Study On The Roles And Mechanism Of IL-34 In The Pathogenesis Of Hashimoto's Thyroiditis

Objective:Hashimoto's Thyroiditis?HT?is an organ-specific autoimmune disease accompanied by a large number of lymphocytes infiltrate in the thyroid tissue,which is One of the most important causes of hypothyroidism.At present,the related research on HT is increasing and the influence of inflammatory factors on HT plays an important role.IL-34 has been discovered as a ligand for CSF-1R,and Current research indicates that IL-34 is involved in neuronal protection,bone degenerative diseases,delayed hypersensitivity,infection,tumor and organ transplantation.In addition,IL-34 has effects on the development of inflammatory diseases such as inflammatory bowel disease?IBD?and rheumatoid arthritis?RA?.Our previous studies suggest that IL-34 mRNA levels in thyroid tissue of patients with Hashimoto's thyroiditis are significantly lower than those of non-Hashimoto thyroiditis,suggesting that IL-34 may be involved in the development of HT.This study aims to explore the mechanism of IL-34 in HT.Methods:Thyroid tissues were obtained from 24 patients with HT who were undergoing thyroidectomies for treatment.Non-HT thyroid tissues collected from 23 paracancerous tissues from patients without HT as controls.Serum was obtained from 35 patients with HT and 21 healthy controls.Immunohistochemical method to observe the expression of IL-34 in thyroid tissue.The expression of IL-34 mRNA in thyroid tissue was detected by real-time quantitative PCR?RT-PCR?.The expression of IL-34 protein in thyroid tissue was detected by Western Bolt.The level of IL-34 in serum was detected by enzyme-linked immunosorbent assay?ELISA?.Apoptotic thyrocytes were detected by flow cytometry and observation of the change of thyrocyte apoptosis rate after stimulated by IL-34.The expression of Bcl-2/Bax in thyrocytes stimulated by IL34 was detected by RT-PCR and Western Blot.Inhibition of CSF-1R by IL-34 stimulation,observation of changes in STAT3 phosphorylation levels in primary thyroid cells,in addition,inhibition of STAT3 in thyrocytes,to observe the effect of IL-34 on the expression of Bcl-2/Bax.Nthy-ori 3-1 were treated with Interferon-??IFN-??and the expression of IL-34 in Nthy-ori 3-1 cells was observed by RT-PCR and Western Blot.NOD.H-2^h4 mice at 4 weeks of age with a mean weight of 20 g were randomlydivided into the control and spontaneous autoimmune thyroiditis?SAT?groups.Mice in the control group?16 mice,8 per time-point?were given sterile water.SAT group?16mice,8 per time-point?were given 0.05%mg/L NaI in their drinking water for 8 weeks or 16 weeks.Each group of mice was killed at 8 and 16 weeks after grouping.All of the animal study procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were performed according to the institutional ethical guidelines.Thyroid tissues were used for HE staining?32 mice,8 per group?,RT-PCR?32mice,8 mice per group?and Western blotting?32mice,8 per group?.Results:1.IL-34 is expressed in human thyroid follicular epithelial cells.We performed IHC analysis and measured the expression of IL-34 in human thyroid tissue.We detected IL-34 expression in follicular epithelial cells in patients without HT.In contrast,IL-34expressed in thyroid follicular epithelial cells and infiltrating lymphocytes in the thyroid tissue of patients with HT.2.IL-34 protein expression in the thyroid tissue of the HT group was significantly lower than that of the non-HT group,Similarly,IL-34 mRNA expression in the thyroid tissue of the HT group was significantly lower than that of the non-HT group and a negative correlation was observed between the mRNA expression of IL-34 and both TPOAb?r=-0.368,p=0.015?and TgAb?r=-0.356,p=0.019?.3.Serum IL-34 levels in the HT group were significantly lower than those in the healthy control group?p<0.05?,and a significant negative correlation was observed between IL-34 and TgAb?r=-0.502,p=0.021?.4.Thyroid follicles in the control group had a uniform morphology and moderate size.In the SAT groups,thyroid follicles were significantly enlarged,exhibited different morphologies,and contained a large amount of lymphocyte infiltration.5.IL-34 expression was significantly lower in the SAT groups than in the control groups,but there was no difference between the groups treated with NaI water for16 weeks and 8 weeks by western blot and RT-PCR.6.IL-34 expression decreased after Nthy-ori 3-1 cells were treated with IFN-?,and IL-34 expression was significantly lower in the IFN-?-treated group than in the untreated group?p<0.05?.7.flow cytometric analysis demonstrated that treatment with IL-34 significantly decreased thyrocyte apoptosis compared with control?P<0.05?.Following IL-34 treatment,the protein expression of Bcl-2 increased,and the expression of Bax was decreased,resulting in an increased Bcl-2/Bax ratio?P<0.05?.A similar tendency was discovered in the mRNA expression of the Bcl-2/Bax ratio?P<0.05?.8.As a result of IL-34 treatment,increased p-STAT3 expression was evidently increased in a dose-dependent manner upon exposure IL-34?P<0.05?.Decreased protein expression levels,including p-STAT3/STAT3and Bcl-2/Bax,were observed following treatment with CSF1R inhibitor?P<0.05?and decreased protein and mRNA expression levels of Bcl-2/Bax,were observed following treatment with STAT3 inhibitor?P<0.05?.Conclusion:1.our study confirmed the presence of IL-34 in thyroid tissue.In patients with HT,IL-34 expression was significantly reduced in thyroid tissue,and IL-34 mRNA expression in thyroid tissue was negatively correlated with TgAb and TPOAb.Serum IL-34 levels in patients with HT were also significantly reduced,and serum IL-34 levels were negatively correlated with TgAb.IL-34 can reduce the apoptosis of thyrocytes in HT.In addition,high-dose INF-?stimulation of Nthy-ori 3-1 cells can cause a significant reduction in IL-34 expression.Decreased expression of IL-34 in thyroid tissue also occurred in SAT mice.These results suggest that IL-34may be a potential indicator for evaluating thyrocyte damage.2.IL-34 could promote apoptosis resistance in thyrocytes in HT and may be achieved by CSF-1R/STAT3/p-STAT3 signaling.