银杏叶提取物对三氯乙烯诱导的人角质形成细胞NO合成的影响

Effect of Ginkgo Biloba Extract on Trichloroethylene-induced Nitric Oxide Synthesis in Human Keratinocytes

背景与目的:观察银杏叶提取物(ginkgo biloba extract,GBE)对三氯乙烯(trichloroethylene,TCE)诱导的人角质形成细胞(kenatinocyte,KC)一氧化氮(NO)合成和一氧化氮合酶(NOS)基因表达及活性的影响,为探讨TCE职业性皮炎的机制及保护因子提供理论依据。材料与方法:分离的KC用无血清培养基进行原代培养至80%以上融合时,加入不同浓度的GBE预适应2h后再用2.0mmol/L的TCE染毒4h,以不含染毒的培养基作为对照组。根据试剂盒的方法检测NO含量和NOS活力,同时用RT_PCR的方法检测细胞i NOS mRNA的表达情况。结果:2.0mmol/L的TCE处理组NO含量和i NOS活力分别为(41.22±5.45)μmol/L和(0.53±0.07)U/4×105cell,明显高于对照组(24.20±3.72)μmol/L和(0.31±0.03)U/4×105cell。而TCE对KC cNOS活力无影响。10、50和100mg/L GBE保护组未见NO含量的明显下降,150mg/L GBE则能够拮抗2.0mmol/L TCE所致的NO水平和i NOS活力的升高。RT_PCR结果显示GBE的预处理可以抑制TCE诱导的KC i NOS mRNA的过表达。结论:TCE通过诱导KCiNOS基因上调产生大量的NO,GBE对TCE诱导的KCi NOS活力的升高以及iNOS基因表达的上调有抑制作用,也进一步抑制了NO的过量生成,可能对TCE皮肤损害具有保护作用。

BACKGROUND＆AIM： To observe the antagonism of ginkgo biloba extract（GBE） to trichloroethylene （TCE）-induced elevation of nitric oxide, nitric oxide synthase activity and up-regulation of iNOS mRNA in keratinocyte; also to furl.her explore effective protective agent for toxic dermatitis caused by TCE. MATERIALS AND METHODS： Primary cultured normal human keratinocytes in serum-free medium were treated with different concentrations of TCE for 4 h or pre-incubated with different concentrations of GBE for 2 h then with 2.0 mmol/L of TCE. Levels of NO generation and activity of iNOS and cNOS wrere measured according to the kit protocols,whilst RT-PCR was used to determine expression of iNOS mRNA.RESULTS..2.0 mmol/L of TCE could dramatically elevate NO generation and iNOS activity but not cNOS activity. While 10 rag/L, 50 mg/L and 100 mg/L of GBE could partially decrease NO level and iNOS activity induced by 2.0 mmol/L of TCE, 150 mg/L of GBE could fully attenuate the elevated NO level and iNOS activity, iNOS mRNA expression was up-regulated by 2.0 mmol/L of TCE and significantly inhibited by 150 mg/L of GBE. CONCLUSION： Trichloroethylene could up-regulate iNOS gene in keratinocyte, then generate large amount of NO. GBE could inhibit TCE-induced elevation of NO level and iNOS activity, as well as the induced iNOS mRNA up-regulation. Our in vitro study demonstrated that NO may be involved in dermato-toxicity of trichloroethylene and GBE can be a potential protective agent working through NO pathway.