Endothelial dysfunction in rat adjuvant-induced arthritis - Vascular superoxide production by NAD(P)H oxidase and uncoupled endothelial nitric oxide synthase
Endothelial dysfunction in rat adjuvant-induced arthritis - Vascular superoxide production by NAD(P)H oxidase and uncoupled endothelial nitric oxide synthase
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DOI：
10.1002/art.21891
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发表时间：
2006-06-01
影响因子：
--
通讯作者：
Kashihara, Naoki
中科院分区：
文献类型：
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作者：
Haruna, Yoshisuke;Morita, Yoshitaka;Kashihara, Naoki
目标。研究大鼠佐剂性关节炎（AIA）的内皮功能和血管氧化应激水平，考虑到类风湿关节炎（RA）和血管加速疾病之间的关联。取AIA大鼠和对照大鼠制备胸主动脉环。去甲肾上腺素预收缩后，测定乙酰胆碱对血管的舒张反应。采用Western blotting法测定AIA大鼠主动脉中4-羟基-2壬烯醛（HNE）和硝基酪氨酸的含量。主动脉匀浆与各种底物一起培养产生超氧化物的酶，并通过荧光氧化将二氢乙啶氧化为乙啶来评估超氧化物的产生。实时逆转录聚合酶链反应和Western blotting检测主动脉内皮型一氧化氮合酶（eNOS）的表达。采用高效液相色谱法测定eNOS关键辅助因子四氢生物蝶呤（BH4）的血清水平。与对照组大鼠相比，AIA大鼠主动脉环内皮依赖性松弛明显降低。在AIA大鼠主动脉中HNE和硝基酪氨酸的含量增加，表明活性氧过量产生。用NADH或l -精氨酸（eNOS的底物）孵育AIA大鼠主动脉匀浆，可显著增加超氧化物的产生。内皮细胞NOS在AIA大鼠主动脉中高表达。AIA患者血清BH4水平明显降低。用BH4治疗AIA可逆转内皮功能障碍，表明BH4的缺乏可能有助于enos的解偶联。类风湿关节炎的血管功能障碍可以在动物中部分模拟。NAD(P)H氧化酶和未偶联的eNOS导致血管氧化应激增加，可能参与AIA的内皮功能障碍。
Objective. To investigate endothelial function and levels of vascular oxidative stress in rat adjuvant-induced arthritis (AIA), in view of mounting evidence for an association between rheumatoid arthritis (RA) and accelerated vascular disease.Methods. Thoracic aortic rings were prepared from AIA and control rats. After preconstriction by norepinephrine, the vasodilatory response to acetylcholine was determined. The amounts of 4-hydroxy-2nonenal (HNE) and nitrotyrosine in AIA rat aortas were measured by Western blotting. Homogenates of the aortas were incubated with various substrates for superoxide-producing enzymes, and superoxide production was assessed by fluorogenic oxidation of dihydroethidium to ethidium. Expression of endothelial nitric oxide synthase (eNOS) in aortas was examined by real-time reverse transcriptase-polymerase chain reaction and Western blotting. Serum levels of tetrahydrobiopterin (BH4), a critical eNOS cofactor, were determined by high-performance liquid chromatography.Results. Endothelium-dependent relaxation of the aortic ring was significantly depressed in AIA rats compared with control rats. The amounts of HNE and nitrotyrosine were increased in AIA rat aortas, indicating overproduction of reactive oxygen species. Incubation of AIA rat aorta homogenates with NADH or L-arginine, a substrate of eNOS, resulted in a significant increase in superoxide production. Endothelial NOS was highly expressed in AIA rat aortas. Serum levels of BH4 were significantly lower in AIA. Treatment of AIA with BH4 reversed the endothelial dysfunction, suggesting that its deficiency may contribute to the uncoupling of eNOS.Conclusion. Vascular dysfunction in RA can be partially modeled in animals. NAD(P)H oxidase and uncoupled eNOS are responsible for the increase in vascular oxidative stress, which is likely to be involved in the endothelial dysfunction in AIA.