Establishment Of Animal Models Of AITD And The Study Of Anti-inflammatory Medication For AITD

Objective To establish animal models of Graves’ disease (GD) and thyroid associated orphthamopathy (TAO), and to explore the immunosuppressive effect of luteolin on murine experimental autoimmune thyroiditis (EAT) through inhibiting IL-6/STAT1 and STAT3 signaling pathway.Methods Part1:20 8-week-old BALB/c mice were divided into 3 groups:T289 (n=10), pcDNA3.1 (n=5) and Control (n=5). Mice in T289 and pcDNA3.1 groups were given genetic immunization using recombinant plasmid pcDNA3.1-TSHR289 overexpressing TSHR A subunit, immediately after which all mice were given in vivo electroporation. Injection and electroporation were performed every 3 weeks for 4 times. One week after the last immunization, mice in all groups were subjected to in vivo magnetic nuclear resonance (MRI) scans of the eyes and frontal region of the brain on a 7.0T MRI scanner. Two weeks after the last immunization, all mice were sacrificed, after which HE staining, immunohistochemistry (IHC), and Alcian blue staining of thyroid glands and orbital tissues were performed. Serum T4 and TSHR antibodies were examined using ELISA. Part 2:30 8-week-old C57BL/6 mice were dividied into 4 groups:TG (n=10), Luteolin (n=10), DEX (n=5) and Control (n=5). For the induction of autoimmune thyroiditis,100μg of porcine thyroglobulin (pTG) was emulsified 1:1 in 100μl complete Freund’s adjuvant (CFA). All mice except control ones were injected subcutaneous at the base of tails with the emulsion on Day 0. The second subcutaneous injection was given on Day 14 using the same amount of pTg in incomplete Freund’s adjuvant (IFA). After the second immunization, Luteolin and DEX mice were given daily intraperitoneal injection of luteolin (10mg/kg/day) and dexamethasone (5mg/kg/day), respectively, while control mice were given PBS instead. All mice were sacrificed on Day 21. Serum T4 and TG antibodies were examined using ELISA. HE staining and immunohistochemistry of thyroid glands were performed. Meanwhile, cultured Raw264.7 cells were stimulated by human IFN-γ (10ng/ml) overnight and then luteolin(20μmol/l) for 6h. After that, western blot was performed in order to explore the effect of luteolin on the expression of an inflammatory marker COX2, transcription factors STAT1 and STAT3 which are the downstream of IL-6 signaling pathway.Results Part 1:We have successfully established an animal model of GD and TAO. The immune mice demonstrated goiter (about 90%), increased serum T4 and TRAb levels (about 85.7%), typical features of hyperthyroid glands in pathology. Moreover, widening of palpebral fissure (about 30%), orbital muscle swelling, pathologically lymphocytic infiltration in orbital tissues (about 50%) and hyaluronic acid distributed between orbital muscle fibers were observed (about 30%) in the immune animals compared with the age-matched controls. Part 2:The classic experimental autoimmune thyroiditis (EAT) model was established successfully. About 40% of TG mice demonstrated goiter compared to controls. HE examination of thyroid glands of TG mice showed significant infiltration of monocytes between follicles compared with controls, while in thyroid glands of luteolin mice almost no momocytic infiltration was observed. Moreover, IHC results of thyroids demonstrated significant higher expression of p-STAT3 (Y705) and p-STAT1 (Y701) in TG mice, while much lower expression in luteolin-treated mice. Besides, western blot of Raw264.7 cells showed luteolin significantly inhibit the increased expression of COX2, p-STAT1 (Y701) and p-STAT3 (Y705) induced by IFN-y treatment, while total STAT1 and total STAT3 remain unchanged, demonstrating the immunosuppressive effect of luteolin by inhibiting IL-6/STAT1 and STAT3 signaling pathway in vitro.Conclusion In the present study, we have successfully established an animal model of GD and TAO, providing a very useful tool for future study of pathogenesis and novel therapies for GD and TAO. The treatment of luteolin has showed significant immunosurpressive effect and reduced the incidence of autoimmune thyroiditis in mice, laying the foundation of developing luteolin as an early intervention for autoimmune thyroiditis. However, more mechanisms of this drug remained to be discussed in the future.