Efficacy And Mechanism Of Exosome Inhibitors And Dihydroartemisinin In AIT Animal Models

Objective: Autoimmune thyroiditis(AIT)is a typical organ-specific autoimmune disease.It is caused by genetic and environmental factors that cause immune homeostasis.The production of specific antibodies against autoantigens is a pathological feature that causes disease occur.Multiple studies have confirmed the existence of T cell differentiation and functional imbalance in patients with AIT,which is characterized by the increase in Th1 and Th17 expression with regulatory T cell dysfunction.Exosomes are like a signal carrier "storage pool" in the body.They contain effective immune stimulating substances and have the function of immune stimulation and immune regulation.GW4869 is an exosomal inhibitor,which can inhibit the secretion of exosomes in vivo or in vitro.Dihydroartemisinin(DHA)is a derivative of natural artemisinin.In addition to its significant antimalarial effect,it also has anti-inflammatory and immune-regulating effects.It has been found to be involved in the regulation of T cell immunity in different types of diseases.The process can affect many key links such as T cell activation,proliferation,differentiation,apoptosis,and subpopulation function.In summary,we suppose that exosomes are involved in the pathogenesis of autoimmune thyroiditis.Therefore,we applied exosomal inhibitors in AIT animal models to prevent exosome secretion in vivo to confirm the role of exosomes in the pathogenesis of autoimmune thyroiditis.At the same time,dihydroartemisinin was used to observe whether it could reduce the secretion of exosomes and improve autoimmune thyroiditis.Methods: Part I: 4-week-old NOD.H-2h4 mice were randomly divided into 4 groups: CON: sterile deionized water feeding;HI: 0.05% sodium iodide water feeding;HI+GW4869: feeding with 0.05% sodium iodide water,and intraperitoneal injection of GW4869 2.5mg/kg each mouse,every other day;CON+GW4869: feeding with deionized water,meanwhile Intraperitoneal injection of GW4869 2.5mg/kg per mouse,every other day.The CON and HI group were intraperitoneally injected with a dose of normal saline containing DMSO once every other day.All mice were sacrificed at 12 weeks of age.Part II: 4-week-old NOD.H-2h4 mice were randomly divided into CON(sterile deionized water feeding)and HI(0.05% sodium iodide water feeding),at 8 weeks of age The HI group was randomly divided into 4 groups: the HI group(0.05% sodium iodide water),the DHA(H)group(0.05% sodium iodide water+DHA 50 mg/kg),and the DHA(M)group(0.05% iodine Sodium water+DHA 25mg/kg),DHA(L)group(0.05% sodium iodide water+DHA 12.5mg/kg),the DHA group was given the corresponding dose of DHA,and the HI group and the CON group were given equal doses containing DMSO saline was administered intragastrically once a day.All mice were sacrificed at 12 weeks of age.The serum was collected to measure the serum Tg Ab and the exosomes level.The thyroid was taken and weighed,the thyroid relative weight was calculated,and the inflammatory status of the mouse was evaluated.The spleen was opened under aseptic conditions and weighed.The spleen relative weight was calculated and the proportion of T lymphocyte subgroups was analyzed by flow cytometry.Total RNA was extracted from the spleen,and the expression of the corresponding cytokines Th1(T-bet,IFN-?),Th17(ROR-? t,IL-17),and Treg(Foxp3)m RNA were detected by real-time quantitative PCR.Results: 1.Intraperitoneal injection of GW4869: 1)HE staining of frozen sections of thyroid found that compared with the CON group and CON+GW4869 group,the thyroid inflammation score of mice in the HI group and the HI+GW4869 group was significantly increased(P <0.0001),There was no significant difference between the HI group and the HI+GW4869 group.2)The serum Tg Ab titers of the four groups of mice were detected by ELISA.Compared with the HI group,the serum Tg Ab levels of the CON group and the CON+GW4869 group were significantly reduced(P <0.05),but there was no significant difference between the HI group and the HI+GW4869 group;3)The ratio of T lymphocyte subsets in the spleen of the four groups was detected by flow cytometry,and the ratio of Th2 and Treg lymphocytes in the four groups were found There were no significant differences between them.Compared with the normal group,the ratio of Th1 and Th17 cells was increased in the HI group and the HI+GW4869 group(P <0.05),but there was no significant difference between the two groups.Compared with the CON + GW4869 group,the Th17 cell ratio increased in the HI group and the HI+GW4869 group(P <0.05).2.intragastrically of DHA: 1)Calculating the thyroid relative weight found that compared with the CON group,the thyroid relative weight of the HI group and the DHA(L)group increased significantly(P <0.001 and P <0.05);compared with the HI group,the thyroid relative weight of the DHA(H)group decreased(P <0.05);although there was no significant difference in thyroid relative weight between the DHA groups,there was an increasing trend with the decrease of the dose.Compared with the HI group,HE staining scores of thyroid frozen sections showed that the thyroid inflammation scores of mice in the CON group and the DHA groups were significantly decreased(P <0.01).Compared with the CON group,the thyroid inflammation score of the mice in the DHA(L)group increased(P <0.05);2)Tg Ab level in the serum of the five groups of mice were measured by ELISA.The serum Tg Ab level of the CON?DHA(H)and DHA(M)group was significantly reduced(P <0.05),and the Tg Ab level gradually increased with the decrease of the DHA dose.3)The spleen relative weight of the CON?DHA(H)and DHA(M)group were significantly decreased,and the spleen relative weight decreased with the increase of the DHA dose;4)Flow cytometry was used to detect the ratio of T lymphocytes in the spleen of the five groups of mice,although no significant difference was found,but compared with the HI group,the ratio of Th1 and Th17 cells decreased in the CON and the DHA groups,and the Treg cells showed an increasing trend.As the DHA dose increased,the ratio of Th1 cells gradually decreased.Conclusion: 1.GW4869 did not reduce the serum Tg Ab level of AIT mice and improve thyroid inflammation,and there was no significant difference in the ratio of T lymphocyte subgroups.2.The serum Tg Ab level of mice treated with DHA decreased,thyroid lymphocyte infiltration improved,and thyroid and spleen relative weight decreased in a dose-dependent manner.These results indicate that treatment with DHA improves AIT by suppressing immunity and lymphocyte infiltration Condition of mice.At the same time,DHA also reduced the ratio of Th1 and Th17 cells in AIT mice,increased Treg cells,and proved that DHA can improve AIT by regulating the T lymphocyte imbalance.