Research In Activation Of Wnt Signaling Pathway In Autoimmune Uveitis

Objective Autoimmune uveitis is one of the leading causes of blindness.It mainly affect adult working people,leading to heavy social and financial burden.The pathogenesis of uveitis is not fully clear.Experimental autoimmune uveoretinitis(EAU)is a useful animal model for studying the pathogenesis of human autoimmune uveitis which is believed to have simillar pathological features.Wnt/β-catenin signaling pathway plays a key role in heredity,development,proliferation and differentiation and also participates in the process of inflammation.The pathogenesis of a variety of autoimmune diseases involves the abnormal activation or inhibition of Wnt/β-catenin signaling pathway.However,the change of Wnt/β-catenin signaling pathway in autoimmune uveitis has been rarely reported.We investigated the state of Wnt/β-catenin pathway in the EAU model in this study,thereby to provide new ideas for the pathogenesis of autoimmune uveitis and new targets for the treatment of uveitis.Methods 1.Induction of EAU model: The interphotoreceptor retinoid-binding protein 651-670(IRBP 651-670)was emulsified with Complete Freund’s adjuvant(CFA),containing Mycobacterium tuberculosis H37 RA.The emulsion was injected subcutaneously into both sides of the back of 8-10 weeks old female C57BL/6 mouse.The diphtherin was injected intraperitoneally into the mouse.2.Clinical observation: Clinical signs of inflammation were examined by indirect ophthalmoscope from the day 6 after immunization and scored according to Caspi criteria every other day.Meanwhile we used optical coherence tomography(OCT)to monitor the retinal changes.3.The separation of the retina: After anesthesia,mouse eyeballs were removed by cutting off optic nerve with curved tweezer.Scleral shell was opened cutting along limbal conjunctival.Remove cornea,iris,lens and vitreous then separate retina gently.4.Real time PCR(RT-PCR)were used to detect the transcriptional levels of Wnt related cytokines(TCF,LEF,AXIN2,MYC and Cyclin D1)in the retina.5.Western blot were used to detect the translational levels of β-catenin and LRP6 in the retina.6.Enzyme linked immunosorbent assay(ELISA)were used to detect some Wnt pathway inhibitors(DKK1,PDEF and Kallistatin)in the plasma.Results 1.Mouse models of EAU were successfully established and the clinical signs of inflammation were scored according to Caspi criteria.2.RT-PCR showed that the transcript levels of TCF,AXIN2 and MYC were significantly elevated in the retina of EAU mouse compared with WT mouse.3.Western blot demonstrated the protein expression levels of β-catenin and LRP6 were higher in the retina of EAU mouse than WT mouse.4.ELISA manifested the expression levels of DKK1 and PDEF were significantly higher in the plasma of EAU mouse than WT mouse.Conclusion 1.The pathogenesis of autoimmune uveitis involves the abnormal activation of Wnt/β-catenin signaling pathway.2.Wnt/β-catenin signal pathway may become a novel therapeutic target for autoimmune uveitis.