| Background: Autoimmune thyroiditis(Autoimmune thyroiditis,AIT)is an autoimmune disease caused by differences in family inheritance,living environment and individual immunity.The main pathological change is that the thyroid tissue is flooded with a large number of lymphocytes,and specific antibodies of thyroid autoantigen can be detected in patients’ blood[1].Macrophages are one of the constituent cells of the innate immune system.When the polarization of macrophages in the body is out of balance,the homeostasis of the internal environment will change [2],which may trigger a series of diseases.Notch signaling pathway is one of the important classic signaling pathways for macrophage activation [3].At present,there is no relevant report on the polarization effect of Notch signaling pathway on AIT macrophages at home and abroad.In this study,we used iodine-induced NOD.H-2h4 mouse AIT model,made Notch signal blocked and overexpressed in vitro,to explore whether there is macrophage polarization change,whether Notch signaling pathway is overexpressed in autoimmune thyroiditis and the correlation between Notch signaling and macrophage polarization in AIT mice.Methods: Animal experiment:4-week-old female NOD.H-2h4 mice were randomly divided into two groups(Control group and SAT group).The control group was fed with sterile deionized water,and SAT group was given 0.05% sodium iodide water.Mice were killed at 2nd,4th,8th,12 th and 16 th week respectively.The thyroid,serum,spleen and abdominal lavage fluid were taken.The weight of the thyroid was recorded,the serum Tg Ab titer was measured by ELISA,the degree of lymphatic infiltration of thyroid cells was observed by HE staining,and the distribution of macrophages in the abdominal cavity and spleen was detected by flow cytometry.Vitro experiment:1.Bone marrow of mice in the SAT group and the control group was taken and cultured with colony stimulating factor.The expression level of Notch signaling pathway m RNA was detected by RT-PCR,and the expression level of Notch signaling pathway protein was detected by Western blot.2.Blockage and activation experiment of Notch signal:4-week-old female NOD.H-2h4 mice were randomly divided into 2 groups.The control group was fed with sterile deionized water,and the SAT group was fed with 0.05% sodium iodide water for 16 weeks.At 16 th week,the bone marrow was taken and implanted in a 6-well plate.After induction and culture with colony-stimulating factor for7 days,different treatment factors were added to the culture medium to induce polarized macrophages.They were divided into control group,M1 polarization group(1,2,3),and M2 polarization group(4,5,6).After 24 hours of stimulation,the distribution of macrophage populations was detected by flow cytometry,and the protein expression level was detected by Western blot.Control group: +DMSO;M1 polarization group 1:LPS+IFN-γ;M1 polarization group 2:LPS+IFN-γ+Jagged1(Notch ligand);M1 polarization group 3:LPS+IFN-γ+DAPT(Notch blocker);M2 polarization group 4:IL-4;M2 polarization group 5:IL-4+Jagged1(Notch ligand);M2 polarization group 6:IL-4+DAPT(Notch blocker)Results: The thyroid weight,serum TgAb level and the infiltration degree of thyroid lymphocytes of NOD.H-2h4 mice in SAT group were significantly higher than those in control group at 8th,12 th and 16 th week after high iodine water feeding,and there was statistically significant difference.There was no statistically significant difference between groups of mice after 2 and 4 weeks of iodine intervention.Compared Control group with SAT group at 8th,12 th and16th week,M1 macrophages(F4 The ratio of/80+CD11c+)in the spleen and abdominal cavity increased significantly,and the ratio of M2 type macrophages(F4/80+CD206+)decreased significantly in NOD.H-2h4 mice’s F4/80+ macrophages.With the increase of iodine feeding time,this change trend became more prominent and had significant statistical significance.Compared with the control mice fed with sterile water in the same period of time,the proportion of M1 and M2 macrophages in mice induced by high iodine for 2 and 4 weeks did not increase or decrease significantly and had no statistical difference.At the 8th,12 th and 16 th week of high-iodine water feeding,the m RNA expression of Notch signal-related molecules Notch1,Notch3,Jagged1,Socs3,Delta1,Hey1,Hes1 in the bone marrow macrophages of mice in the SAT group was higher than those in the control group of the same age and there was significant statistical differences.However,there was no such change in the SAT group mice induced by iodine for 2 and 4 weeks,and there was no statistical difference.From 8th to 16 th week,the protein expressions of Notch signaling pathway key proteins Jagged1,Hey1 and Hes1 in bone marrow macrophages in the SAT group were significantly higher than those in the control group,and the difference is statistically significant.In vitro experiments of overexpression and blockage of Notch signal:no matter the SAT group or Control group,in the M1 polarization group,M1 macrophages and Jagged1,Hey1 and Hes1 protein’s expression were significantly higher in the LPS+IFN-γ+Jagged1 group than those in LPS+IFN-γ group,while the protein expression of Jagged1,Hey1,and Hes1 and the expression of M1 macrophages were significantly lower in the LPS+IFN-γ+DAPT(Notch blocker)group than those in the LPS+IFN-γ group.In the M2 polarization group,the expression of M2 macrophages in the IL-4+Jagged1 group was lower than that in the IL-4 group,and the expression of M2 macrophages in the IL-4+DAPT group was higher than that in the IL-4 group;the protein expression levels of Jagged1,Hey1 and Hes1 in the IL-4+Jagged1(Notch ligand)group were higher than those in the IL-4 group and IL-4+DAPT(Notch blocker)group.The expression of key proteins in the Notch signaling pathway in the M1/M2 polarization group of the SAT group was significantly higher than that of the Control group,and the difference was statistically significant.Conclusions:1.NOD.H-2h4 mice developed autoimmune thyroiditis after being fed with0.05% sodium iodide water for 8 weeks.2.The expression of M1 type macrophages in the abdominal cavity and spleen of autoimmune thyroiditis mice significantly increased,and the expression of M2 type macrophages significantly decreased.3.Among molecules related to Notch signal in bone marrow macrophages of AIT mice,the m RNA levels of Notch1,Notch3,Jagged1,Socs3,Delta1,Hey1,Hes1,and the protein levels of Jagged1,Hey1,and Hes1 were significantly higher than those of the control group of the same age,which suggested Notch signaling pathway is involved in the occurrence and development of autoimmune thyroiditis.4.After the Notch signaling pathway was activated,the expression of M1 macrophages increased,the protein expression of Jagged1,Hey1,and Hes1 increased,and the expression of M2 macrophages decreased in the mouse bone marrow.After the Notch signaling pathway was inhibited,the expression of M1 macrophages decreased,the protein expression of Jagged1,Hey1 and Hes1 decreased,and the expression of M2 macrophages increased,which suggested that the Notch signaling pathway regulates the polarization of macrophages and participates in autoimmune thyroiditis. |
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