Tilakaratne A, Soory Mena
King's College London Dental Institute, Denmark Hill, London SE5 9RW, UK.
Infect Disord Drug Targets. 2014;14(1):14-22. doi: 10.2174/1871526514666140827101231.
Investigation of osteoblastic responses to oxidative stress, induced by C-reactive protein (CRP) and IL-6 and ameliorating effects of doxycycline (Dox); using assays for 5-alpha dihydrotestosterone (DHT) as an antioxidant marker of healing. IL-6 and CRP are risk markers of periodontitis and prevalent comorbidities in periodontitis subjects.
Confluent monolayer cultures of osteoblasts were incubated with radiolabelled testosterone (14C-T) as substrate, in the presence or absence (Control) of pre-determined optimal concentrations of CRP, IL-6, Dox; alone and in combination (n=8) for 24h in MEM. The eluent was solvent-extracted for steroid metabolites. They were separated using TLC in a benzene/ acetone solvent system 4:1 v/v; and quantified using radioisotope scanning. The identity of formed metabolites was confirmed using the mobility of cold standards added to the samples and disclosed in iodine. Further confirmation of the authenticity of DHT was carried out by combined gas chromatrography-mass spectrometry, after derivatization to pentafluorobenzyloxime trimethyl silyl ether.
The yields of DHT from 14C-testosterone showed 2-fold and 1.8-fold- inhibition in response to IL-6 and CRP respectively and 28% stimulation in response to Dox, via the 5-alpha reductase pathway. The combination of IL-6 + CRP showed a 2-fold reduction in the yields of DHT, elevated to control values when combined with Dox (n=8; p<0.001). Yields of 4-androstenedione showed an inverse relationship to those of DHT, in response to the agents tested, in keeping with the 17-beta hydroxysteroid dehydrogenase pathway.
Inhibition of DHT synthesis in osteoblasts by IL-6 and CRP was overcome by doxycycline. Oxidative actions of IL-6 and CRP; and antioxidant actions of Dox are reinforced by the metabolic yields of DHT in response to agents tested. Using a novel metabolically active model allows closer extrapolation to in vivo conditions; in the context of adjunctive therapeutic applications for periodontitis and prevalent comorbidities.
研究成骨细胞对由C反应蛋白（CRP）和白细胞介素-6（IL-6）诱导的氧化应激的反应，以及强力霉素（Dox）的改善作用；使用5-α二氢睾酮（DHT）检测作为愈合的抗氧化标志物。IL-6和CRP是牙周炎的风险标志物以及牙周炎患者中常见的合并症。
将汇合的成骨细胞单层培养物与放射性标记的睾酮（14C-T）作为底物一起孵育，在存在或不存在（对照）预先确定的最佳浓度的CRP、IL-6、Dox的情况下；单独及联合使用（n = 8），在MEM中孵育24小时。洗脱液用溶剂萃取类固醇代谢物。它们在苯/丙酮溶剂系统4:1 v/v中使用薄层层析进行分离；并使用放射性同位素扫描进行定量。通过向样品中添加冷标准品的迁移率并在碘中显色来确认形成的代谢物的身份。在衍生为五氟苄基肟三甲基硅醚后，通过气相色谱-质谱联用进一步确认DHT的真实性。
通过5-α还原酶途径，来自14C-睾酮的DHT产量在对IL-6和CRP的反应中分别显示出2倍和1.8倍的抑制，在对Dox的反应中显示出28%的刺激。IL-6 + CRP的组合显示DHT产量降低了2倍，与Dox联合时升高至对照值（n = 8；p < 0.001）。在对所测试的试剂的反应中，并与17-β羟类固醇脱氢酶途径一致，4-雄烯二酮的产量与DHT的产量呈反比关系。
强力霉素克服了IL-6和CRP对成骨细胞中DHT合成的抑制。IL-6和CRP的氧化作用；以及Dox的抗氧化作用通过对所测试试剂的反应中DHT的代谢产量得到加强。使用一种新型的代谢活性模型可以更接近地推断体内情况；在牙周炎和常见合并症的辅助治疗应用背景下。
