Lennikov Anton, Kitaichi Nobuyoshi, Noda Kousuke, Ando Ryo, Dong Zhenyu, Fukuhara Junichi, Kinoshita Satoshi, Namba Kenichi, Mizutani Miho, Fujikawa Tomoyuki, Itai Akiko, Ohno Shigeaki, Ishida Susumu
Laboratory of Ocular Cell Biology and Visual Science, Department of Ophthalmology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
Mol Vis. 2012;18:2586-97. Epub 2012 Oct 20.
Endotoxin-induced uveitis (EIU) is an animal model for acute ocular inflammation. Several substances play major roles in the development of inflammatory changes in EIU, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. These inflammatory cytokines trigger the degradation of IκB by activating IκB kinases (IKKs). Released nuclear factor kappaB (NFκB) subsequently translocates to the nucleus, where NFκB expresses its proinflammatory function. IMD-0354, N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide, selectively inhibits IKKβ, particularly when induced by proinflammatory cytokines, such as TNF-α and IL-1β. In the present study, we examined whether IKKβ inhibition has therapeutic effects on EIU by using IMD-0354 and its prodrug IMD-1041.
Six-week-old male Lewis rats were used. EIU was induced with subcutaneous injections of 200 μg of lipopolysaccharide (LPS) from Escherichia coli that had been diluted in 0.1 ml of phosphate-buffered saline. IMD-0354 was administered intraperitoneally at 30, 10, 3, or 0 mg/kg, suspended in 1.0 ml of 0.5% carboxymethyl cellulose sodium. The prodrug IMD-1041 (100 mg/kg) was also administered orally. The rats were euthanized 24 h after LPS injection, and EIU severity was evaluated histologically. The number of infiltrating cells and the protein, TNF-α, and monocyte chemoattractant protein-1 (MCP-1) concentrations in the aqueous humor were determined. TNF-α and MCP-1 concentrations were quantified with enzyme-linked immunosorbent assay. Eye sections were also stained with anti-NFκB and phosphorylated I-κBα antibodies.
The number of infiltrating cells in aqueous humor was 53.6±9.8×10(5), 72.5±17.0×10(5), 127.25±32.0×10(5), and 132.0±25.0×10(5) cells/ml in rats treated with 30, 10, 3, or 0 mg/kg of IMD-0354, respectively. The total protein concentrations of aqueous humor were 92.6±3.1 mg/ml, 101.5±6.8 mg/ml, 112.6±1.9 mg/ml, and 117.33±1.8 mg/ml in rats treated with 30, 10, 3, and 0 mg/kg of IMD-0354, respectively. Infiltrating cells and protein concentrations were significantly decreased by treatment with IMD-0354 (p<0.01). IMD-0354 treatment significantly reduced the concentration of TNF-α (p<0.05) and MCP-1 (p<0.01) in aqueous humor. The number of NFκB positive nuclei was reduced when treated with IMD-0354. Furthermore, IMD-0354-treated EIU rats showed only background levels of phosphorylated I-κBα; however, it was strongly expressed in the iris-ciliary body cell cytoplasm of the IMD-0354 untreated EIU rats. Oral administration of IMD-1041 also decreased the cell number (p<0.01) and protein concentration (p<0.05) of aqueous humor in EIU.
Acute uveitis was ameliorated by inhibition of IKKβ in rats. IMD-0354 and its prodrug IMD-1041 seem to be promising candidates for treating intraocular inflammation/uveitis.
内毒素诱导性葡萄膜炎（EIU）是一种急性眼部炎症的动物模型。几种物质在EIU炎症变化的发展中起主要作用，包括肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β和IL-6。这些炎性细胞因子通过激活IκB激酶（IKK）触发IκB的降解。释放的核因子κB（NFκB）随后转移至细胞核，在细胞核中NFκB发挥其促炎功能。IMD-0354，N-（3,5-双三氟甲基苯基）-5-氯-2-羟基苯甲酰胺，选择性抑制IKKβ，特别是在由促炎细胞因子如TNF-α和IL-1β诱导时。在本研究中，我们使用IMD-0354及其前药IMD-1041研究了IKKβ抑制对EIU是否具有治疗作用。
使用6周龄雄性Lewis大鼠。通过皮下注射200μg稀释于0.1ml磷酸盐缓冲盐水中的大肠杆菌脂多糖（LPS）诱导EIU。IMD-0354以30、10、3或0mg/kg腹腔注射，悬浮于1.0ml 0.5%羧甲基纤维素钠中。前药IMD-1041（100mg/kg）也经口服给药。LPS注射后24小时对大鼠实施安乐死，并通过组织学评估EIU严重程度。测定房水中浸润细胞数量以及蛋白质、TNF-α和单核细胞趋化蛋白-1（MCP-1）浓度。TNF-α和MCP-1浓度通过酶联免疫吸附测定法定量。眼组织切片也用抗NFκB和磷酸化I-κBα抗体染色。
用30、10、3或0mg/kg IMD-0354治疗的大鼠房水中浸润细胞数量分别为53.6±9.8×10⁵、72.5±17.0×10⁵、127.25±32.0×10⁵和132.0±25.0×10⁵个细胞/ml。用30、10、3和0mg/kg IMD-0354治疗的大鼠房水总蛋白浓度分别为92.6±3.1mg/ml、101.5±6.8mg/ml、112.6±1.9mg/ml和117.33±1.8mg/ml。用IMD-0354治疗可显著降低浸润细胞数量和蛋白浓度（p<0.01）。IMD-0354治疗可显著降低房水中TNF-α（p<0.05）和MCP-1（p<0.01）浓度。用IMD-0354治疗时NFκB阳性细胞核数量减少。此外，经IMD-0354治疗的EIU大鼠仅显示磷酸化I-κBα的背景水平；然而，在未经IMD-0354治疗的EIU大鼠的虹膜睫状体细胞质中其强烈表达。口服IMD-1041也可减少EIU大鼠房水中的细胞数量（p<0.01）和蛋白浓度（p<0.05）。
抑制大鼠IKKβ可改善急性葡萄膜炎。IMD-0354及其前药IMD-1041似乎是治疗眼内炎症/葡萄膜炎的有前景的候选药物。
