肝缺血再灌注致大鼠脑损伤时诱导型一氧化氮合酶表达的变化

Changes in iNOS expression in brain injury induced by hepatic ischemia-reperfusion in rats

目的评价肝缺血再灌注致大鼠脑损伤时诱导型一氧化氮合酶（iNOS）表达的变化。方法健康雄性Wistar大鼠32只，体重240～280g，随机分为假手术组（S组，n=8）和肝缺血再灌注组（IR组，n=24）。S组仅开腹分离肝动脉、门静脉和胆管，游离脾静脉，40min后切脾，关腹；IR组通过夹闭肝动脉、门静脉和胆管建立大鼠脾静脉．股静脉转流下全肝缺血再灌注模型，全肝缺血40min后再灌注，同时切脾。S组断头处死大鼠，IR组分别于再灌注3、6、24h时断头处死8只大鼠，取脑组织，采用硝酸还原酶法测定一氧化氮（NO）含量，黄嘌呤氧化酶法测定超氧化物歧化酶（SOD）活性，硫代巴比妥酸比色法测定丙二醛（MDA）含量，Western blot法检测硝化酪氨酸（NT）表达，RT-PCR法检测iNOSmRNA表达。结果与s组比较，IR组再灌注6、24h时脑组织NO及MDA的含量升高，NT及iNOSmRNA的表达上调，再灌注6h时SOD活性降低（P〈0．05或0．01）；与再灌注3h时比较，再灌注6、24h时IR组脑组织NO及MDA的含量升高，SOD活性降低（P〈0．05）。结论肝缺血再灌注时大鼠脑组织iNOS表达上调，产生大量NO，生成OONO^-，导致脑损伤。

Objective To investigate the changes in iNOS expression in brain injury induced by hepatic ischemia-reperfusion （IR） in rats. Methods Thirty-two male Wistar rats weighing 240-280 g were randomly divided into 2 groups： sham operation group （group S, n = 8） and group IR（ n = 24）. The hepatic IR was induced by clamping the hepatic artery and portal vein according to the method described by LONG et al. In group IR the rats were killed at 3, 6 and 24 h of reperfusion after 40 min hepatic ischemia （8 rats at each time point）. The rats in group S were also killed. The brains were removed for determination of NO content （by nitrate reductase assay）, SOD activity （by xanthine oxidase method）, MDA content（by calorimetric method）, nitrotyrosine （NT） expression （by Western blot）, and iNOS mRNA expression （by RT-PCR）. Results Compared with group S, cerebral NO and MDA content were significantly increased at 6 and 24 h of reperfusion, expression of cerebral NT and iNOS mRNA up-regulated and SOD activity decreased at 6 h of reperfusiou in group IR （P 〈 0.05 or 0.01 ）. Cerebral NO and MDA content were significantly higher and SOD activity lower at 6 and 24 h of reperfusion than at 3 h of reperfusion in group IR （ P 〈 0.05）. Conclusion The expression of iNOS in brain tissues is up-regulated after hepatic IR and it produces a great amount of NO inducing brain injury through peroxynitrite （ ONOO^- ）.