Department of Morphology, Federal University of Ceará, Fortaleza, Brazil.
BMC Gastroenterol. 2011 Aug 16;11:90. doi: 10.1186/1471-230X-11-90.
Methotrexate treatment has been associated to intestinal epithelial damage. Studies have suggested an important role of nitric oxide in such injury. The aim of this study was to investigate the role of nitric oxide (NO), specifically iNOS on the pathogenesis of methotrexate (MTX)-induced intestinal mucositis.
Intestinal mucositis was carried out by three subcutaneous MTX injections (2.5 mg/kg) in Wistar rats and in inducible nitric oxide synthase knock-out (iNOS-/-) and wild-type (iNOS+/+) mice. Rats were treated intraperitoneally with the NOS inhibitors aminoguanidine (AG; 10 mg/Kg) or L-NAME (20 mg/Kg), one hour before MTX injection and daily until sacrifice, on the fifth day. The jejunum was harvested to investigate the expression of Ki67, iNOS and nitrotyrosine by immunohistochemistry and cell death by TUNEL. The neutrophil activity by myeloperoxidase (MPO) assay was performed in the three small intestine segments.
AG and L-NAME significantly reduced villus and crypt damages, inflammatory alterations, cell death, MPO activity, and nitrotyrosine immunostaining due to MTX challenge. The treatment with AG, but not L-NAME, prevented the inhibitory effect of MTX on cell proliferation. MTX induced increased expression of iNOS detected by immunohistochemistry. MTX did not cause significant inflammation in the iNOS-/- mice.
These results suggest an important role of NO, via activation of iNOS, in the pathogenesis of intestinal mucositis.
甲氨蝶呤治疗与肠上皮损伤有关。研究表明，一氧化氮在这种损伤中起重要作用。本研究旨在探讨一氧化氮（NO），特别是诱导型一氧化氮合酶（iNOS）在甲氨蝶呤（MTX）诱导的肠黏膜炎发病机制中的作用。
通过给 Wistar 大鼠和诱导型一氧化氮合酶敲除（iNOS-/-）及野生型（iNOS+/+）小鼠进行三次皮下 MTX 注射（2.5mg/kg）来诱导肠黏膜炎。在 MTX 注射前 1 小时，大鼠通过腹腔内给予一氧化氮合酶抑制剂氨基胍（AG；10mg/Kg）或 L-NAME（20mg/Kg）治疗，并在第 5 天处死前每日治疗，直到处死。采集空肠组织，通过免疫组织化学法检测 Ki67、iNOS 和硝基酪氨酸的表达，通过 TUNEL 检测细胞死亡。通过髓过氧化物酶（MPO）测定法在三个小肠段中检测中性粒细胞活性。
AG 和 L-NAME 显著减轻了 MTX 引起的绒毛和隐窝损伤、炎症改变、细胞死亡、MPO 活性和硝基酪氨酸免疫染色。AG 治疗，但不是 L-NAME 治疗，可预防 MTX 对细胞增殖的抑制作用。MTX 诱导的 iNOS 免疫组化表达增加。MTX 未在 iNOS-/-小鼠中引起明显的炎症。
这些结果表明，NO 通过激活 iNOS，在肠黏膜炎的发病机制中起重要作用。
