Inducible Nitric Oxide Synthase和Cationic Amino Acid Transporter在Endotoxin-Induced Uveitis的表現

Abstract

研究目的：研究cationic amino acid transporter和inducible nitric oxide synthase在endotoxin-induced uveitis中的表現。 研究方法：endotoxin-induced uveitis主要是將200 μg的lipopolysaccharide注射到Lewis rats的footpad中來誘導出uveitis。我們使用bortezomib (velcade)在注射lipopolysaccharide 30分鐘前進行腹腔內注射，作為proteasome inhibitor來研究nuclear factor-kappa B被抑制時的情形。Lewis rats在注射lipopolysaccharide 24 小時後被犧牲，並將其眼部組織取出研究。我們以RT-PCR和Western blotting研究inducible nitric oxide synthase以及cationic amino acid transporter之蛋白還有他們的mRNA在iris-ciliary body的表現；此外，我們利用ELISA 來測定aqueous中nitric oxide的量。另一方面，我們將iris及ciliary的組織切片進行 immunochemical staining來研究inducible nitric oxide synthase以及cationic amino acid transporter的表現，並研究velcade對它們的抑制效果。關於nuclear factor-kappa B的活性則是透過electrophoretic mobility shift assay來測定。除了動物模式之外，我們還利用RAW 264.7 cell這樣的細胞研究模式來驗證我們的理論。 結果：Lewis rats在注射velcade之後，在組織切片所觀察到的inflammation程度下降，aqueous中NO的production也減少。我們的研究結果也顯示出inducible nitric oxide synthase以及cationic amino acid transporter-2的表現量及其mRNA的表現量都有受到velcade的抑制。透過electrophoretic mobility shift assay也證實了nuclear factor-kappa B的活性受到velcade的抑制。另一方面，在RAW 264.7 cell的研究上也得到同樣的結果。

結論：我們證實了cationic amino acid transporter在眼內組織的存在，也發現nuclear factor-kappa B對於inducible nitric oxide synthase以及cationic amino acid transporter-2的表現都有促進的效果。而nuclear factor-kappa B、inducible nitric oxide synthase、cationic amino acid transporter-2以及nitric oxide都是葡萄膜炎的發炎反應中不可或缺的重要因子。

Purpose：To examine the expression of cationic amino acid transporter (CAT) and inducible nitric oxide synthase (iNOS) in endotoxin-induced uveitis (EIU). Methods：EIU was induced by a footpad injection of 200 μg LPS in Lewis rats. Proteasome inhibitor bortezomib (valcade) was injected intraperitoneally 30 minutes before the LPS administration. The rats were sacrificed 24 hours later and the eyes were enucleated. The expression of iNOS and CAT mRNA and protein in the iris and ciliary body was determined by RT-PCR and Western blotting. The production of NO in aqueous was measured by ELISA. Immunochemical staining of the iris and ciliary body was performed to evaluate the inhibitory effect of valcade. The activity of NF-κB was assessed by electrophoretic mobility shift assay (EMSA). In addition to animal model, we also used RAW264.7 cells as an animal model to investigate our hypothesis. Results： Proteasome inhibitor valcade abrogated the inflammation of EIU as demonstrated by immunochemical stain and the decreased NO production in aqueous. The expression of iNOS and CAT mRNA in EIU mouse eyes were suppressed by valcade. Supershift assay revealed that NF-κB activation was responsible for iNOS and CAT induction. Similar results were found in cellular model study. Conclusions：We confirmed the existence of CAT in intraocular tissue. NF-κB is an essential transcription factor not only for the induction of iNOS, but also for the up-regulation of CAT-2. The simultaneous up-regulation of CAT-2 and iNOS may play an important role in the pathogenesis of EIU.