ABSTRACT
Objective To explore the anti-oxidation stress effect of hydrogen-rich water (HW) on thyroid associated ophthalmopathy (TAO). Methods The orbital fibroblasts were derived from orbital adipose connective tissue of TAO patients and cultured in vitro. The cells were treated with different concentrations of hydrogen peroxide (H2O2) for 18 h, and the proliferation ability was detected by CCK-8 method to determine the appropriate H2O2 concentration. The cells were divided into four groups: blank group (normal culture), H2O2 group (treating cells with H2O2 for 18 h), HW+H2O2 group (culturing cells using culture media containing HW for 72 h in combination with H2O2 for 18 h), dexamethasone + H2O2 group (treating cells using dexamethasone 1 μmol/L for 72 h in combination with H2O2 for 18 h). After culturing for 72 h in each group, the fluorescence intensity of reactive oxygen species (ROS) was measured by flow cytometry, and the contents of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected by ELISA. Results We successfully cultured orbital fibroblasts derived from orbital adipose connective tissues of TAO patients. The higher concentration of H2O2, the greater inhibition effect on the proliferation ability of the orbital fibroblasts from TAO patients, and we finally chose 100 μmol/L H2O2. After 72 h of cell culture, the contents of MDA, SOD, and GSHPx and the fluorescence intensity of ROS were (1.63±0.29), (5.06±0.24), (3.94±0.29), and (2.34±0.24) nmol/mL, (10.51±0.32), (2.41±0.23), (5.58±0.29), and (7.98±0.15) U/mL, (107.79±1.06), (21.07±0.92), (49.19±6.75), and (76.33±4.70) U/mL and 18 275.82±521.05, 92 524.81±2 097.01, 54 460.87±572.64, and 35 961.37±540.61 in the blank, H2O2, HW+H2O2 and dexamethasone+H2O2 groups, respectively. Statistic analysis results showed that both HW and dexamethasone could significantly inhibit oxidative stress induced by H2O2 in orbital fibroblasts (all P<0.01), and the inhibitory effects of dexamethasone were significantly more obvious than those of HW (all P<0.01). Conclusio HW may be a treatment option for TAO through anti-oxidant stress.
ABSTRACT
Background The pathogenic mechanism of thyroid associated ophthalmopathy (TAO) is still unclear,which is considered to be an autoimmune disease.It is confirmed that interleukin-17A (IL-17A) plays an important role in the occurrence and development of many autoimmune diseases.It is unclear that whether IL-17A participates in the pathogenesis of TAO.Objective This study was to explore whether IL-17A secreted by coculture system of peripheral blood mononuclear cells (PBMCs) and orbital fibroblasts (OFs) participates in the pathogenesis of TAO and its possible mechanism.Methods Periphery blood and orbital connective tissue were obtained from 12 patients with TAO and 8 patients who received prosthesis implantation for eyeball atrophy in Xiangya Hospital during April to December 2014.PBMCs were isolated by density gradient centrifugation,and OFs were cultured by explant culture method.The purity of T leukomonocyte in PBMCs was tested by flow cytometry,and OFs were identified by Giemsa staining and immunochemistry.OFs and PMBCs were incubated into 96-well plate in a 1:20 proportion to establish co-culture system.Different concentrations of phytagglutinin (PHA) (0,1.0,2.5,5.0,10.0 μg/ml) was added for 72 hours,and IL-6,IL-17A levels in the co-culture system supernatant and IL-17A receptor (IL-17RA) of the total cell membranes in the co-culture system were assayed by ELISA.The differences of IL-6,IL-17A,IL-17RA levels in co-culture system were compared between the TAO group and control group.Results The mean purity of T leukomonocyte in PBMCs was (81.10±0.21)％ in the TAO group and (80.05 ±0.38)％ in the control group respectively,with no significant difference between them(t =0.923,P＞0.05).Cultured OFs showed the positive response for Vimentin expression and Giemsa staining.After stimulated by 1.0 μg/ml PHA,the proliferation of both PBMCs and OFs were increased in the co-culture system.Apoptosis exist in PBMCs and the number of OFs decreased when PHA was higher than 1.0 μg/ml.The growth of PBMCs and OFs was faster in the TAO group than that in the control group in the same concentration of PHA.The contents of IL-6,IL-17A and IL-17RA in co-culture system were significantly different among various concentrations of PHA subgroups (IL-6:Fgroup =12.561,P=0.000;F ion =23.356,P =0.001.IL-17A:Fgroup =12.037,P =0.000;Fconcentration =19.206,P=0.000.IL-17RA:Fgroup =16.216,P=0.000;Fconcentraction =4.627,P=0.018).The production of IL-6,IL-17A and IL-17RA reached peak in both TAO group and the control group after 1.0 μg/ml PHA stimulated.However,the concentrations of IL-6,IL-17A and IL-17RA reduced with the increase of PHA concentration.The concentrations of IL-6,IL-17A and IL-17RA in co-culture system were significantly higher in the TAO group than those in the control group under the stimulation of the same concentration of PHA (all at P＜0.05).Conclusions The co-culture system of PBMCs and OFs stimulated with PHA can be the imitation of TAO pathogenesis in vitro,and PHA can amplify its immune reaction to imitate TAO pathogenic processes intuitively.The IL-6,IL-17A and IL-17RA secreted by PBMCs and induced by PHA are increased in TAO patients,implying that IL-17A participates in the pathogenesis of TAO through magnifying cellular immune response and inflammatory reaction.
ABSTRACT
Objective To investigate the effects of soluble CD40L (sCD40L) on proliferation of orbital fibroblasts (OFs) and the expression of three types of hyaluronan synthase (HAS) in vitro, so as to explore the role of sCD40L in the pathogenesis of thyroid-associated ophthalmopathy (TAO). Methods OFs obtained from 5 patients with TAO and 3 normal controls were primarily culutred. The effect of different concentrations of sCD40L (6.25,12.5,25,50,100 and 200 ng/mL) on proliferation of OFs of different sources were examined by MTS after 48 h exposure. OFs were also cultured with different concentrations of sCD40L (12.5, 25, 50 and 100 ng/mL) for 3,6,12 and 24 h, and then the expression levels of HAS 1-3 mRNA were determined by real-time RT-PCR. Results Treatment with sCD40L at concentrations higher than 25 ng/mL for 48 h obviously promoted the proliferation of OFs in patients with TAO (P<0.05). In contrast, treatment with sCD40L only at concentrations higher than 50 ng/mL for 48 h could promote proliferation of OFs from normal control, and the effect was comparatively weak. HAS3 mRNA expression of OFs in TAO patients was increased after exposed to sCD40L (P<0.05), and the increase was in a concentration- and time-dependent manner. Conclusion sCD40L can promote the proliferation of OFs and expression of HAS3 mRNA in patients with TAO, which implies that sCD40L plays an important role in the pathogenesis of TAO.
ABSTRACT
The aim of the present study was to identify a new candidate anti-inflammatory compound for use in the active stage of thyroid-associated ophthalmopathy (TAO). Benzylideneacetophenone compound JC3 [(2E)-3-(4-hydroxy-3-methoxyphenyl)phenylpro-2-en-l-one] was synthesized based on a structural modification of yakuchinone B, a constituent of the seeds of Alpinia oxyphylla, which belongs to the ginger family (Zingiberaceae), has been widely used in folk medicine as an anti-inflammatory phytochemical. Orbital fibroblasts were primarily cultured from patients with TAO, and the potential of JC3 to suppress the interferon (IFN)-gamma-induced protein (IP)-10/CXCL10 production in these cells was determined. IFN-gamma strongly increased the level of IP-10/CXCL10 in orbital fibroblasts from patients with TAO. JC3 exerted a significant inhibitory effect on the IFN-gamma-induced increase in IP-10/CXCL10 in a dose-dependent manner; its potency was greater than that of an identical concentration of yakuchinone B with no toxicity to cells at the concentration range used. Moreover, the constructed dimer and trimer polystructures of JC3, showed greater potency than JC3 in suppressing the IFN-gamma-induced production of IP-10/CXCL10. JC3 significantly attenuated the IP-10/CXCL10 mRNA expression induced by IFN-gamma, and a gel-shift assay showed that JC3 suppressed IFN-gamma-induced DNA binding of signal transducer and activator of transcription-1 (STAT-1) in TAO orbital fibroblasts. Our results provide initial evidence that the JC3 compound reduces the levels of IP-10/CXCL10 protein and mRNA induced by IFN-gamma in orbital fibroblasts of TAO patients. Therefore, JC3 might be considered as a future candidate for therapeutic application in TAO that exerts its effects by modulating the pathogenic mechanisms in orbital fibroblasts.