| 摘要: |
| 目的 探讨香烟烟雾提取物（CSE）及尼古丁对甲状腺相关眼病（TAO）氧化应激的影响，并以NF-κB为靶点探索其可能的分子机制。方法 取5例无吸烟史的TAO患者和3例无吸烟史的因其他原因行眼外科手术的患者（对照患者）的眼外肌组织，原代培养眼外肌来源的眼眶成纤维细胞（OFs）。将OFs经10% CSE、0.5 μg/mL尼古丁、10 μg/mL尼古丁分别作用，即为CSE组、低浓度尼古丁组和高浓度尼古丁组。分别培养24、72 h后分别采用MTT法、ELISA法和分光光度计法检测各组细胞活性、8-羟脱氧鸟苷（8-OHdG）水平及丙二醛（MDA）含量；24 h后采用酶标仪法检测活性氧（ROS）水平，RT-PCR法检测NF-κB mRNA水平。结果 干预24及72 h后，TAO患者及对照患者OFs的细胞活力在CSE作用下均下降（P<0.05），且TAO患者对OFs更敏感。高浓度尼古丁干预24 h后，TAO患者OFs的8-OHdG水平增加（P<0.05）；低浓度尼古丁干预72 h时，OFs的8-OHdG水平也增加（P<0.05）。TAO患者及对照患者OFs经CSE或不同浓度尼古丁干预72 h后的MDA含量均增加（P<0.01）。高浓度尼古丁干预24 h后，TAO患者OFs中ROS水平上升（P<0.01）。CSE干预24 h后，TAO患者OFs中NF-κB mRNA的表达上升（P<0.05）；而对照患者OFs NF-κB mRNA的表达经CSE及高浓度尼古丁干预后下降（P<0.05）。结论 吸烟可通过激活TAO患者OFs的氧化应激参与TAO的病理过程，即使吸烟量较少，经时间累积也可能发挥作用，其机制可能是通过上调NF-κB mRNA的表达来实现的。 |
| 关键词: 吸烟 尼古丁 氧化性应激 NF-κB 甲状腺相关眼病 |
| DOI：10.16781/j.0258-879x.2016.10.1239 |
| 投稿时间：2016-04-18修订日期：2016-07-03 |
| 基金项目:国家自然科学基金（81371056）. |
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| Cigarette smoke extract and nicotine induce oxidative stress and up-regulate NF-κB expression in orbital fibroblasts of patients with thyroid-associated opthalmopathy |
| WANG Qiu-hong,ZENG Cheng-cheng,MOU Pei,GAO Lian-di,SHEN Ya,WEI Rui-li* |
(Department of Ophthalmology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
*Corresponding author) |
| Abstract: |
| Objective To discuss the impact of cigarette smoke extract (CSE) and nicotine on the oxidative stress in thyroid-associated ophthalmopathy (TAO) and to explore the possible molecular mechanism involving NF-κB signal pathway. Methods The orbital fibroblasts (OFs) were originally generated from 5 patients without smoking history and 3 volunteers without smoking history and were identified by using immunohistochemistry. Primary cultured extraocular muscle OFs were stimulated with 10% CSE (10% CSE group), 0.5 μg/mL nicotine (low-concentration nicotine group) or 10 μg/mL nicotine (high-concentration nicotine group). MTT assay, ELISA and spectrophotometer method were used to determine the changes of cell activity, 8-hydroxydeoxyguanosine (8-OHdG) levels and malondialdehyde (MDA) contents after intervention for 24 h and 72 h. ELISA was used to determine reactive oxygen species (ROS) level after 24 h culture, while RT-RCR was used to determine NF-κB mRNA expression. Results After intervention for 24 h and 72 h, the cell activities of OFs were significantly decreased after intervention with CSE in both TAO patients and healthy volunteers (P<0.05), with the OFs of TAO patients being more sensitive. After intervention for 24 h, the 8-OHdG level was significantly increased in TAO patients' OFs treated with high-concentration nicotine (P<0.05); for a longer intervention duration of 72 h, the 8-OHdG level was also significantly increased in the group treated with low-concentration nicotine (P<0.05). After intervention with CSE, low- and high-concentration nicotine for 72 h, MDA contents were increased significantly in both OFs groups (P<0.01). After intervention for 24 h, the ROS content was significantly increased in the TAO patients' OFs treated with high-concentration nicotine than in the untreated group (P<0.01). After intervention for 24 h, the expression of NF-κB mRNA was significantly up-regulated after stimulation with CSE in the OFs of TAO patients than that in the untreated group (P<0.05); while the expression of NF-κB mRNA was significantly decreased in OFs stimulated with CSE and high-concentration nicotine compared with untreated group (P<0.05). Conclusion Smoking may influence TAO pathological process through stimulating oxidative stress in OFs of TAO patients, and the stimulation may accumulate and sustain even if the smoking is slight, which may be related to up-regulation of NF-κB mRNA expression. |
| Key words: smoking nicotine oxidative stress NF-κB thyroid-associated ophthalmopathy |
