Effect Of Hydrogen Water On The Orbital Fibroblasts Of Patients With Thyroid Associated Ophthalmopathy In Vitro

PurposeThyroid-associated ophthalmopathy?TAO?is an autoimmune disease,but the pathogenesis is not yet completely understood.The current clinical and basic studies have found that oxidative stress play a role in the pathogenesis of TAO,and experimental anti-oxidative treatment has also achieved ideal therapeutic effect on mild and moderate TAO.Hydrogen water as an antioxidant has been widely studied for its almost non-toxic side effects.A large number of medical and biological research have shown that hydrogen water can effectively treat various organ damage caused by oxidative stress through anti-oxidation,anti-apoptotic,anti-inflammatory effect.The primary cultured orbital fibroblasts derived from orbital adipose connective tissue is adopted in this paper to study the effect of hydrogen rich water on the orbital fibroblasts of normal people and the TAO patients in vitro.In this experiment,hydrogen water was compared with dexamethasone.After oxidative damage of the orbital fibroblasts with hydrogen peroxide?H₂O₂?,we detected various indicators of oxidative stress including reactive oxygen species?ROS?,malondialdehyde?MDA?,superoxide dismutase?SOD?and glutathione peroxidase?GSH-PX?.Furthermore,we detected the apoptosis of orbital fibroblasts,the proliferative ability and the content of hyaluronic acid?HA?in the cells.Methods1.Primary culture of orbital adipose connective tissue in TAO patients and normal people was used to obtain stable passage of orbital fibroblasts.Then morphological and immunohistochemical methods were used to identify whether the cells were orbital fibroblasts.2.We selected different concentrations of H₂O₂ to treat orbital fibroblasts in normal people and TAO patients.CCK-8 assay was used to detect the proliferation ability of cells after 18h,and the appropriate H₂O₂ concentration was selected for subsequent experiments.3.Orbital fibroblasts from normal subjects and TAO patients were divided into several groups:blank group,oxidative injury model group,hydrogen water+model group,dexamethasone+model group.The proliferation ability of 0,24h,48h and 72h of orbital fibroblasts in each group was detected by CCK-8.4.The early apoptosis rate,the late apoptosis rate and the necrosis rate of orbital fibroblasts in each groups were detected by flow cytometry at 72 hours.5.The content of MDA and GSH-PX of orbital fibroblasts in each groups were measured by spectrophotometer at 72 hours.6.The content of SOD of orbital fibroblasts in each groups were measured by enzyme labeling method at 72 hours.7.The content of ROS of orbital fibroblasts in each groups were measured by flow cytometry at 72 hours.8.The orbital fibroblasts of TAO patients were divided into three groups:blank group,hydrogen water group and dexamethasone group.The content of HA of orbital fibroblasts in each group were detected by ELISA at 72 hours.Results1.In this study,We successfully cultured stable passaged orbital fibroblasts derived from orbital adipose connective tissue and identified it as orbital fibroblasts through morphological and immunohistochemical method and established a cell system for subsequent experiments successfully.2.Different concentrations of H₂O₂ acted on the orbital fibroblasts 18 hours.According to their effect on cell proliferation,we finally chose 100?mol/L H2O2 as the follow-up drug.3.The cell proliferation ability of the hydrogen water+model group and the dexamethasone+model group in 24h,48h and 72h was significantly higher than that of the oxidative damage model group.4.Compared with the oxidative damage group,the hydrogen water and dexamethasone could significantly reduce the late apoptosis rate and the necrosis rate after the action of 72h.5.Hydrogen water and dexamethasone can significantly reduce the MDA content of the oxidative damaged cells and increase the GSH-PX content of Oxidative damaged cells after the action of 72h.6.Hydrogen water and dexamethasone could significantly increase the SOD content of the oxidative damaged cells after the action of 72h.7.Hydrogen water and dexamethasone can significantly reduce the ROS content of the oxidative damaged cells after the action of 72h.8.The content of HA in orbital fibroblasts of TAO patients was significantly higher than that of normal persons.Hydrogen water and dexamethasone can significantly reduce the HA content of orbital fibroblasts in TAO patients after the action of 72h.ConclusionsThe proliferation ability of orbital fibroblasts in TAO patients is higher than that of normal people,and hydrogen water and dexamethasone all increase the proliferation ability of orbital fibroblasts after oxidative damage from H₂O₂.There is no significant difference in the rate of apoptosis and necrosis of the orbital fibroblasts of TAO patients and normal people,but after adding hydrogen water and dexamethasone to the orbital fibroblasts after oxidative injury,the late apoptosis rate and necrosis rate of TAO patients'cells were significantly higher than those of normal persons.Both hydrogen water and dexamethasone can reduce the content of MDA and ROS and increase the content of SOD and GSH-PX in cells after oxidative damage to inhibit oxidative stress.Finally,the content of HA in OFs of TAO patients could be reduced both by hydrogen rich water and dexamethasone.