Division of Genetics and Development, Toronto Western Research Institute, 399 Bathurst Street, Toronto, M5T 2S8, Canada.
Arthritis Res Ther. 2012 Jun 20;14(3):R150. doi: 10.1186/ar3886.
Effective treatment of reactive arthritis would ideally achieve both control of inflammation and eradication of persisting arthritogenic pathogens. We use a model of experimental Chlamydia trachomatis-induced arthritis (CtIA) to evaluate the effectiveness of nafamostat mesilate (NM), a serine protease inhibitor with complement-modifying effects and anticoagulant properties. To date clinical use of NM has largely been in Asia and has been primarily confined to inflammatory states such as pancreatitis.
In vitro studies examined inhibition of Chlamydia proliferation using fibroblast cell lines as targets and phase contrast microscopy. In vivo studies used an established protocol, experimental CtIA, induced in Lewis rats by injection of synoviocyte-packaged C. trachomatis. NM was dissolved in water and administered by daily intraperitoneal injection at a dose of 10 mg/kg beginning the day prior to the administration of Chlamydia. Readouts in vivo included (i) joint swelling, (ii) histopathology scoring of severity of arthritis, (iii) host clearance of the pathogen (by ELISA, the IDEIA PCE Chlamydia).
NM exerted a dose-dependent inhibition of chlamydial proliferation in vitro. Without NM, the mean number of inclusion bodies (IB) per well was 17,886 (± 1415). At 5 μg/mL NM, there were 8,490 (± 756) IB, at 25 μg/mL NM there were 35 IB and at 50 μg/mL NM no IB was observed. Chlamydial antigens in each well along the concentration gradient were assayed by ELISA, demonstrating that at 25 μg/mL NM inhibition of Chlamydia was almost complete. In the experimental arthritis model, joint swelling was significantly reduced with NM treatment: average joint width for the NM-treated animals was 8.55 mm (s.d. ± 0.6578, n = 10) versus 11.18 mm (s.d. ± 0.5672, n = 10) in controls (P < 0.001). Histopathology scoring indicated that NM resulted in a marked attenuation of the inflammatory infiltration and joint damage: mean pathology score in NM-treated animals was 10.9 (± 2.45, n = 11) versus 15.9 (± 1.45, n = 10) in controls (P < 0.0001). With respect to persistence of Chlamydia within the synovial tissues, NM treatment was accompanied by a reduction in the microbial load in the joint: mean optical density (O.D.) for ELISA with NM treatment was 0.05 (± 0.02, n = 4) versus 0.18 (± 0.05, n = 4) in controls (P < 0.001).
NM is a protease inhibitor not previously recognized to possess antimicrobial properties. The present study demonstrates for the first time that NM exerts significant impact on C. trachomatis-induced arthritis and suggests that such approaches may prove clinically useful in chronic reactive arthritis.
理想情况下，反应性关节炎的有效治疗应既能控制炎症，又能消除持续存在的致关节炎病原体。我们使用沙眼衣原体（CtIA）诱导的实验性关节炎模型来评估奈莫司他（NM）的有效性，奈莫司他是一种具有补体调节作用和抗凝特性的丝氨酸蛋白酶抑制剂。迄今为止，NM 的临床应用主要在亚洲，主要局限于胰腺炎等炎症状态。
体外研究使用成纤维细胞系作为靶标，通过相差显微镜观察抑制衣原体增殖的效果。体内研究使用已建立的实验性 CtIA 模型，通过注射含有衣原体的滑膜细胞包被物在刘易斯大鼠中诱导关节炎。NM 溶解在水中，在注射衣原体前一天开始，每天腹腔注射 10mg/kg 的剂量。体内研究的结果包括：（i）关节肿胀，（ii）关节炎严重程度的组织病理学评分，（iii）宿主清除病原体（通过 ELISA，IDEIA PCE 衣原体）。
NM 在体外表现出对衣原体增殖的剂量依赖性抑制作用。没有 NM，每个孔的包涵体（IB）数量为 17,886（±1415）。在 5μg/mL NM 时，有 8,490（±756）个 IB，在 25μg/mL NM 时，有 35 个 IB，在 50μg/mL NM 时没有观察到 IB。在每个孔的浓度梯度上用 ELISA 检测衣原体抗原，证明在 25μg/mL NM 时，衣原体的抑制作用几乎完全。在实验性关节炎模型中，NM 治疗显著减轻了关节肿胀：NM 治疗组动物的平均关节宽度为 8.55mm（s.d. ±0.6578，n=10），而对照组为 11.18mm（s.d. ±0.5672，n=10）（P<0.001）。组织病理学评分表明 NM 导致炎症浸润和关节损伤明显减轻：NM 治疗组动物的平均病理评分（10.9±2.45，n=11）明显低于对照组（15.9±1.45，n=10）（P<0.0001）。关于衣原体在滑膜组织中的持续存在，NM 治疗伴随着关节中微生物负荷的降低：NM 治疗的平均光密度（O.D.）为 0.05（±0.02，n=4），而对照组为 0.18（±0.05，n=4）（P<0.001）。
NM 是一种以前未被认为具有抗菌特性的蛋白酶抑制剂。本研究首次证明 NM 对沙眼衣原体诱导的关节炎有显著影响，并表明这种方法可能在慢性反应性关节炎的临床治疗中具有实用价值。
