Inhibition of nuclear factor kappa B activation and inducible nitric oxide synthase transcription by prolonged exposure to high glucose in the human keratinocyte cell line HaCaT
Inhibition of nuclear factor kappa B activation and inducible nitric oxide synthase transcription by prolonged exposure to high glucose in the human keratinocyte cell line HaCaT
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DOI：
10.1111/j.0007-0963.2004.05867.x
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发表时间：
2004-04-01
影响因子：
10.3
通讯作者：
Kosaka, H
中科院分区：
文献类型：
--
作者：
Nakai, K;Kubota, Y;Kosaka, H
背景：在人类皮肤中，诱导型一氧化氮合酶(INOS)似乎是伤口愈合过程中的关键酶，在预防感染方面具有重要作用。我们推测，糖尿病皮肤并发症，如伤口愈合延迟和皮肤感染，是由于皮肤角质形成细胞中高糖改变iNOS活性所致。目的观察高糖对人角质形成细胞系(HaCaT)中iNOS活性的影响。用Griess试剂检测细胞培养上清液中一氧化氮(NO)终产物亚硝酸盐的含量，用高效液相色谱法测定细胞内一氧化氮合酶辅助因子四氢生物蝶呤(BH4)的含量，用逆转录聚合酶链式反应(RT-PCR)分析iNOS mRNA的表达水平，用酶免疫法测定核因子-kappaB(NF-kappaB)的DNA结合活性。但长期(10d)高糖暴露可下调NF-kappaB结合活性，抑制iNOS转录和活性。结论高糖预处理10d可下调NF-kappaB活性，抑制iNOS转录和NO生成，提示糖尿病患者皮肤感染和创面愈合均存在NO合成不足。
Background In human skin, inducible nitric oxide synthase (iNOS) appears to be a key enzyme during wound healing and has roles in protection from infection. We speculated that diabetic skin complications such as delayed wound healing and skin infection were due to iNOS activity altered by high glucose in skin keratinocytes.Objectives The purpose of this study was to see how high levels of glucose affect iNOS activity in the human keratinocyte cell line (HaCaT).Methods HaCaT cells were exposed to high glucose for 1 day or 10 days. We measured nitric oxide (NO) end product nitrite in the culture medium using the Griess reagent, and intracellular tetrahydrobiopterin (BH4, a cofactor of NOS) content by using high-performance liquid chromatography, analysed the expression level of iNOS mRNA by the reverse transcriptase-polymerase chain reaction method and evaluated the DNA binding activity of nuclear factor kappa B (NF-kappaB) by enzyme-immunoassay.Results Short-term exposure (1 day) to a high level of glucose increased BH4 and iNOS activity at the post-translational level. However, long-term exposure (10 days) to high glucose downregulates NF-kappaB binding activity and inhibits iNOS transcription and its activity.Conclusions Pretreatment with high glucose for 10 days down-regulated NF-kappaB activity and inhibited iNOS transcription and NO production, implying the involvement of a deficiency in NO synthesis in both skin infection and impaired wound healing in diabetic patients.