Cilostazol在Streptozotocin誘發之糖尿病動物模式中抑制視網膜發炎之療效與機轉

Abstract

1.研究背景： 在已開發中國家的工作人口中，糖尿病視網膜病變仍然是造成失明及視力不良的首要原因。新的證據指出糖尿病視網膜病變與慢性發炎性疾病有共通性，而且發炎作用在糖尿病視網膜病變中佔有舉足輕重的角色。在糖尿病的情況中，白血球變得較無法塑形，造成白血球滯留於視網膜，視網膜血管中細胞黏著分子intercellular adhesion molecule-1 (ICAM-1) 的表現也會增加，會影響內皮細胞作用及視網膜血管灌流狀況，造成血管新生暨通透性增加。發炎的視網膜組織會釋放出更多黏著分子、 cytokine及 chemokine以招募更多淋巴球聚集。在增殖性糖尿病視網膜病變病人的玻璃體中也被證實可偵測出發炎媒介物包括monocyte chemoattractant protein-1 (MCP-1) 及fracktalkine (FKN) 的增加。 氧化壓力 (oxidative stress) 增加會產生過量的活性氧 (reactive oxygen species，ROS)，被認為是導致糖尿病視網膜病變的關鍵所在。適當的使用氧化劑可以藉由抑制動物體內的發炎作用，而減緩糖尿病視網膜病變；這些反應可能是透過清除自由基的能力以及增加抗氧化防禦酵素的功能來減少NF-κB的活化、白血球阻滯以及ROS的形成。 Cilostazol是phosphodiesterase 3 (PDE3) 的抑制劑，可以抑制血小板的聚集以及血管擴張而達到動脈血壓下降的效果。目前的研究顯示cilostazol也有潛在的抗氧化及抗發炎的功能，在視網膜短暫缺血後，cilostazol可以藉由抑制cytokines、 黏著分子以及其他發炎媒介物，來減緩白血球與內皮細胞作用以及血小板所造成的組織傷害。同時，在高血壓合併第二型糖尿病的病人身上也被證實cilostazol可以減少免疫負荷以及氧化壓力。 我們假設在糖尿病視網膜病變的致病機轉中， cilostazol 可以透過阻斷NF-κB 活化來減少眾多發炎媒介物及黏著分子的表現，包括 MCP-1, FKN 及ICAM-1。Cilostazol的治療也可以使得糖尿病視網膜中抗氧化防禦酵素的活性增加以及相對應的減少DNA氧化物 (8-hydroxy-2'-deoxyguanosine；8OH-dG)、蛋白質氧化物(nitrotyrosine)與脂肪氧化物(acrolein)。我們將會評估在streptozotocin (STZ) 誘發之糖尿病大鼠模式這些發炎媒介物與氧化壓力的biomarker的表現程度，同時建立起 cilostazol的治療模式來證實 cilostazol在糖尿病視網膜病變中可能有抗發炎及抗氧化的效果。 2.材料及方法： 本研究採用30隻六週大的Wistar雌性大鼠，體重介於 200-250公克。再將其分為正常對照組 (n=10) 及糖尿病組 (n=20)。 20隻糖尿病組大鼠經腹腔內注射60 mg/kg劑量的STZ 來誘發糖尿病。糖尿病大鼠再任意分為兩組：其中10 隻給予兩個月的胃內灌注cilosatzol (18 mg/kg/day)，其餘10隻給予兩個月的胃內灌注等量的0.9%生理食鹽水。每週測量所有大鼠的體重及血糖值變化。八週後，每隻大鼠的眼球被取下以取得新鮮的視網膜組織檢體來作研究。為了評估ICAM-1、 MCP-1及FKN在視網膜的表現，我們採用Polymerase chain reaction ( PCR ) 、Western blot analysis和 Immunohistochemical staining（IHC）的方法。而Enzyme-linked immunosorbent assay ( ELISA ) 是用來直接測量ICAM-1、MCP-1及FKN在前房及玻璃體中的濃度。NF-κB在視網膜的活性是使用IHC和Enzyme-linked immunosobent assay（EMSA）的方法來評估。此外，為了評估視網膜中氧化壓力的變化，我們使用IHC的方法來檢查8OH-dG、nitrotyrosine和acrolein在視網膜的含量。 3.結果與討論： 相較於未治療的糖尿病大鼠，在糖尿病大鼠身上給予八週的 cilostazol治療並不會影響到血糖值。 Cilostazol 會抑制ICAM-1、 MCP-1 及 FKN 的 mRNA 和 蛋白質在視網膜的表現，cilostazol 也會抑制發炎作用而減少 ICAM-1、MCP-1及FKN 在前房和玻璃體內的濃度。根據 IHC 和 EMSA 的結果， cilostazol 同樣地也抑制了NF-κB在視網膜中的活性。因過度氧化而產生的8OH-dG、 nitrotyrosine 與 acrolein，在 cilostazol 治療後糖尿病大鼠中視網膜的含量，都有減少的趨勢。 4.結論： 本實驗動物模式證實 cilostazol 的確有保護視網膜的功效，會減少在大鼠之糖尿病視網膜病變中的發炎作用，視網膜中NF-κB的活性受到抑制，受到NF-κB調控的發炎相關物質ICAM-1、MCP-1及FKN的表現也會下降。Cilostazol已被廣泛被研究運用於糖尿病的併發症治療，包括動物模式中的血管病變、神經病變、腎臟病變及視網膜病變等。相信在不久將來，cilostazol有機會成為減少糖尿病患者視力喪失的輔助治療藥物。

1.Background: Among working adults in developed countries, diabetic retinopathy (DR) remains the leading cause of blindness and visual impairment. New evidence indicates that DR shares similarities with chronic inflammatory disease, and inflammation may play a central role in the development of DR. In diabetes, leukocytes become less deformable and retinal leukostasis increases, corresponding to the fact that the expression of cellular adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) are increased in retinal vasculature, which affects retinal endothe¬lial function, retinal perfusion, angiogen¬esis and vascular permeability. The inflamed retina tissues release increased levels of adhesion molecules, cytokines and chemokines for leukocyte recruitment. The concentrations of inflammatory mediators such as monocyte chemoattractant protein-1 (MCP-1) and fracktalkine (FKN) are reported to be increased in the vitreous of patients with proliferative diabetic retinopathy. It has been suggested that increased oxidative stress, which generates excess reactive oxygen species (ROS), is a key point in the pathogenesis of DR. The use of appropriate antioxidants may have potential to inhibit the inflammatory changes in the development of DR in animals, including activation of NF-κB, leukostasis, and formation of ROS with the ability of scavenging free radicals, or increasing the antioxidants defense enzyme capabilities. Cilostazol, is a phosphodiesterase 3 (PDE3) inhibitor which results in inhibition of platelet aggregation and vasodilation leading to reduction in arterial pressure. Among recent studies, potent anti-inflammatory and anti-oxidative effects of cilostazol were shown through the suppression of cytokines, adhesion molecules, and other inflammatory mediators leading to leukocyte-endothelial cell interactions and platelet-mediated tissue damage after transient retina ischemia. Besides, it has been reported that cilostazol could reduce inflammatory burden and oxidative stress in hypertensive type 2 diabetes mellitus patients. We hypothesized that cilostazol inhibits the expression of various inflammatory mediators and adhesion molecules such as MCP-1, FKN and ICAM-1 by blocking NF-κB activation in the development of DR. Treatment with cilostazol could also increase the activity of antioxidants defense enzymes and reduce levels of oxidatively modified DNA (8-hydroxy-2'-deoxyguanosine), oxidatively protein (nitrotyrosine) and oxidative lipids (acrolein) in the diabetic retina. We will assess retinal expression of these inflammatory mediators and oxidative stress biomarkers in streptozotocin (STZ) induced diabetic rat model and set up cilostazol treatment model to define the possible anti-inflammatory and anti-oxidative effect of cilostazol in DR. 2.Material and methods: Thirty 6 weeks old female Wistar rats with the body weight about 200-250g were divided into a normal control group (n=10) and a STZ-induce diabetic group (n=20). Twenty rats were intraperitoneally injected with STZ to induce diabetes. Eight weeks later, the eyeball of each rat was taken out to obtain fresh retina tissues. We performed polymerase chain reaction (PCR), western blot analysis, hematoxylin and Eosin (H&E) and immunohistochemical staining to assess ICAM-1, MCP-1 and FKN in the retina of rats. The concentrations of ICAM-1, MCP-1 and FKN in the aqueous humor and vitreous cavity were examined by enzyme-linked immunosorbent assay (ELISA). The nuclear factor (NF)-κB activity was assessed by IHC and electrophoresis mobility shift assay (EMSA). Besides, the contents of 8-OHdG, nitrotyrosine and acrolein in the retina were measured by IHC in order to evaluate the change of oxidative stress in the diabetic retina. 3.Results and discussions: The administration of cilostazol for eight weeks in diabetic rats did not alter the blood glucose levels compared with untreated diabetic rats. Cilostazol inhibits the increase of ICAM-1, MCP-1 and FKN mRNA and protein expression in the retina, as well as the increase of ICAM-1, MCP-1 and FKN contents in the aqueous humor and vitreous cavity. Consistent with these findings, cilostazol attenuated the enhanced activation of NF-κB in diabetic rats by IHC and EMSA findings. The levels of oxidatively modified DNA (8-OHdG), nitrotyrosine and oxidative lipids (acrolein) were also diminished in the cilostazol-treated diabetic group. 4.Conclusions: Cilostazol reduces inflammatory reactions and oxidative stress in the development of DR. The anti-inflammatory effects of cilostazol is supposed to be mediated by the inhibition of NF-κB activity, and the subsequent decrease in inflammatory mediators such as ICAM-1, MCP-1 and FKN expression in the retina. The effects of cilostazol had been widely studied in the treatment of diabetic vasculopathy, neuropathy, nephropathy and retinopathy of the animal model. In the future, cilostazol may be clinically applied in the treatment of diabetic patients to avoid progressive visual loss.