This study aims to explore the effects of Buzhong Yiqi Decoction(BYD) on the cyclic guanosine monophosphate-adenosine monophosphate synthase(cGAS)-stimulator of interferon genes(STING) signaling pathway in the mouse model of autoimmune thyroiditis(AIT) and the mechanism of BYD in alleviating the immune injury. Forty-eight NOD.H-2~(h4) mice were assigned into normal, model, low-, medium-, and high-dose BYD, and selenium yeast tablets groups(n=8). Mice of 8 weeks old were treated with 0.05% sodium iodide solution for 8 weeks for the modeling of AIT and then administrated with corresponding drugs by gavage for 8 weeks before sampling. High performance liquid chromatography was employed to measure the astragaloside Ⅳ content in BYD. Hematoxylin-eosin staining was employed to observe the pathological changes in the mouse thyroid tissue. Enzyme-linked immunosorbent assay was employed to measure the serum levels of thyroid peroxidase antibody(TPO-Ab), thyroglobulin antibody(TgAb), and interferon-γ(IFN-γ). Flow cytometry was employed to detect the distribution of T cell subsets in the spleen. The immunohistochemical method was used to detect the expression of cGAS, STING, TANK-binding kinase 1(TBK1), and interferon regulatory factor 3(IRF3). Real-time PCR and Western blot were employed to determine the mRNA and protein levels, respectively, of markers related to the cGAS-STING signaling pathway in the thyroid tissue. The results showed that the content of astragaloside Ⅳ in BYD was(7.06±0.08) mg·mL~(-1). Compared with the normal group, the model group showed disrupted structures of thyroid follicular epithelial cells, massive infiltration of lymphocytes, and elevated levels of TgAb and TPO-Ab. Compared with the model group, the four treatment groups showed intact epithelial cells, reduced lymphocyte infiltration, and lowered levels of TgAb and TPO-Ab. Compared with the normal group, the model group showed increases in the proportions of Th1 and Th17 cells, a decrease in the proportion of Th2 cells, and an increase in the IFN-γ level. Compared with the model group, the four treatment groups presented decreased proportions of Th1 and Th17 cells and lowered levels of IFN-γ, and the medium-dose BYD group showed an increase in the proportion of Th2 cells. Compared with the normal group, the modeling up-regulated the mRNA levels of cGAS, STING, TBK1, and IRF3 and the protein levels of cGAS, p-STING, p-TBK1, and p-IRF3. Compared with the model group, the four treatment groups showed reduced levels of cGAS, STING, TBK1, and IRF3-positive products, down-regulated mRNA levels of cGAS, STING, and TBK1, and down-regulated protein levels of cGAS and p-STING. The high-dose BYD group showed down-regulations in the mRNA level of IRF3 and the protein levels of p-TBK1 and p-IRF3. The above results indicate that BYD can repair the imbalance of T cell subsets, alleviate immune injury, and reduce thyroid lymphocyte infiltration in AIT mice by inhibiting the cGAS-STING signaling pathway.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Signal Transduction/drug effects* ; Thyroiditis, Autoimmune/metabolism* ; Mice ; Membrane Proteins/metabolism* ; Mice, Inbred NOD ; Humans ; Female ; Nucleotidyltransferases/metabolism* ; Male ; Disease Models, Animal

OBJECTIVETo explore the regulatory mechanism of Xiaoyin Recipe () on the T helper 1/T helper 2 (Th1/Th2) immune balance.

METHODSThirty-six experimental animals were divided into three groups, 12 rats in each group: blank control group (B group), negative control group (N group), and Xiaoyin Recipe treatment group (T group). The latter two groups received immunization of experimental autoimmune thyroiditis (EAT), and T group were treated with Xiaoyin Recipe for a month. Then, the expression of Th1-Th2-related genes in peripheral blood mononuclear cells (PBMCs) were screened with Oligo GEArray Rat Th1-Th2-Th3 Microarray. The expressions of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), T-box expressed in T-cells (T-bet), and GATA-binding protein-3 (GATA-3) were detected by real-time polymerase chain reaction (RT-PCR).

RESULTSGene array screening showed that compared to N group, in T group after Xiaoyin Recipe treatment, 3 genes were upregulated in EAT rats, including interleukin-27 receptor alpha (IL-27rα), glomulin (Glmn), and GATA-3, while 38 genes were downregulated, such as CD28, IL-18, signal transducer, and activator of transcription 1 (STAT1), T-bet, TNF receptor superfamily member 4 (TNFRSF4), TNF ligand superfamily member 5 (TNFSF5), and TNF receptor superfamily member 5 (TNFRSF5). While RT-PCR showed that there was an increased level of TNF-α mRNA (P<0.01), an elevated ratio of T-bet/GATA-3, and a decreased level of IL-10 mRNA in PBMC of N and T group compared to B group (P <0.01); and after treatment with Xiaoyin Recipe, IL-10 mRNA level increased (P <0.01), while TNF-α mRNA level and T-bet/GATA-3 ratio decreased in T group compared to N group (P <0.01).

CONCLUSIONXiaoyin Recipe for psoriasis could induce a Th1/Th2 balance drift toward Th2 in PBMC of EAT rats and thus improve the conditions.

Animals ; Autoantibodies ; blood ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; GATA3 Transcription Factor ; genetics ; metabolism ; Gene Expression Regulation ; drug effects ; Interleukin-10 ; genetics ; metabolism ; Psoriasis ; blood ; drug therapy ; immunology ; pathology ; Rats ; Rats, Wistar ; T-Box Domain Proteins ; genetics ; metabolism ; Th1-Th2 Balance ; drug effects ; Th2 Cells ; drug effects ; immunology ; Thyroiditis, Autoimmune ; blood ; drug therapy ; immunology ; pathology ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

OBJECTIVETo characterize the morphological features of thyroid papillary microcarcinoma (PMC) and assess the significance of expression of CK19, HBME-1, Galectin-3, CD56 and p63 in differential diagnosis of PMC from benign thyroid lesions.

METHODSClinicopathologic features of 78 cases PMC were reviewed. Immunohistochemical analysis of CK19, HBME-1, Galectin-3, CD56, and p63 in 78 cases of PMC and 48 cases of benign thyroid lesions (18 cases of papillary hyperplasia, 17 cases of nodular goiter and 13 cases of lymphocytic thyroiditis) was conducted. The patients were followed up for from 6 to 269 months after surgical operation.

RESULTS69 cases nuclear atypia and overlapping nuclei (88.5%), 67 cases nuclear grooves (85.9%), 50 cases nuclear pseudoinclusions (64.1%) and 60 cases papillary architecture (76.9%) were detected in 78 cases of PMC. Moderate to strong co-expression of CK19, HBME-1 and galectin-3 was observed in 98.0% (50/51) in the PMC group but in none of the benign disease group. The expression of CD56 and p63 was negative in both groups. In the postoperative follow-up period of 6-269 months, 7 cases (9.0%) developed intrathyroid recurrence, 3 cases (3.8%) developed lymph node metastasis, no distant metastasis or death was observed. In 12 cases (15.4%) the PMC lesion smaller than 3 mm in diameter was not found by frozen section diagnosis.

CONCLUSIONSOverlapping nuclei, nuclear atypia, polar disorder, ground glass nuclei, nuclear grooves and nuclear pseudoinclusions are most important for the diagnosis of PMC with or without papillary architecture. The appearance of definite interstitial invasion, interstitial sclerosis and true complex papillary architecture are more helpful to make right diagnosis. Intraoperative frozen section is of limited value for a reliable diagnosis of PMC in diameter < or = 3 mm. Moderate to strong co-expression of CK19, HBME-1 and Galectin-3 is a very useful indicator for differential diagnosis of PMC from benign thyroid lesions.

Adult ; Biomarkers, Tumor ; metabolism ; CD56 Antigen ; metabolism ; Carcinoma, Papillary ; diagnosis ; metabolism ; pathology ; surgery ; Cell Nucleus ; pathology ; Diagnosis, Differential ; Female ; Follow-Up Studies ; Galectin 3 ; metabolism ; Goiter, Nodular ; metabolism ; pathology ; Humans ; Hyperplasia ; Keratin-19 ; metabolism ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Thyroid Gland ; metabolism ; pathology ; Thyroid Neoplasms ; diagnosis ; metabolism ; pathology ; surgery ; Thyroidectomy ; methods ; Thyroiditis, Autoimmune ; metabolism ; pathology ; Transcription Factors ; metabolism ; Tumor Suppressor Proteins ; metabolism

OBJECTIVETo observe the effect of 1,25(OH)2D3 on thyroid inflammation and Th1/Th2 cells in rats with experimental autoimmune thyroiditis (EAT).

METHODSForty-eight female Wistar rats were randomly divided into 4 groups, namely the prevention group treated with 1, 25-(OH)2D3 from 0 to the 6th week (n=10), treatment group with 1,25(OH)2D3 treatment from the 2nd to the 8th week (n=10) after immune sensitization, positive control group (n=12) and the negative control group (n=16). All the rats were challenged with porcine thyroglobulin for immune sensitization until the 6th or 8th week except for those in the negative control group. In the prevention group and treatment group, the rats received 1,25(OH)2D3 at 5 microg/kg by intraperitoneal injection every other day, while those in the positive and negative control groups were given peanut oil instead. The thyroid pathologies, serum autoantibody level and cytokine levels were examined after the treatments.

RESULTSThe thyroid gland remained structurally intact in the negative control group. In the positive control group, the thyroid showed obvious inflammatory change with structural disruption and even disappearance of the thyroid follicle. The structure of the thyroid gland follicles was intact in the prevention group and treatment group. No significant differences were found in the autoantibody and cytokine levels between the prevention group and negative control group (P>0.05). Compared with the positive control groups, the autoantibody and IFN-gamma and IL-12 levels decreased significantly in the treatment group, but the levels of IL-4 and IL-10 were markedly increased (P<0.05).

CONCLUSION1,25(OH)2D3 given before the establishment of the EAT model helps maintain structural integrity of the thyroid gland and normal levels of the antibodies and cytokines in rats. 1,25(OH)2D3 can ameliorate the pathological changes of the thyroid gland and correct the cytokine disequilibrium in rats with EAT.

Animals ; Autoantibodies ; blood ; Female ; Interferon-gamma ; blood ; Interleukin-10 ; blood ; Interleukin-12 ; blood ; Rats ; Rats, Wistar ; Th1 Cells ; cytology ; Th2 Cells ; cytology ; Thyroiditis, Autoimmune ; drug therapy ; metabolism ; Vitamin D ; analogs & derivatives ; therapeutic use

OBJECTIVETo study the effect of different selenium intake on the expression of apoptosis protein Fas/FasL in experimental autoimmune thyroiditis (EAT) rats' thyroid with adequate iodine.

METHODSThirty-two female lewis rats were divided stochastically into 4 groups as C group, M group, Se(+) + M group, Se(-) + M group, respectively, and pretreated with feedstuffs containing different concentrations of selenium (Se(+) + M group 2 mg/kg, C and M group 0.20 mg/kg, Se(-) + M group 0.02 mg/kg, respectively) for two weeks, and immunized the rats with porcine thyroglobulin (pTg) to establish an EAT model. The thyroid gland was sampled, embedded in mineral wax and sliced, and the expression of Fas/FasL was measured with immunohistochemistry.

RESULTSBoth the expressions of Fas and FasL of EAT rats were significantly increased as compared with control group. The expression of Fas in rats' thyroid follicular cells with EAT was down-regulated as the increased selenium intake (optical density: 0.059 +/- 0.006), the expression of Fas of Se(+) + M group (0.036 +/- 0.004) was significantly inhibited (q = 11.591, P = 0.000), and expression of Fas was lower in the Se(+) + M group than Se(-) + M group (0.050 +/- 0.005) (q = 7.055 , P = 0.000). Effect of selenium on FasL was not identified.

CONCLUSIONIncreasing the intake of selenium might decrease the expression of Fas on thyroid follicular cells and restrain the development of EAT.

Animals ; Apoptosis ; Fas Ligand Protein ; biosynthesis ; Female ; Rats ; Rats, Inbred Lew ; Selenium ; pharmacology ; therapeutic use ; Thyroid Gland ; cytology ; metabolism ; Thyroiditis, Autoimmune ; drug therapy ; metabolism

BACKGROUNDPrevious studies showed that the role of Fas ligand (FasL) is not consistent in the pathogenesis of autoimmune thyroiditis. This study was designed to investigate the effects of FasL on the pathogenesis of experimental autoimmune thyroiditis (EAT) using CMV-human FasL (hFasL) transgenic mice.

METHODSTransgenic mice ubiquitously expressing hFasL were used as an animal model of EAT by injection of porcine thyroglobulin (pTg). Expression of hFasL was detected by RT-PCR and Western blot. The activity of hFasL transgenic thyrocytes killing Jurket cells was determined. CMV-hFasL transgenic mice and wild type (WT) mice were immunized with pTg and killed 28 days later to evaluate the lymphocytic infiltration of their thyroids. The number of CD4+ and CD8+ lymphocytes from the spleen was detected using FACS. The serum interferon-gamma (IFN-gamma) concentration was measured by ELISA.

RESULTShFasL expression in the thyroid of CMV-hFasL transgenic mice was confirmed. After co-incubation of Jurket thymocytes with thyroid tissues of CMV-hFasL transgenic mice, the percentage of apoptotic cells in the CMV-hFasL transgenic thyroid group was significantly higher than that of the control WT thyroid group [(23.4 +/- 4.3)% vs (6.6 +/- 2.5)%, P < 0.01]. On day 28 after immunization with pTg, the infiltration index of lymphocytes in thyroids of the CMV-hFasL transgenic mice was significantly lower than that of the WT mice [(1.0 +/- 0.5) vs (2.1 +/- 0.7), P < 0.001]. Moreover, the number of CD4+ and CD8+ lymphocytes of the spleen and serum IFN-gamma concentration were significantly decreased in the CMV-hFasL transgenic mice.

CONCLUSIONSFasL plays an important role in the pathogenesis of autoimmune thyroiditis. Transgenic mice ubiquitously expressing hFasL may strongly inhibit lymphocytic infiltration of the thyroid of EAT and ameliorate the course of this disease.

Animals ; Blotting, Western ; CD4-CD8 Ratio ; Cytomegalovirus ; genetics ; Fas Ligand Protein ; Female ; Humans ; Interferon-gamma ; blood ; Jurkat Cells ; Membrane Glycoproteins ; physiology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Promoter Regions, Genetic ; Reverse Transcriptase Polymerase Chain Reaction ; Thyroid Gland ; metabolism ; Thyroiditis, Autoimmune ; immunology ; prevention & control

PURPOSE:Chronic autoimmune thyroiditis is the most common thyroid disease in children and adolescents, and also is the most common cause of hypothyroidism in children and adolescents. We studied the clinical manifestations and natural courses of chronic autoimmune thyroiditis in children and adolescents. METHODS:We analyzed retrospectively twenty one children and adolescents who were diagnosed as chronic autoimmune thyroiditis before 15 years of age. The diagnoses were made from July 1997 to February 2004 at Ajou Univesity Hospital. We reviewed the clinical manifestations and thyroid functions of these patients. RESULTS:Nineteen patients were female and two were male. The mean age at diagnosis was 9.95+/-3.1 years, the mean duration of follow up was 23.52+/-22.98 months. At diagnosis, the most common chief complaint was goiter (15 patients/21 patients) and the others were short stature, easy fatigue, cardiac murmur and weight loss. Eighteen patients had goiters at diagnosis. Four had familial history of thyroid disease, Three of them had maternal history of thyroid disease and one had paternal history of thyroid disease. At diagnosis, Seven patients were euthyroid and four patients were hyperthyroid, eighteen patients had antithyroglobulin antibody, seventeen patients had antimicrosomal antibody, fourteen patients had both of them. At last follow-up, eight patients were euthyroid and ten patients were hypothyroid, three patients were hyperthyroid. CONCLUSION: Chronic autoimmune thyroiditis in children and adolescents can have the various important influences on the metabolism including the growth. The clinical manifestations and thyroid functions should be monitored in children and adolescents with chronic autoimmune thyroiditis.
Adolescent* ; Child* ; Diagnosis ; Fatigue ; Female ; Follow-Up Studies ; Goiter ; Heart Murmurs ; Humans ; Hypothyroidism ; Male ; Metabolism ; Retrospective Studies ; Thyroid Diseases ; Thyroid Gland ; Thyroiditis, Autoimmune* ; Weight Loss

Apoptosis is responsible for the loss of thyrocytes in autoimmune thyroiditis. Recent investigations into the pathogenesis of apoptosis have revealed that the important roles of suicide molecules expression on both thyrocytes and cytotoxic T-lymphocytes. To study the mechanism of thyrocyte loss in various forms of thyroiditis, we evaluated in situ expression patterns of CD40, Fas, and Fas-L on thyrocytes and infiltrating inflammatory cells by immunohistochemical staining of thyroid samples obtained from 49 patients (Graves' disease, n=10 : Hashimoto's thyroiditis, n=14; nonspecific lymphocytic thyroiditis, n=11; subacute granulomatous thyroiditis, n=11; normal, n=3). The role of cytotoxic T-lymphocytes was also evaluated by analyzing the expression of granzyme B along with their phenotypic characteristics. CD40 was not expressed on thyrocytes of normal controls while they showed a diffuse expression of Fas and a scattered focal expression of Fas-L. The plump thyrocytes proximal to the inflammatory infiltrates showed more intense expressions of these three molecules in various forms of thyroiditis and a close correlation was found between CD40 and Fas-L expression on thyrocytes. Unlike Fas, which was expressed on infiltrating lymphocytes in all groups, Fas-L was not expressed on infiltrating lymphocytes, except those in subacute granulomatous thyroiditis. Granzyme B expressing activated cytotoxic T-lymphocytes occupied a negligible proportion of CD8+ T-lymphocytes in various forms of thyroiditis, and no difference was found in terms of their proportions according to the type of thyroiditis. These results show the acquisition of CD40, Fas and Fas-L molecules on thyrocytes proximal to inflammatory cell aggregates and the negligible expression of granzyme B and Fas-L on the infiltrating lymphocytes, and suggest that Fas and Fas-L mediated apoptosis of thyrocytes (fratricide) may be more important than T cell-mediated cytotoxicity in various forms of thyroiditis.
Antigens, CD40/*metabolism ; Antigens, CD95/metabolism ; Apoptosis/*physiology ; Graves' Disease/*metabolism/pathology ; Human ; Membrane Glycoproteins/metabolism ; Reference Values ; Thyroiditis, Autoimmune/*metabolism/pathology

To determine the content of thyroglobulin in oxyphilic cells of the thyroid, which have been considered as non-thyroglobulin producing cells, the degree of stainability of the various oxyphilic cells for thyroglobulin was compared with that of non-oxyphilic follicular cells in either same or different lesion. A total of 13 oxyphilic lesions, including three follicular adenomas containing oxyphilic cell nodules, four pure oxyphilic cell adenomas, and six Hashimoto's thyroiditis were compared with 16 of non-oxyphilic lesions such as, seven follicular adenomas, four chronic lymphocytic thyroiditis, and five Graves' disease. Many oxyphilic cells stained positively for thyroglobulin regardless of their morphologic variation, but less intensely than the usual follicular cells in follicular adenomas, chronic lymphocytic thyroiditis, and Graves' disease. The stainability of oxyphilic cells for thyroglogulin did not show any significant correlation with morphologic features, whereas in follicular adenomas, the non-oxyphilic follicular cells forming microfollicles stained less strongly for thyroglobulin than the same cells lining large mature follicles in a reproducible way. With above findings, we concluded that oxyphilic cells maintain the functional activity in terms of thyroglobulin synthesis, although the content of the thyroglobulin is less than that of non-oxyphilic colloid forming follicular cells.
Adenoma/*metabolism/pathology ; Graves Disease/*metabolism/pathology ; Humans ; Staining and Labeling ; Thyroglobulin/*biosynthesis ; Thyroid Neoplasms/pathology ; Thyroiditis, Autoimmune/*metabolism/pathology