Dooley A, Low S Y, Holmes A, Kidane A G, Abraham D J, Black C M, Bruckdorfer K R
Department of Biochemistry and Molecular Biology, Royal Free Campus, University College Medical School, London NW3 2PF, UK.
Rheumatology (Oxford). 2008 Mar;47(3):272-80. doi: 10.1093/rheumatology/kem303. Epub 2008 Jan 30.
Nitric oxide (*NO) is an important physiological signalling molecule and a potent vasodilator. We have previously demonstrated abnormal *NO metabolism in the plasma of patients with systemic sclerosis (SSc; scleroderma), a disease that features vascular dysfunction as well as collagen overproduction and fibrosis. The aim of the present study was to examine nitric oxide synthase (NOS) expression and activity and assess the potential role of antioxidants in the scleroderma-like syndrome of the tight-skin 1 (TSK-1/+) mouse, an experimental animal model for fibrosis.
Skin, lung or plasma was taken from TSK-1/+ (n = 15) and wild-type (WT; n = 12) littermate mice. Type 1 collagen, endothelial NOS (eNOS), haemoxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) protein and gene expression were determined by western blot and reverse transcriptase-polymerase chain reaction. eNOS expression was further determined by immunohistochemistry. NOS activity was evaluated by conversion of [14C] L-arginine to [14C] L-citrulline. Levels of circulating plasma nitrite/nitrate (NO(x)) were also measured. Total antioxidant activity was evaluated by ABTS+ production (ABTS = 2,2'-azino-bis-[3-ethylbenz-thiazoline-6-sulphonic acid).
In the skin, eNOS was present in the epidermal layer, hair follicles and also in the endothelial cells lining the blood vessels. Expression of both the eNOS protein and gene was significantly reduced in TSK-1/+ skin tissue, while type 1 collagen protein was elevated compared with WT. Furthermore, there was decreased NOS activity in TSK-1/+ skin tissue; however, there was no measurable difference in plasma NO(x). Correspondingly, the protective antioxidant enzyme HO-1 and the associated transcription factor Nrf2 showed reduced protein and gene expression levels in TSK-1/+ skin, while there was also less total antioxidant activity. In TSK-1/+ lung tissue, however, we observed no difference in collagen protein expression, *NO metabolism or HO-1 expression and total antioxidant activity compared with WT.
The findings suggest that there is also abnormal *NO metabolism in the TSK-1/+ mouse model of fibrosis, particularly in the skin, while expression and activity of protective antioxidants are reduced. The TSK-1/+ mouse may also be useful for testing treatments that target vascular endothelial cell function in patients with SSc.
一氧化氮（NO）是一种重要的生理信号分子和强效血管舒张剂。我们之前已证明系统性硬化症（SSc；硬皮病）患者血浆中存在异常的NO代谢，该疾病具有血管功能障碍以及胶原蛋白过度产生和纤维化的特征。本研究的目的是检测一氧化氮合酶（NOS）的表达和活性，并评估抗氧化剂在紧皮1（TSK-1/+）小鼠硬皮病样综合征中的潜在作用，TSK-1/+小鼠是一种纤维化实验动物模型。
从TSK-1/+（n = 15）和野生型（WT；n = 12）同窝小鼠中获取皮肤、肺或血浆。通过蛋白质印迹法和逆转录聚合酶链反应测定Ⅰ型胶原蛋白、内皮型一氧化氮合酶（eNOS）、血红素加氧酶-1（HO-1）和核因子红细胞2相关因子2（Nrf2）的蛋白质和基因表达。通过免疫组织化学进一步测定eNOS表达。通过将[14C]L-精氨酸转化为[14C]L-瓜氨酸来评估NOS活性。还测量了循环血浆中亚硝酸盐/硝酸盐（NO(x)）的水平。通过ABTS+生成（ABTS = 2,2'-联氮-双-[3-乙基苯并噻唑啉-6-磺酸]）评估总抗氧化活性。
在皮肤中，eNOS存在于表皮层、毛囊以及血管内衬的内皮细胞中。TSK-1/+皮肤组织中eNOS蛋白和基因的表达均显著降低，而与WT相比，Ⅰ型胶原蛋白蛋白升高。此外，TSK-1/+皮肤组织中的NOS活性降低；然而，血浆NO(x)没有可测量的差异。相应地，保护性抗氧化酶HO-1和相关转录因子Nrf2在TSK-1/+皮肤中的蛋白和基因表达水平降低，同时总抗氧化活性也较低。然而，与WT相比，在TSK-1/+肺组织中，我们未观察到胶原蛋白蛋白表达、*NO代谢或HO-1表达及总抗氧化活性的差异。
研究结果表明，在TSK-1/+纤维化小鼠模型中也存在异常的*NO代谢，尤其是在皮肤中，同时保护性抗氧化剂的表达和活性降低。TSK-1/+小鼠也可能有助于测试针对SSc患者血管内皮细胞功能的治疗方法。
