Cigarette smoke extract and nicotine induce oxidative stress and up-regulate NF-κB expression in orbital fibroblasts of patients with thyroid-associated opthalmopathy / 第二军医大学学报

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 immunohis to chemistry. 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 inTAO patients7 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 patients7 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 NFκB 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 NFκB mRNA expression.