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Objective:To investigate correlation between neutrophil extracellular traps(NETs) formation and T cell subsets in mice with experimental autoimmune thyroiditis(EAT) and the impact of active vitamin D intervention.Methods:Six-week-old female BALB/c mice were randomly divided into Control group, EAT group and 1, 25 dihydroxy vitamin D 3[1, 25(OH) 2D 3] treatment group(VitD group; n=6/group). HE staining was used to observe thyroid pathology. Plasma thyroglobulin antibody(TGAb), thyroid peroxidase antibody(TPOAb), and 1, 25(OH) 2D 3 were measured by ELISA. Peripheral NETs formation, Th1, Th2, and Th17 cell ratio from spleen were measured by flow cytometry. Correlation between NETs formation rate and Th1, Th2, and Th17 cell ratio was analyzed. Results:Compared with Control group, mice in EAT group had significantly increased thyroid inflammation scores, thyroiditis morbidity, TPOAb, TGAb levels, NETs formation rate, Th2(CD4 + IL-4 + or CD4 + IL-13 + )and Th17 cell proportions( P were <0.001, 0.002, 0.007, <0.001, <0.001, 0.003, 0.001, and 0.002, respectively), and significant decreased 1, 25(OH) 2D 3, Th1 cell proportions, Th1/Th2(CD4 + IL-4 + ), Th1/Th2(CD4 + IL-13 + ), and Th1/Th17 ratios( P were 0.010, 0.018, 0.010, 0.005, and 0.007, respectively). Compared with the EAT group, the VitD group had lower thyroid inflammation scores, TPOAb, TGAb levels, NETs formation rate, Th2(CD4 + IL-4 + or CD4 + IL-13 + ) and Th17 cell proportions( P were 0.044, 0.007, <0.001, 0.001, 0.014, 0.008, and 0.001, respectively), and significant higher Th1 cell ratio, Th1/Th2(CD4 + IL-13 + ) and Th1/Th17 ratio( P were 0.011, 0.009, and 0.003, respectively). The Th1/Th2(CD4 + IL-4 + ) was not significantly increased in VitD group compared with EAT group( P=0.174). NETs formation rate was positively correlated with Th2(CD4 + IL-4 + or CD4 + IL-13 + ) and Th17 cell proportion( r were 0.65, 0.59, and 0.61; and P were 0.004, 0.010, and 0.007, respectively), but not with Th1 cell proportion( r=-0.47, P=0.051). Conclusion:EAT mice were more prone to NETs formation. Active vitamin D may relieve immune imbalance with increased Th2 and Th17 cell ratio and decreased Th1 cell ratio by reducing the formation of NETs in EAT mice. Vitamin D played the protective role in thyroid by reducing thyroid pathological damage and thyroid autoantibody levels, and relived overall lymphocyte imbalance.
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Objective:To investigate the molecular mechanism of excessive iodine induced experimental autoimmune thyroiditis (EAT) in mice.Methods:Sixty female non-obese diabetic (NOD) mice were selected and divided into 5 groups according to body weight [(25 ± 3) g] via the random number table method, with 12 mice in each group: control group (group A), 10-fold high iodine group (group B), 100-fold high iodine group (group C), 1 000-fold high iodine group (group D) and 1 000-fold high iodine combined with polyinosinic acid-polycytidylic acid [Poly (I:C)] group (group E). The experiment period was 16 weeks. Mice in each group drank purified water with sodium iodine (NaI) content of 0.000, 0.005, 0.050, 0.500 and 0.500 mg/L, respectively; mice in group E were intraperitoneally injected with Poly (I:C) at week 7 and week 15, respectively. At the end of the 16th week, mice were dissected and blood samples and thyroid tissue were taken. The levels of serum thyroid function indexes [thyroid stimulating hormone (TSH), free triiodothyronine (FT 3), free thyroxine (FT 4), and thyroid peroxidase antibody (TPOAb)] were detected by enzyme-linked immunosorbent assay (ELISA); the pathological changes of thyroid tissue were observed after hematoxylin-eosin (HE) staining; differentially expressed genes in thyroid tissue were detected by RNA-sequencing (RNA-seq), and analyzed by KEGG pathway; mRNA and protein levels of p38, intercellular adhesion molecule-1 (ICAM-1) and chemokine 10 (CXCL10) in thyroid tissue were detected by real-time fluorescence quantitative PCR (qPCR) and Western blotting, respectively. Results:There were statistically significant differences in serum levels of TSH (ng/ml: 6.53 ± 0.86, 6.61 ± 0.82, 7.68 ± 0.55, 7.93 ± 0.60, 8.73 ± 1.60), FT 3 (pg/ml: 59.35 ± 10.16, 53.73 ± 10.96, 46.19 ± 8.03, 41.01 ± 8.67, 34.21 ± 11.75), FT 4 (pg/ml: 136.74 ± 10.06, 124.33 ± 14.34, 101.80 ± 6.78, 91.37 ± 6.75, 73.29 ± 17.31), and TPOAb (U/ml: 130.81 ± 24.53, 145.47 ± 28.89, 166.52 ± 41.59, 199.78 ± 42.19, 201.99 ± 44.03) among the 5 groups of mice ( F = 4.77, 4.96, 23.12, 3.68, P < 0.05). Compared with group A, the serum TSH levels of mice in groups C, D and E were higher, the levels of FT 3 and FT 4 in groups B, C, D and E were lower, and the levels of TPOAb in groups D and E were higher, and the differences were statistically significant ( P < 0.05). HE staining showed that the thyroid follicle lesion in groups D and E was serious, and the EAT phenotype appeared in both groups. The differentially expressed genes were analyzed by KEGG pathway. Compared with group A, 8 metabolic pathways related to thyroid autoimmunity and inflammation were found in groups B, C, D and E. Further analysis found that 3 genes appeared in multiple pathways, namely p38, ICAM-1 and CXCL10. There were significant differences in the mRNA levels of p38, ICAM-1 and CXCL10 in thyroid tissue of the 5 groups of mice ( F = 14.77, 12.76, 16.39, P < 0.05); compared with group A, the mRNA levels of p38 in groups B, C, D and E were higher, and the mRNA levels of ICAM-1 and CXCL10 in groups C, D and E were higher ( P < 0.05). There were significant differences in the protein levels of p38, ICAM-1 and CXCL10 in thyroid tissue of the 5 groups of mice ( F = 7.97, 73.86, 18.02, P < 0.05); compared with group A, the protein levels of ICAM-1 and CXCL10 in groups B, C, D and E were higher ( P < 0.05). Conclusion:Excessive iodine promotes the occurrence and development of EAT in mice by up-regulating the expressions of p38 and ICAM-1 genes that are closely related to thyroid autoimmune and inflammatory responses.
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Objective To investigate the therapeutic effect of CTLA4Ig gene on experimental autoimmune thyroiditis (EAT) by the use of portable synthetic costimulatory molecules of cytotoxic T lymphocyte antigen 4 (CTLA-4) antagonist (CTLA4Ig) eukaryotic expression vector.Methods Thirty C57BL/6J female mice were divided into three groups,named EAT model group (EAT,n =10),CTLA4Ig-treatment group (CTLA4Ig-EAT,n =10) and control group(n =10).At 28 day after first immunization,plasmids mixture with pCI or pCI/CTLA4Ig were injected into thyroid tissues of EAT and CTLA4Ig-EAT by surgery,respectively.Serum,thyroid tissues and spleens were collected as samples.Thyroid autoantibody and expression of interleukin (Th)1,Th2 related cytokinesby were measured by real-time quantitative PCR and ELISA.Results Compared with EAT group,the expression of CTLA-4 in thyroid of CTLA4Ig-EAT group was elevated double folds (P =0.038),and the expression of Th1 cytokine interferon γ and intercellular adhesion molecule-1 decreased significantly (P =0.016,0.042).Meanwhile,Th2 cytokine IL-4 was increased after CTLA4Ig treatment (P =0.044).The same changes were seen in spleen tissues and serum.There was no significant difference in terms of TPOAb between EAT and treated group.Conclusion Local thyroid injection of CTLA4Ig gene shows the therapeutic effect to same degree on EAT through adjusting the underlying Th1/Th2 imbalance.
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Objective To evaluate the therapeutic effect of Xiaoying Ruanjian Decoction on experimental autoimmune thyroiditis (EAT) from the levels of autoantibodies, interleukin-1, tumor necrosis factor-? and apoptosis-regulated proteins Fas/Fasl of EAT rats, and investigate the mechanism from the relation between cytokines and apoptosis-regulated proteins. Methods Sixty SD female rats of SPF level were divided into normal group and model group. An animal model of EAT in rats was developed by injecting thyroglobulin mixed with Freund’s adjuvant immunization and drinking periodate water. The rats modeled were divided into Chinese medicine group, western medicine group and model group. The levels of autoantibodies, interleukin-1, tumor necrosis factor-? were detected with enzyme-linked immunosorbent assay. The level change of Fas/Fasl in the thyroid of rat was observed with immunohistochemical staining. Results Ruanjian Xiaoying Decoction significantly reduced the levels of autoantibody, interleukin-1, tumor necrosis factor-? and lowered the expression of Fas and Fasl in thyroid cells. Conclusion Ruanjian Xiaoying Decoction alleviates the levels of apoptosis-regulated proteins Fas/Fasl by decreasing the relative expression of antibodies and cytokines in serum levels, which plays a role in reducing apoptosis of thyroid cells.
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AIM: To evaluate the therapeutic effect of Jianing Decoction (Rhizoma sparganii, Rhizoma curcumae, Radix bupleuri, Fructus schisandrae chinensis, Spuama manis, Radix polygoni multifrori, Spica prunellae, Flos chrysanthemi indici, Fructus ligustri lucidi) (JN) on autoimmune thyroiditis. METHODS: An animal model of experimental autoimmune thyroiditis (EAT) in rats was developed by using thyroglobulin of porcine (PTg). These animals were divided into JN group, triptolide (TP) group and control group. Pathological changes were observed in the thyroid tissues and serum thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibodies (TGAb) were determined by the method of radiorimmuoussay. RESULTS: The serum TGAb and TPOAb were significantly higher in EAT model group than those in normal group (P
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Objective To explore the effects of different levels of selenium nutrition on thyroid function,liver deiodinase type Ⅰ(DⅠ) and brain deiodinase type Ⅱ(DⅡ) activities in experimental autoimmune thyroiditis (EAT) rats. Methods A total of 32 female Lewis rats were randomly divided into four groups that included control group,model group,EAT with selenium-supplementation group and EAT with selenium-deficiency group. After two weeks of the basal diet administration,the rats were fed on forage containing different levels of selenium for nine weeks and the total intake of selenium were 4,4,40 and 0.4 ?g/d,respectively. The rats of model group,EAT with selenium-supplementation group and EAT with selenium-deficiency group were induced to establish the model of experimental autoimmune thyroiditis from the third week to eighth week. The pathological change of thyroid was examined,and the levels of selenium in the blood,serum thyroid autoantibody and thyroid hormone,DⅠ and DⅡ activities were measured simultaneously. Results Compared with control group,thyroid autoantibody and thyroid hormone levels significantly increased in model group,EAT with selenium-supplementation group and EAT with selenium-deficiency group (P