Schiekofer S, Rudofsky G, Andrassy M, Schneider J, Chen J, Isermann B, Kanitz M, Elsenhans S, Heinle H, Balletshofer B, Häring H-U, Schleicher E, Nawroth P P, Bierhaus A
Department of Medicine I, University of Heidelberg, Heidelberg, Germany.
Diabetes Obes Metab. 2003 Jul;5(4):251-61. doi: 10.1046/j.1463-1326.2003.00270.x.
Glimepiride has the lowest ratio of insulin release to glucose decrease compared with other sulphonylureas. This prompted us to study in vitro and in vivo in a placebo-controlled study the effect of glimepiride on the redox-sensitive transcription factor nuclear factor-kappa B (NF-kappaB).
Fifteen patients with type 2 diabetes on glibenclamide with a stable HbA1c over the last 6 months were included. After sampling for determination of baseline values, 10 patients were changed to an equivalent dose of glimepiride, while the placebo group was maintained at glibenclamide plus placebo. The glimepiride dose in these patients was adjusted so that no change in glucose control occurred, allowing for direct comparison. The others were kept on glibenclamide and received additional placebo. After 4 weeks of glimepiride or glibenclamide plus placebo, a second blood sample was taken. Mononuclear cells were isolated and assayed in a tissue-culture-independent electrophoretic mobility shift assay (EMSA)-based detection system for NF-kappaB binding activity, and by Western Blot for nuclear localization of NF-kappaB-p65, the cytoplasmic content of IkappaBalpha and the NF-kappaB-controlled haemoxygenase-1. Glimepiride dose-dependent inhibition of carboxymethyllysin (CML) albumin or tumour necrosis factor alpha (TNFalpha)- and H2O2-induced activation of NF-kappaB binding were determined, using isolated peripheral blood mononuclear cells from healthy volunteers, and transcriptional activity of bovine aortic endothelial cells either left untreated or induced with CML albumin incubated with or without glimepiride. Furthermore, in-vitro studies were implemented to demonstrate radical quenching properties of glimepiride in the cell-free 2,2'-azo-bis(2-aminopropane)-dihydrochloride system.
Baseline glucose and HbA1c remained stable in the patients switched from glibenclamide to a corresponding dose of glimepiride or kept on glibenclamide plus placebo. While in the group of patients only taking glibenclamide plus placebo the NF-kappaB binding activity did not change significantly (p = 0.58), the NF-kappaB binding activity in the group of patients taking glimepiride was reduced from 19.3 relative NF-kappaB-p65-equivalents to 15.5 relative NF-kappaB-p65-equivalents (p = 0.04). The nuclear translocation of NF-kappaB-p65 was reduced from 100% at baseline to 58% after 4 weeks (p = 0.04); the cytoplasmic localization of NF-kappaB-p65 increased from 100% to 129% (p = 0.03) and the cytoplasmic content of IkappaBalpha increased from 100% to 109% (p = 0.06). The redox-sensitive haemoxygenase-1 antigen was reduced from 100% to 82% (p = 0.04). To prove directly that glimepiride reduces NF-kappaB activation, we isolated peripheral blood mononuclear cells (PBMC) from healthy volunteers. In vitro, glimepiride reduced TNFalpha-(1 nmol/l) and CML albumin (800 nmol/l)-induced NF-kappaB activation dose dependently, being half maximal at 120 micromol/l. H2O2-mediated NF-kappaB activation was only partially reduced. In addition, glimepiride reduced NF-kappaB-dependent gene expression using a NF-kappaB-driven luciferase reporter system. Finally, a cell-free detection system showed that glimepiride has radical quenching properties.
Glimepiride can affect the activation of the redox-sensitive transcription factor NF-kappaB in vitro and in vivo.
与其他磺脲类药物相比，格列美脲的胰岛素释放与血糖降低的比率最低。这促使我们在一项安慰剂对照研究中，对格列美脲在体外和体内对氧化还原敏感转录因子核因子-κB（NF-κB）的作用进行研究。
纳入15例服用格列本脲且在过去6个月内糖化血红蛋白（HbA1c）稳定的2型糖尿病患者。在采集样本测定基线值后，10例患者换用等效剂量的格列美脲，而安慰剂组继续服用格列本脲加安慰剂。调整这些患者的格列美脲剂量，使血糖控制无变化，以便进行直接比较。其余患者继续服用格列本脲并额外给予安慰剂。在服用格列美脲或格列本脲加安慰剂4周后，采集第二份血样。分离单核细胞，并在基于组织培养非依赖性电泳迁移率变动分析（EMSA）的检测系统中检测NF-κB结合活性，通过蛋白质免疫印迹法检测NF-κB-p65的核定位、IκBα的细胞质含量以及NF-κB调控的血红素加氧酶-1。使用健康志愿者的外周血单个核细胞，测定格列美脲对羧甲基赖氨酸（CML）白蛋白或肿瘤坏死因子α（TNFα）以及H2O2诱导的NF-κB结合激活的剂量依赖性抑制作用，以及牛主动脉内皮细胞在未处理或用CML白蛋白诱导后，在有或无格列美脲孵育情况下的转录活性。此外，进行体外研究以证明格列美脲在无细胞的2,2'-偶氮二（2-氨基丙烷）二盐酸盐系统中的自由基淬灭特性。
从格列本脲换用相应剂量格列美脲或继续服用格列本脲加安慰剂的患者，其基线血糖和HbA1c保持稳定。仅服用格列本脲加安慰剂的患者组中，NF-κB结合活性无显著变化（p = 0.58），而服用格列美脲的患者组中，NF-κB结合活性从19.3相对NF-κB-p65等效物降至15.5相对NF-κB-p65等效物（p = 0.04）。NF-κB-p65的核转位从基线时的100%降至4周后的58%（p = 0.04）；NF-κB-p65的细胞质定位从100%增加至129%（p = 0.03），IκBα的细胞质含量从100%增加至109%（p = 0.06）。氧化还原敏感的血红素加氧酶-1抗原从100%降至82%（p = 0.04）。为直接证明格列美脲可降低NF-κB激活，我们从健康志愿者中分离外周血单个核细胞（PBMC）。在体外，格列美脲可剂量依赖性地降低TNFα（1 nmol/L）和CML白蛋白（800 nmol/L）诱导的NF-κB激活，在120 μmol/L时达到半数最大效应。H2O2介导的NF-κB激活仅部分降低。此外，格列美脲使用NF-κB驱动的荧光素酶报告系统降低了NF-κB依赖性基因表达。最后，无细胞检测系统表明格列美脲具有自由基淬灭特性。
格列美脲在体外和体内均可影响氧化还原敏感转录因子NF-κB的激活。
