Tinti Federico, Soory Mena
King's College London Dental Institute, Guy's Dental Hospital, London SE1 9RT, UK.
Redox Biol. 2013 Dec 10;2:36-43. doi: 10.1016/j.redox.2013.11.008. eCollection 2013.
This study aims to validate pro-oxidant actions of nicotine (N), using hydrogen peroxide (H2O2) and the antioxidant glutathione (G) in an in vitro model of human gingival fibroblasts (HGF) and human oral periosteal fibroblasts (HPF); radiolabelled androgens are used as biomarkers of redox status. Oxidative stress is an important mediator of inflammatory repair. The androgen metabolite 5α-dihydrotestosterone (DHT) is an effective biomarker of oxidative stress and healing.
6 Cell-lines of HGF and HPF established in confluent monolayer culture were incubated in Eagle's MEM using 14C-testosterone and 14C-4-androstendione as substrate; in conjunction with effective concentrations of N, G and H2O2 established at N250, G3 μg/ml and 3%H2O2 w/w, 0.5 μl/ml. Combinations of H2O2G and H2O2GN were used in order to compare the oxidative effects of N/H2O2 and their responses to glutathione. At 24 h, the medium was solvent extracted, evaporated to dryness and subjected to TLC in a benzene/acetone solvent system 4:1 v/v for the separation of metabolites. The separated metabolites were quantified using a radioisotope scanner.
The mean trends of 6 cell-lines for both substrates and each cell type demonstrated that the yield of the main metabolite DHT was significantly reduced by N and H2O2 alone (2-fold, n=6; p<0.01). The inhibition caused by H2O2 was overcome by the antioxidant glutathione in the combination H2O2G, to values similar to those of controls (n=6; p<0.01). It is relevant that when N was added to this neutralized combination, the decrease in yields of DHT triggered by N were comparable to those induced by H2O2; and retaining the positive effect of G.
Oxidative stress mediated by H2O2 was overcome by glutathione and recurred when nicotine was added, suggestive of a pro- oxidant role for nicotine. Androgen biomarkers are a sensitive index of oxidative stress which affects wound healing.
本研究旨在利用过氧化氢（H₂O₂）和抗氧化剂谷胱甘肽（G），在人牙龈成纤维细胞（HGF）和人口腔骨膜成纤维细胞（HPF）的体外模型中验证尼古丁（N）的促氧化作用；放射性标记的雄激素用作氧化还原状态的生物标志物。氧化应激是炎症修复的重要介质。雄激素代谢物5α-二氢睾酮（DHT）是氧化应激和愈合的有效生物标志物。
将在汇合单层培养中建立的6种HGF和HPF细胞系在伊格尔氏MEM培养基中培养，使用¹⁴C-睾酮和¹⁴C-4-雄烯二酮作为底物；结合在N250、G3μg/ml和3%H₂O₂ w/w、0.5μl/ml确定的有效浓度的N、G和H₂O₂。使用H₂O₂G和H₂O₂GN的组合来比较N/H₂O₂的氧化作用及其对谷胱甘肽的反应。在24小时时，对培养基进行溶剂萃取，蒸发至干，并在苯/丙酮溶剂系统4:1 v/v中进行薄层层析以分离代谢物。使用放射性同位素扫描仪对分离的代谢物进行定量。
两种底物和每种细胞类型的6种细胞系的平均趋势表明，单独使用N和H₂O₂可使主要代谢物DHT的产量显著降低（2倍，n = 6；p < 0.01）。在组合H₂O₂G中，抗氧化剂谷胱甘肽克服了H₂O₂引起的抑制作用，使其值与对照组相似（n = 6；p < 0.01）。重要的是，当将N添加到这种中和组合中时，N引发的DHT产量下降与H₂O₂诱导的下降相当；并保留了G的积极作用。
谷胱甘肽克服了由H₂O₂介导的氧化应激，当添加尼古丁时氧化应激复发，提示尼古丁具有促氧化作用。雄激素生物标志物是影响伤口愈合的氧化应激的敏感指标。
