Hemorrhage)與黃色脂質性滲出物 (Yellow lipid exudates)，或是因為血管病變而產生動脈瘤 (Aneurysm)之情形；在 DR 後期，可以明顯地發現不正常血管新生 (Neovascularization)及黃斑部水腫 (Macular edema)的狀況。目前臨床治療方面僅針對後期的不正常血管新生，利用 anti-VEGF 藥物或是手術等方式進行治療。本論文所使用的藥物 Aza-PBHA (AzP)，是由台北醫學大學藥學系王惠珀教授實驗室提供的新穎組蛋白去乙醯酶抑制劑 (Histone deacetylase inhibitor, HDACi)。本實驗室先前研究顯示，AzP 可以有效治療由化療藥物所引起的口腔粘膜潰瘍與腸道黏膜炎，也能夠促進傷口之癒合。在本論文中，活體動物實驗部分，分別利用單處理 Streptozotocin (STZ)誘導小鼠產生糖尿病，以及同時合併 STZ 與缺氧處理的方式，觀察小鼠視網膜之變化。在僅處以 STZ 的實驗中，在約莫一個月左右，於螢光血管造影 (FFA)結果發現小鼠視網膜形成類似滲漏的病變，且透過 AzP 腹腔注射治療後有延緩的現象，然而小鼠個體差異性大，並非每次實驗都能夠明顯發現滲漏現象；配合光學相干斷層掃描圖 (OCT)觀察在糖尿病條件下之視網膜，未發現有明顯之結構破壞；利用 ELISA 觀察小鼠血清中 IL-6 之濃度，會隨著糖尿病狀態而增加；將小鼠眼球組織均質化後觀察其蛋白表現，iNOS 在糖尿病環境下表現量增加，且在6 AzP 治療下則有減少趨勢。為了加速疾病之發展，將小鼠處以 STZ 與缺氧條件觀察，發現滲漏現象提早表現，以及發現表示深層滲漏之黃白色斑塊，OCT 結果發現病灶部位之內神經層 (INL)有明顯變薄的情形；使用 ERG 觀察發現，糖尿病小鼠 a、b 波均有波幅減少及延遲現象，而在 AzP 給予下 ERG 結果均較疾病組正常。顯示此動物模型能夠讓視網膜病變之病程加速，有利於疾病之觀察與藥物篩檢。離體細胞實驗部分，最初發現 AzP 在 RPE 細胞具有較高的容許劑量，且能夠抑制單獨使用缺氧誘導之 HIF1α的表現量。首先使用高糖/缺氧方式模擬 DR 環境，結果發現所造成的影響不足以模擬先前在動物上觀察到之結果，因此選擇再合併外加 IL-1β方式處理細胞。利用高糖/缺氧/IL-1β處理後的 RPE 細胞，其通透性有最明顯之改變，且在 AzP 保護之下可以回復。在西方墨點法觀察發現 Azp 可以抑制由高糖/缺氧/IL-1β所誘導之 iNOS、HIF1α的上升，又以 HIF1α的抑制效果較為明顯。ZO1 與 Occludin 在三條件處理之下有下降的趨勢，Azp 治療之下則對兩者沒有影響。磷酸化蛋白表現部分探討 AzP 對 HIF1α的調控機制，結果發現 AzP 對於缺氧所誘導之 mTOR、Akt 與 p70 之磷酸化有抑制的趨勢，顯示 AzP 是透過 mTOR/Akt/p70 之路徑來調控 HIF-1α蛋白表現。綜合以上實驗之結果，利用 STZ 合併缺氧處理適合在建立 DR 之動物模型，並且推論 AzP 能夠保護眼球組織細胞對抗因糖尿病所導致的缺氧與發炎的現象，進而能夠延緩糖尿病視網膜病變的惡化，具有發展成為治療 DR 之潛力藥物。

關鍵字

組蛋白去乙醯酶抑制劑；糖尿病視網膜病變；缺氧狀態；發炎狀態；缺氧性誘導因子；誘導型一氧化氮合成酶

並列摘要

In many developed countries, diabetic retinopathy (DR), one of the complications caused by DM, is a leading cause of vision loss in working-aged adults. DR can be divided into two phases, the early phase and the late phase, also known as “Non-proliferative DR” (NPDR) and “Proliferative DR” (PDR). The classic retinal microvascular signs of NPDR are hemorrhages, yellow lipid exudates, or aneurysms due to microvascular pathological changes. In PDR, abnormal neovascularization and macular edema can be observed in the retina. AzP, a novel Histone deacetylase inhibitor (HDACi), was provided by Dr. Hui-Po Wang’s lab. In previous study, we found that AzP can heal mucositis induced by chemotherapy drug (5-fluorouracil), and it can also enhance wound healing ratio. Our research is divided into two parts: in vivo study and in vitro study. In the part of in vivo study, streptozotocin (STZ) was used to induce diabetic condition in C57BL/6 mice, combined with or without hypoxia to mimic DR. The results of STZ alone DR animal model revealed that leakage occurred in mice retina in 1 month observation by using fundus fluorescein angiography (FFA) and optic coherence tomography (OCT). However, the circumstances varied between different batches of examination and different mice. Electroretinography (ERG) showed the retinal neurons were certainly injured by the influence of diabetic condition. Interleulin-6 (IL-6) in serum and inducible nitric oxide (iNOS) in eye tissue were both upregulated by diabetes. The results of STZ combined hypoxia DR animal model showed the acceleration of progress. Leakage, white cotton spot, and deep white lesions was discovered by FFA. OCT showed the thinning of inner nuclear layer (INL) and the thickening of outer retinal layer. The results of ERG matched the observation on OCT. The Amplitude and implicit time of waves were affected by diabetic/hypoxia 8 condition, whereas it showed a more normal pattern in the treatment of AzP. In the part of in vitro study, the results suggested that AzP has no changes to the viability of RPE cells. AzP could decrease HIF-1α protein expression induced by hypoxia. In order to simulate DR condition, firstly we used high glucose coupled with hypoxia. However, western blot showed that this condition cannot mimic the same pattern in animal model, so IL-1β was selected for the purpose of building the DR cell model. In transwell permeability assay, the permeability of RPE cells was significantly increased in high glucose/hypoxia/IL-1β condition, and AzP could reverse this situation. Western blot revealed that HIF-1α and iNOS were increased, while zona occludens protein 1 (ZO1) and occludin were decreased. To know how AzP regulate the expression of HIF-α, we investigated the related protein. Prolyl hydroxylase domain-containing protein 2 (PHD2) and von Hippel-Lindau protein (vHL) were decreased in hypoxia condition, and recovered by treating with AzP. The phosphorylation of mTOR, Akt, p70 was increased by hypoxia, whereas AzP could recover this situation, suggested that AzP may influence HIF-1α protein expression by the mTOR/Akt/p70 pathway. To sum up, the STZ combined hypoxia animal model is suitable for DR study, and AzP can lowering the worsen of DR, which has a good potential for treating DR.