McKenzie S M, Doe W F, Buffinton G D
Division of Molecular Medicine, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.
Gut. 1999 Feb;44(2):180-5. doi: 10.1136/gut.44.2.180.
Reactive oxygen and nitrogen derived species produced by activated neutrophils have been implicated in the damage of mucosal proteins including the inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the active inflammatory lesion in patients with inflammatory bowel disease (IBD). This study investigated the efficacy of currently used IBD therapeutics to prevent injury mediated by reactive oxygen and nitrogen derived species.
GAPDH activity of human colon epithelial cells was used as a sensitive indicator of injury produced by reactive oxygen and nitrogen derived species. HCT116 cells (10(6)/ml phosphate buffered saline; 37 degrees C) were incubated in the presence of 5-aminosalicylic acid (5-ASA), 6-mercaptopurine, methylprednisolone, or metronidazole before exposure to H2O2, HOCl, or NO in vitro. HCT116 cell GAPDH enzyme activity was determined by standard procedures. Cell free reactions between 5-ASA and HOCl were analysed by spectrophotometry and fluorimetry to characterise the mechanism of oxidant scavenging.
GAPDH activity of HCT116 cells was inhibited by the oxidants tested: the concentration that produced 50% inhibition (IC50) was 44.5 (2.1) microM for HOCl, 379.8 (21.3) microM for H2O2, and 685.8 (103.8) microM for NO (means (SEM)). 5-ASA was the only therapeutic compound tested to show efficacy (p<0. 05) against HOCl mediated inhibition of enzyme activity; however, it was ineffective against H2O2 and NO mediated inhibition of GAPDH. Methylprednisolone, metronidazole, and the thiol-containing 6-mercaptopurine were ineffective against all oxidants. Studies at ratios of HOCl:5-ASA achievable in the mucosa showed direct scavenging to be the mechanism of protection of GAPDH activity. Mixing 5-ASA and HOCl before addition to the cells resulted in significantly greater protection of GAPDH activity than when HOCl was added to cells preincubated with 5-ASA. The addition of 5-ASA after HOCl exposure did not restore GAPDH activity.
Therapies based on 5-ASA may play a direct role in scavenging the potent neutrophil oxidant HOCl, thereby protecting mucosal GAPDH from oxidative inhibition. These findings suggest that strategies for the further development of new HOCl scavenging compounds may be useful in the treatment of IBD.
活化的中性粒细胞产生的活性氧和氮衍生物质与黏膜蛋白损伤有关，包括炎症性肠病（IBD）患者活动性炎症病变中甘油醛 - 3 - 磷酸脱氢酶（GAPDH）的抑制。本研究调查了目前使用的IBD治疗药物预防活性氧和氮衍生物质介导的损伤的疗效。
人结肠上皮细胞的GAPDH活性用作活性氧和氮衍生物质产生损伤的敏感指标。在体外将HCT116细胞（10⁶/ml磷酸盐缓冲盐水；37℃）在5 - 氨基水杨酸（5 - ASA）、6 - 巯基嘌呤、甲基泼尼松龙或甲硝唑存在下孵育，然后暴露于H₂O₂、HOCl或NO。通过标准程序测定HCT116细胞的GAPDH酶活性。通过分光光度法和荧光法分析5 - ASA与HOCl之间的无细胞反应，以表征清除氧化剂的机制。
测试的氧化剂抑制了HCT116细胞的GAPDH活性：产生50%抑制（IC₅₀）的浓度，HOCl为44.5（2.1）μM，H₂O₂为379.8（21.3）μM，NO为685.8（103.8）μM（均值（标准误））。5 - ASA是测试的唯一显示出对HOCl介导的酶活性抑制有疗效（p<0.05）的治疗化合物；然而，它对H₂O₂和NO介导的GAPDH抑制无效。甲基泼尼松龙、甲硝唑和含硫醇的6 - 巯基嘌呤对所有氧化剂均无效。在黏膜中可达到的HOCl:5 - ASA比例下的研究表明，直接清除是保护GAPDH活性的机制。在将5 - ASA和HOCl添加到细胞之前混合，比将HOCl添加到预先用5 - ASA孵育的细胞中能更显著地保护GAPDH活性。在HOCl暴露后添加5 - ASA不能恢复GAPDH活性。
基于5 - ASA的疗法可能在清除强效中性粒细胞氧化剂HOCl中起直接作用，从而保护黏膜GAPDH免受氧化抑制。这些发现表明，进一步开发新的HOCl清除化合物的策略可能对IBD的治疗有用。
