Study On The Role Of Toll Like Receptor 9 And Its Signaling Pathway In The Pathogenesis Of Thyroid Associated Ophthalmopathy

Purpose.Thyroid associated ophthalmopathy(TAO)is the most common type of orbital disease in daily life.Current research consider that it is an autoimmune disease that is associated with thyroid dysfunction and often associated with Graves' disease(GD).Foreign studies show that the incidence of Graves disease per million population is 210/year,the male to female ratio is 1:3.9,the peak age is 40-60 years,and the incidence of TAO is 42.2/million/year,which is about Graves hyperthyroidism is 20.1%.At present,the exact pathogenesis of TAO is still not yet clear.Currently,there are many existing TAO treatment measures for symptomatic treatment,which can only control the condition but still lack an effective targeted drug.Meanwhile,because the premise of TAO treatment is the correct clinical stage,the current clinical CAS score system is subjective,especially for ophthalmologists and general practitioners who are not specialized in TAO diagnosis and treatment.Foreign studies have found that Toll-like receptor 9(TLR9)activation can promote plasmacytoid DC(pDC)secretion of IFN-?,and SLE is closely related to the disease activity and prognosis.Our previous study found that the expression of TLR9 in peripheral mononuclear cells in active TAO patients is significantly higher than that in the normal population.Therefore,we speculate whether TLR9 is also involved in the pathogenesis of TAO.To test this hypothesis,we have verified whether TLR9 is highly expressed by orbital fibroblasts(OFs)in patients with active TAO and compare it with stable TAO.The difference in TLR9 expression is found between the two.To further explore the relationship between TLR9 and OFs proliferation and release of inflammatory factors,to understand the role of TLR9 in the pathogenesis of TAO,and provide basis and direction for the search for TAO therapeutic targets.Methods.In this study,OFs is isolated and purified from the orbital adipose connective tissue of active TAO patients,inactive TAO patients,and normal persons performing orbital decompression surgery to improve appearance,who is excluded from immunological disease.Quantitative Real-time PCR(qRT-PCR)is used to analyze the difference of TLR9 mRNA expression in each group.TLR9-specific ligands,artificially synthesized olitaodeoxynucleotides containing CpG motifs(CpG-ODN),is used to stimulate groups of cells.qRT-PCR is used to detect IL-6,TNF-?,ICAM-1,IFN-? mRNA expression levels of the trend of changesat 0,6,12 and 24 hours after stimulation.Then wetake CCK8 method to determine thechanges of cells' OD value in each group,after stimulatedby different concentrations with different time,to calculate cell viability and find the relationship between TLR9 and OFS proliferation.Finally,the expression level of TLR9 mRNA is down-regulated by specific small interfering RNA(siRNA).qRT-PCR is used to detect the expression of TLR9,IL-6,IFN-?and ICAM-1 mRNA in the active group,with the purpose ofverifying wether TLR9 is in correlation with the secretionofinflammatory factors IL-6,ICAM-1,IFN-?.Results.1.The groups of OFs are successfully obtained by the tissue culture,the morphological and immunohistochemical identification is consistent with the characteristics of fibroblasts.2.The expression level of TLR9 mRNA in the OFs of active TAO group is significantly higher than that in the normal control group,while it was down-regulated in the inactive TAO group.3.After stimulated by CpG-ODN,the cell viability of OFs in active TAO group is increased significantly,but there is no significant change in inactive TAO group and normal control group.This effect appeared after 48 h of CpG-ODN stimulation,and there was dose dependence,which was most obvious at 200 nM.4.After stimulated by CpG-ODN,the expression levels of inflammatory cytokines(IL-6,TNF-?,ICAM-1,IFN-?)mRNA in OFs of active TAO group were increased to varying degrees,while the levels in OFs of inactive TAO group wereincreased slightly or even been down-regulated.5.After down-regulating the expression of TLR9 mRNA inOFs of active TAO group by siRNA,the expression levels of related inflammatory factors(IL-6,ICAM-1,IFN-?)mRNAalso show a downward trend,showing a positive correlation.Conclusions.1.Tissue block culture method to obtain OFs is reliable and effective.2.TLR9 is highly expressed in OFs from patients with active TAO,while it's relatively low in inactive group,suggesting that TLR9 may be involved in the TAO active inflammatory response.3.After activation of TLR9 with specific ligands,the cell viability of OFsis significantly increased,suggesting that activation of TLR9 may be involved in the pathological process of extraocular muscle hyperplasia and carbumal adipose tissue hyperactivity in pations with TAO.4.After activation of TLR9,active cytokines,such as IL-6,TNF-?,ICAM-1,IFN-?,can be induced to synthesize and secrete in OFs,indicating that TLR9 may be involved in the process of active ocular inflammatory reaction in TAO pations.