Yang Shu-Long, Chen Li-jun, Kong Yin, Xu Dan, Lou Yi-Jia
Institute of Pharmacology and Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China.
Pharmacology. 2007;80(1):11-20. doi: 10.1159/000102595. Epub 2007 May 10.
Leukotriene (LT) C4 (LTC4) synthesis enzymes including LTC4 synthase (LTC4S), microsomal glutathione S-transferase (MGST) 2 and MGST3 can all conjugate LTA4 and reduced glutathione (GSH) to form LTC4, which is related to hepatic ischemia/reperfusion (I/R) injury. The relationship between nitric oxide (NO) and cysteinyl LTs has been shown in previous studies. However, the mechanisms of NO action on gene expression of LTC4 synthesis enzymes are still largely unclear during hepatic I/R. Adult male Sprague-Dawley rats were divided into 5 groups: a sham group (control), an I/R group, and sodium nitroprusside (SNP, 2.5, 5 and 10 microg/kg/min)+I/R groups. Livers were subjected to 60 min of partial hepatic ischemia followed by 5 h of reperfusion, saline or SNP (2.5, 5 and 10 microg/kg/min) administered intravenously. The mRNA levels of LTC4 synthesis enzymes, inducible NO synthase (iNOS) and endothelial No synthase (eNOS) in rat liver tissue were examined by RT-PCR; the protein expressions of NF-kappaB p65, p50 and IkappaBalpha in liver cell lysates and nuclear extracts were detected by Western blot analysis, and serum NO2. levels were also evaluated. Serum NO2. levels, the protein expressions of NF-kappaB p65 and p50 in the nucleus extract, and hepatic mRNA expressions of LTC4S and iNOS were decreased while hepatic mRNA of eNOS was increased in the SNP (5 and 10 microg/kg/min)+I/R groups when compared with those in the I/R group. SNP (2.5 microg/kg/min) promoted the mRNA expressions of both MGST2 and MGST3, whereas SNP (10 microg/kg/min) increased MGST2 mRNA but decreased MGST3 mRNA compared to those in I/R group. Compared with control, the mRNA expression of MGST2 and MGST3 were elevated in SNP (2.5 microg/kg/min)+I/R group, MGST3 mRNA was significantly declined in the SNP (5 and 10 microg/kg/min)+I/R groups. Immunohistochemistry staining revealed that I/R liver exhibited strong cytoplasmic and nuclear staining for NF-kappaB p65, but the livers of the SNP (2.5 microg/kg/min)+I/R group presented slight cytoplasmic and nuclear staining. But IkappaBalpha protein in all groups remains unchanged. It was concluded that SNP downregulated LTC4S mRNA expression by inhibiting NF-kappaB activation independent of IkappaBalpha, but appeared to have a dual influence on the mRNA expressions of MGST2 and MGST3 by other signaling pathways during hepatic I/R injury.
白三烯（LT）C4（LTC4）合成酶，包括LTC4合酶（LTC4S）、微粒体谷胱甘肽S - 转移酶（MGST）2和MGST3，均可使白三烯A4（LTA4）与还原型谷胱甘肽（GSH）结合形成LTC4，这与肝脏缺血/再灌注（I/R）损伤有关。先前的研究已表明一氧化氮（NO）与半胱氨酰白三烯之间的关系。然而，在肝脏I/R过程中，NO作用于LTC4合成酶基因表达的机制仍不清楚。成年雄性Sprague - Dawley大鼠分为5组：假手术组（对照组）、I/R组、硝普钠（SNP，2.5、5和10μg/kg/min）+I/R组。对肝脏进行60分钟的部分肝缺血，随后进行5小时的再灌注，静脉注射生理盐水或SNP（2.5、5和10μg/kg/min）。通过逆转录聚合酶链反应（RT - PCR）检测大鼠肝组织中LTC4合成酶、诱导型一氧化氮合酶（iNOS）和内皮型一氧化氮合酶（eNOS）的mRNA水平；通过蛋白质免疫印迹分析检测肝细胞裂解物和核提取物中核因子κB（NF - κB）p65、p50和IκBα的蛋白表达，同时评估血清中NO2 - 的水平。与I/R组相比，SNP（5和10μg/kg/min）+I/R组血清NO2 - 水平、核提取物中NF - κB p65和p50的蛋白表达以及肝脏中LTC4S和iNOS的mRNA表达降低，而eNOS的肝脏mRNA表达增加。SNP（2.5μg/kg/min）促进了MGST2和MGST3的mRNA表达，而与I/R组相比，SNP（10μg/kg/min）增加了MGST2 mRNA但降低了MGST3 mRNA。与对照组相比，SNP（2.5μg/kg/min）+I/R组中MGST2和MGST3的mRNA表达升高，SNP（5和10μg/kg/min）+I/R组中MGST3 mRNA显著下降。免疫组织化学染色显示，I/R组肝脏中NF - κB p65呈现强烈的细胞质和细胞核染色，而SNP（2.5μg/kg/min）+I/R组肝脏呈现轻微的细胞质和细胞核染色。但所有组中IκBα蛋白保持不变。研究得出结论，在肝脏I/R损伤过程中，SNP通过独立于IκBα抑制NF - κB激活而下调LTC4S mRNA表达，但似乎通过其他信号通路对MGST2和MGST3的mRNA表达具有双重影响。
