The Role Of Smoking And Oxidative Stress On The Pathogenesis Of Thyroid Associated Ophthalmopathy

ObjectiveThyroid-associated ophthalmopathy(TAO)is autoimmunity disease with unknown pathogenesis and is a common orbit disease in adults.With diversified clinical manifestations and severe outcomes,the disease has been a problem troubling ophthalmologists for recent years.Smoking has been demonstrated to be a significant risk factor for TAO and found to be related with oxidative stress.It is known to us that oxidative stress plays an important role in the course of TAO.Cigarettes have extremely complicated ingredients,in which nicotine is believed to be the toxic ingredient.Recently,cigarette smoke extractive(CSE)and nicotine are commonly used as stimulating factors in Chinese and foreign studies on smoking for other diseases;orbital fibroblast as the final effector cell of TAO is the classic study model in TAO-concerned studies during recent years.Therefore,the primary culture of orbital fibroblasts derived from extraocular muscle is adopted in this paper to study changes of different oxidative stress indexes,including ROS,MDA and 8-OHdG,after CSE and nicotine acting on orbital fibroblasts of TAO patients and normal persons,exploring whether CSE and nicotine play roles in TAO via oxidative stress and exploring specific dosage relations,and further discussing possible mechanism by detecting the impacts of CSE and nicotine on NF-?B pathway of orbital fibroblast.Method1.The tissue culture method was used for primary cultures of orbital fibroblasts derived from extraocular muscle from TAO patients and normal persons;then immunohistochemistry was adopted for identification.2.MTI was adopted to test activities of 24 h and 72 h cells after orbital fibroblasts being acted by CSE and nicotine.3.The changes of 8-OHdG after CSE and nicotine acting on orbital fibroblasts for 24 h and 72 h were examined by ELISA.4.The 24 h and 72 h changes of MDA after CSE and nicotine acting on orbital fibroblasts were evaluated with spectrophotometer assay.5.After CSE and nicotine acting on orbital fibroblasts for 24 h,the changes of ROS were tested with ELAISA were examined by ELISA.6.RT-PCR was adopted to test expressions of NF-?B-related genes after CSE and nicotine acting on orbital fibroblasts for 24 h.Results1.The tissue culture method was used to successfully achieve primary cultures of fibroblasts derived from extraocular muscle with passage stability.The cultures were identified as orbital fibroblasts.Cell lines were successfully established to be used as cell models for TAO and served following trials.2.It was indicated by MTT results that,for 24 h stimulations,cell activities of TAO orbital fibroblasts were decreased more sharply under CSE stimulation than normal orbital fibroblasts,and nicotine of low-concentration and high-concentration showed similar killing efficiency under 72 h stimulations,suggesting that the damage effects of smoking accumulated with time.3.For 24 h stimulation,nicotine of 10?g/ml significantly elevated 8-OHdG levels in two cell types;for 72 h stimulation,single stimulation with CES and nicotine of low-concentration could increase 8-OHdG,suggesting that nicotine in high dosage facilitated sudden increase of 8-OHdG content while nicotine in low dosage lead to slow accumulation of 8-OHdG.4.The impacts of CSE and nicotine on orbital fibroblasts highly accorded with MTT results;after administrations of both CSE and nicotine of 10?g/ml,the MDA contents were significantly higher than the group with single CSE stimulation and the negative control group.After stimulation for 72 h,observations on intracellular MDA content changes showed that MDA expressions were largely elevated for all stimulation conditions than the negative control group.5.Smoking played a role by increasing the content of ROS,while nicotine in high concentration had most prominent effects.6.In orbital fibroblasts of TAO patients,the level of NF-?B RNA was significantly up-regulated after stimulation with CSE,suggesting that smoking played its role by up-regulating the expression of NF-?B gene.ConclusionSmoking causes elevated oxidative stress indicators,MDA,8-OHdG and ROS,of orbital fibroblasts,and nicotine,as the major toxic ingredient in cigarette,plays its role.Even nicotine in low concentration exerts high effects by accumulation with time.It is suggested that smoking participates in the pathological process of TAO by causing oxidative stress.The possible molecular pathway might be NF-?B,as expressions of NF-?B genes are up-regulated by smoking.