Fenofibrate對於與糖尿病視網膜病變相關之發炎介質的影響-動物模式研究

Effect of Fenofibrate on the Expression of Inflammatory Mediators in Animal Model of Diabetic Retinopathy

王律鈞(Lu-Chun Wang)

指導教授 ： 楊長豪

共同指導教授 ： 楊中美

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.03254

摘要

糖尿病視網膜病變是導致視力喪失的重要疾病之一，其成因與視網膜微血管病變、缺氧及異常的血管新生有關。而與糖尿病視網膜病變相關的介質中，一類是與發炎反應有關的介質，例如monocyte chemoattractant protein-1 (MCP-1)及the chemokines regulated on activation, normal T-cell expressed and presumably secreted (RANTES)/CCL5等，一類則是與血管新生促進因子相關的介質，例如vascular endothelial growth factor (VEGF)。這些介質會透過不同的機轉來影響糖尿病視網膜病變的產生。另外，近年來在代謝症候群及糖尿病方面被研究很多的adiponectin，在整體上對於糖尿病的產生似乎有保護效果，被認為是「好」的細胞激素。然而少數幾篇針對糖尿病視網膜病變的研究，卻發現「血清中」的adiponectin與糖尿病視網膜病變似乎有關聯，但是好是壞尚無定論。而直接採集玻璃體或視網膜檢體的動物及人體實驗迄今則尚未有人做過。
另外一個令我們感興趣的，是Effect of fenofibrate on the need for laser treatment for diabetic retinopathy (FIELD study) 2007年發表在Lencet期刊的研究，發現fenofibrate這種peroxisome proliferator-activated receptor-alpha (PPAR-α) agonist可以降低糖尿病人接受全網膜雷射治療的機會，而機轉仍不清楚。由於有其他研究發現fenofibrate可以在糖尿病大鼠的腎臟組織抑制與nuclear factor-κ B (NF-κB)相關的發炎反應，加上糖尿病視網膜病變是一種與發炎相關的病變，特別是受到NF-κB所調控，這個理論已經被證實。因此，本研究的中心假說如下：
「糖尿病病人因NF-κB所調控的發炎反應的上升，相關的chemokines包括MCP-1、ICAM-1、及FKN都會增加，使得視網膜上產生局部白血球聚集及浸潤，誘發發炎現象及組織缺氧，並使內皮細胞產生移行、導致血管新生，進而使糖尿病視網膜病變惡化。我們的假設使用Fenofibrate來治療糖尿病大鼠，可以抑制NF-κB的活化，連帶抑制它所調控的MCP-1、ICAM-1、及FKN的產生，達到抑制視網膜的局部發炎反應，延緩糖尿病視網膜病變的惡化。」
我們預期在糖尿病大鼠的視網膜上，可以發現NF-κB活化表現，MCP-1、ICAM-1、及FKN也會有表現增加的情形。而服用Fenofibrate的糖尿病大鼠，將可以抑制NF-κB的活化，上述的三種chemokines的表現增加也將受到抑制。
本研究有兩個目的：第一，我們想要以糖尿病大鼠模式，使用fenofibrate治療，研究其對於糖尿病視網膜病變相關的、受NF-κB調控的發炎介質有何影響；第二，利用同一模式，研究adiponectin及其受器在大鼠模式糖尿病視網膜病變扮演什麼角色，fenofibrate又對它們有怎麼樣的作用？
我們的研究方式是將母的Wistar大鼠分成三組，第一組是健康大鼠，作為對照組(normal control)；第二組則是以streptozotocin(STZ)誘發糖尿病模式的大鼠，但不給予fenofibrate治療 (diabetic control)；第三組則是糖尿病大鼠，並每日以灌食方式餵食fenofibrate，研究時間為期三個月。之後將老鼠犧牲並將玻璃體及視網膜取出，用反轉錄-聚合酶連鎖反應（reverse transcription – polymerase chain reaction, RT-PCR）、西方墨點法（Western blotting）、酵素連結免疫吸附分析（enzyme-link immunosorbent assay，ELISA）及免疫組織染色法（immunohistochemistry, IHC）等方式，研究其玻璃體及視網膜上adiponectin、adiponectin receptors、NF-κB P65、intercelullar adhesion molecule (ICAM)-1、MCP-1及fractalkine (FKN)這些chemokines的表現。
研究結果發現，糖尿病大鼠在罹病三個月時，玻璃體及視網膜上，受NF-κB調控的發炎介質，包括MCP-1, FKN,及NFKB P65本身，在mRNA及蛋白質的表現上，跟未罹病的對照組比起來都有明顯的上升。而服用fenofibrate的糖尿病大鼠則可明顯抑制上述這些發炎介質的表現。ICAM-1則僅在蛋白質的表現方面（西方墨點法及免疫組織染色法）呈現上述的趨勢；mRNA的表現在對照組大鼠及糖尿病大鼠差別不大，而服用fenofibrate反而使ICAM的mRNA表現增加。另外。我們也發現糖尿病大鼠的視網膜上adiponectin及其受器的表現跟對照組比較是會增加的，而fenofibrate則可以使其表現稍微下降。實驗過程還發現，服用大劑量的fenofibrate，在糖尿病大鼠有降低血糖的作用。
基於研究結果，NF-κB所調控的發炎介質確實在糖尿病大鼠的玻璃體及視網膜上有表現增加的狀況。fenofibrate則可以抑制NF-κB的活化及減少這些發炎介質，這些發炎反應正是糖尿病視網膜病變產生的重要因素。因此fenofibrate對糖尿病病人而言，可能具有抑制糖尿病視網膜病變的潛力。
這樣的研究結果可以用來解釋FIELD study的結論，服用fenofibrate的病人可以減少糖尿病視網膜病變惡化到需要全網膜雷射光凝固治療的機會可能是透過抑制NF-κB所調控的發炎反應。這也可作為進一步人體試驗的參考。至於我們所發現adiponecitn跟糖尿病視網膜病變的關聯性，由於是第一個這種類型的研究，還需要進一步用其他動物模式或人體研究來加以驗證，並尋找可能的機轉。
我們希望在未來，可以嘗試用不同濃度的fenofibrate及不同的PPAR-α agonist來進行更廣泛的動物實驗、細胞組織培養、及人體實驗。基於這些實驗結果找出治療糖尿病視網膜病變的新方法或是新藥物，讓苦於糖尿病引起視力喪失的病人有重見光明的機會。

關鍵字

非諾貝特 ； 病視網膜病變 ； 脂締素 ； 核因子κB ； 發炎 ； 血管新生

並列摘要

Diabetic retinopathy (DR) is an important cause of vision loss in patients with diabetes. It’s characterized by increased vascular permeability at initial stage, and then progresses to vascular closure with ischemia. At proliferative stage, abnormal new vessels grow on retina and result in vitreous hemorrhage and tractional retinal detachment 1.Recently, many studies aimed to verify cytokines related to DR, and some cytokines had been identified in previous studies.
Cytokines related to angiogenesis was found to be potential factors that induced DR. Vascular endothelial growth factor (VEGF) was proved to be an important cytokine involved in DR in many studies. Another group of chemokines which may induce DR is proinflammatory cytokines. Monocyte chemoattractant protein-1 (MCP-1) and the chemokines regulated on activation, normal T-cell expressed and presumably secreted (RANTES)/CCL5 are proinflammatory cytokines that may induce DR.
Adiponectin, a well-known adipocytokine which is closely related to metabolic syndrome6, had also been studied to find its effect on DR. Controversial results were reported in limited studies. All these studies only survey the relationship between “serum” adiponectin and the severity of DR. The relationship between vitreoretinal adiponectin level and the severity of DR had not been investigated in human or animal studies before.
Besides, results from the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study (Lancet 2007) showed beneficial effects of fenofibrate (peroxisome proliferator-activated receptor-alpha (PPAR-α) agonist) in reducing the requirement for panretinal photocoagulation in patients with DR, and particularly in preventing disease progression in patients with pre-existing diabetic retinopathy. The mechanism is not fully understood. Another study showed that fenofibrate can inhibit inflammatory responses associated with NF-κB pathway in kidneys from diabetic rats. Based on the concept that DR is a NF-κB-related inflammatory disease, we hypothesize fenofibrate may prevent the progression of DR via inhibit NF-κB-related inflammation. We conduct a diabetic rat model study to confirm the above hypothesis. We also want to use the same model to identify the expression of adiponectin and its receptors in diabetic rats’ vitreous and retina.
In our study, female Wistar rats were divided into 3 groups: normal control, diabetic control, and diabetic + fenofibrate (10 in each group). Diabetes is induced by intraperitoneal injection of streptozotocin (STZ). Blood, vitreous, and retina samples of these rats are collected. Enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry (IHC) will be performed to study the expression of adiponectin, adiponectin receptors, NF-κB P65, intercelullar adhesion molecule (ICAM)-1, MCP-1 and fractalkine (FKN).
Our results showed that the mRNA and protein expression of NF-κB P65, MCP-1 and fractalkine (FKN) were significantly higher in diabetic rats than those in normal control. Treatment with fenofibrate inhibited the increased expression of these factors in diabetic rats’ retina. Protein expression (Western blotting and IHC) of ICAM-1 showed similar trend as above. However, diabetic control and normal control have similar level of ICAM-1 in mRNA level. Treatment with fenofibrate can even increase the expression of ICAM-1 mRNA.
Besides, diabetic control had increased expression of adiponectin and adiponectin receptors (R1 and R2) than normal control, both in mRNA and protein levels. Treatment with fenofibrate inhibited the increased expression of adiponectin and its receptors (R1 and R2) in diabetic rats’ retina.
According to the results of our study, inflammatory chemokines mediated by NF-κB had increased expression in diabetic rats, and treatment with fenofibrate can inhibit these reactions. NF-κB-mediated inflammation is a key factor of progression of DR. Fenofibrate therefore has the potential to prevent DR progression. It can also explained the results in FIELD study – taking fenofibrate daily has beneficial effect in reducing the requirement for panretinal photocoagulation in patients with DR.
In the future, we will try different doses of fenofibrate and different kinds of PPAR-α agonists in different settings, including in vitro, in vivo, and clinical trials. We hope we can find new medical treatment for diabetic retinopathy and save these patients’vision.

並列關鍵字

fenofibrate ； diabetic retinopathy ； adiponectin ； nuclear factor-κB ； NF-κB ； inflammation ； angiogenesis

參考文獻

1. Fong, D.S., et al., Retinopathy in Diabetes. Diabetes Care, 2004. 27(90001): p. S84-87.

2. Meleth, A.D., et al., Serum Inflammatory Markers in Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci., 2005. 46(11): p. 4295-4301.

3. Harada, C., et al., Role of monocyte chemotactic protein-1 and nuclear factor kappa B in the pathogenesis of proliferative diabetic retinopathy. Diabetes Research and Clinical Practice, 2006. 74(3): p. 249-256.

4. Simo, R., et al., Angiogenic and Antiangiogenic Factors in Proliferative Diabetic Retinopathy. Current Diabetes Reviews, 2006. 2: p. 71-98.

5. Murugeswari P., et al., Proinflammatory cytokines and angiogenic and anti-angiogenic factors in vitreous of patients with proliferative diabetic retinopathy and eales' disease. Retina, 2008. Jun;28(6): p. 817-24.