| Background:Graves’ disease (GD) is an autoimmune thyroid disease characterized by diffuse goiter, hyperthyroidism and Graves’ ophthalmopathy. Patients with GD have an autoimmune disorder and produce large amounts of thyrotropin receptor antibodies (TRAb), which bind and activate thyrotropin receptors on thyrocytes, leading to excessive cell proliferation and thyroid hormone secretion, presenting goiter and hyperthyroidism in these patients.Excessive hyperplasia in the thyroid of GD is closely related to the mechanism of proliferation. Some researches indicated that the factors correlated with proliferation (including IGF-1, NF-κB, cyclin D1and PCNA) play important roles in the goiter of GD. IGF-1is a pleiotropic polypeptide that plays a critical role in cell growth and proliferation. It has previously been reported that IGF-1promotes cell cycle progression through the up-regulation of cyclin D1expression via the activation of the NF-κB pathway in FRTL thyroid cells. NF-κB is a transcription factor that plays a key role in regulating cell proliferation and cell differentiation. Cyclin D1is likely the most important cell cycle protein in the regulation of cell cycle from G1phase to S phase. PCNA is a cofactor of DNA polymerase delta that is necessary for DNA synthesis in the S phase.Autoimmune disorder is the causative factor of GD. Recent studies showed patients with GD have a dysfunction of lymphocyte subsets, involving that the functional defect of suppressor T cells (Ts) and the inappropriate sensitization of helper T cells (Th). It makes B lymphocytes to produce TRAb. According to the cell surface molecules (CD), T lymphocytes are divided into different lymphocyte subsets. CD3is only expressed on the membranes of all T lymphocytes, so it is used to mark the mature T lymphocytes. The mature T lymphocytes are divided into CD4+T and CD8+T cells by cell surface molecules. CD4+T cells are mainly Th which main roles are to send signals to other types of immune cells. CD8+T cells are mainly Ts and CTL (Cytotoxic lymphocyte). The ratio of CD4+/CD8+reflects the state of immune function in body. T regulatory cells (Treg) are a kind of lymphocytes that express CD4, CD25, and Foxp3, secrete IL-10and TGF-P and inhibit T and B lymphocytes. T regulatory cells play the important role in the pathogenesis of GD.Over the past two decades, antithyroid drugs, such as carbimazole, methimazole, and propylthiouracil, have been the preferred mode of therapy for the treatment of GD. However, there are several disadvantages in antithyroid drug treatment, including a high risk of recurrence, a long course of treatment and the low remission rate of large goiters. Until recently, there is no special antithyroid drug target for the suppression of the thyrocyte proliferation or the regulation of immune response.Dio (Dio), a naturally occurring steroid saponin, is abundantly produced in a yam tuber (Dioscorea sp.) that is traditional Chinese herbs. Over the past three decades, Dio has primarily been studied for the prevention and/or treatment of various diseases, such as cancer, diabetes, obesity and dyslipidemia. Moalic S et al. showed that Dio inhibited the growth of leukemia and various cell lines from solid tumors in vitro and in vivo. The mechanisms of the anticancer activity involve the modulation of proliferation, apoptotic machinery, and the COX/LOX and cholesterol biosynthetic pathways. Dio is a precursor compound to synthesize various steroidal drugs. As a steroid, it has a wide range of effects on immune function, such as inducing the TNF-a secretion, suppressing IgE production, and up-regulating T regulatory cell production. As a folk prescription of traditional Chinese medicine, Dioscorea is used to treat thyroid adenoma and hyperthyroidism. A researcher of traditional Chinese medicine reported that Dioscorea has the therapeutic effect on the immunological goiter in a rat model. Previously, we reported that Dio treatment resulted in thyrocyte G0/G1arrest, reduced the expression of cyclin D1and inhibited the IGF-1-induced proliferation of primary human thyrocytes. The ability of Dio to inhibit thyrocyte proliferation and modulate immune response could be extrapolated to its prospective chemotherapeutic actions against GD.Based on the discoveries above, in this study, we applied Ad-TSHR-289(adenovirus expressing the A subunit of the thyrotropin receptor) mediated mouse model of GD to simulate the phenotype of human disease. We used this model in vivo to examine the effects of Dio on GD.Objectives:1. To evaluate the effect of Dio on treating the goiter in the mouse model of GD induced by Ad-TSHR-289immunization, and explore the mechanism of this effect from the perspective of anti-proliferation.2. To explore the impact of Dio on the immune function in the mouse model of GD.Methods:1. GD induction and Dio treatmentAll mice were randomly divided into the following groups:Norm, High-dose Dio (100mg·kg-1·day-1), Ad-β-gal, GD, GD+Low-dose Dio (20mg·kg-1·day-1), and GD+High-dose Dio (100mg·kg-1·day-1). Mice were injected with Ad-TSHR-289three times at21-day intervals to induce GD. At day21, after the third injection (before Dio treatment), blood samples were collected and tested to verify the GD model. Subsequently, the GD mice received treatment with and without different doses of Dio. The animals were euthanized to obtain blood, thyroid and spleen samples after Dio treatment for12or24days.2. The initial assessment of the side effect of DioThe body weight was measured before euthanization. AST, ALT and Cr levels in the serum were measured by the automatic biochemical analyzer.3. The effect of Dio on goiterThe TT4and TRAb levels in the serum were measured using radioimmunoassay and electrochemiluminescence assay. The thyroid area was measured by Image-Pro Plus6.0software. The thyroid morphology was observed by H&E staining and transmission electron microscopy.4. The mechanism of Dio on the inhibition of thyrocyte proliferationThe proliferating cells in the thyroid were detected by BrdU incorporation assay. The protein expression of IGF-1, NF-κB, cyclin D1and PCNA in the thyroid was detected using Immunohistochemistry. The mRNA expression of IGF-1, cyclin D1and PCNA in the thyroid was detected using Real-time PCR.5. The impact of Dio on immune functionThe expression of CD3, CD4, CD8, CD25and Foxp3on the spleen cells was detected using flow cytometry.Results:1. Diosgenin administration did not cause obvious changes in body weight, AST, ALT and Cr.2. The results of TT4and TRAb:The levels of TT4and TRAb in the GD groups were higher than those in the Norm group (P<0.05). After low and high doses of Dio treatment, the levels of TT4were reduced, compared with the GD group (P<0.01). A significant dose-dependent difference low and high doses of Dio was detected. Surprisingly, neither low-nor high-dose Dio treatment reduced the levels of TRAb in the GD mice.3. Gross inspection revealed that low and high doses of Dio for12and24days relieved the goiter of the GD mice. The thyroid area was measured as the thyroid size using Image Pro-Plus6.0software. The thyroid areas measured in the GD group were larger than those in the Norm group (P<0.01). However, low-and high-dose Dio treatments reduced the thyroid area compared with the GD group (P<0.05). The action of Dio was dose-dependent.4. H&E staining revealed that the thyroids in the GD group were characterized by the predominance of diffuse hypercellularity, hyperplasia and hypertrophy. These pathological abnormalities were alleviated through Dio treatment.5. The electron micrographs revealed that the thyrocytes of the GD group exhibited the characteristics of increased synthesis and secretion activity:these cells showed distorted nuclei, abundant mitochondria, plentiful vesicles of colloid and a rough endoplasmic reticulum with dilated cisternae. These changes were recovered after treatment with high-dose Dio.6. The BrdU incorporation assay showed that the ratio of proliferating thyrocytes labeled by BrdU increased in the GD group, compared with the Norm group (P<0.01). Dio treatment decreased the ratio of proliferating thyrocytes labeled by BrdU, compared with the GD group (P<0.05).7. In immunohistochemical staining, the protein expression of IGF-1, NF-κB, cyclin D1and PCNA was significantly increased in the GD group, compared with the Norm group (P<0.05). When GD mice were treated with Low and High-dose Dio, the protein expression of IGF-1, NF-κB, cyclin D1and PCNA was significantly reduced (P<0.05). The Dio-mediated inhibition of the expression of these proliferation-associated proteins was also dose-dependent.8. In Real-time PCR, the mRNA expression of IGF-1, cyclin D1and PCNA was significantly increased in the GD group, compared with the Norm group (P<0.01). However, the mRNA expression of IGF-1, cyclin D1and PCNA was significantly reduced by Low and High-dose Dio treatments, compared with the GD group (P<0.05). The Dio-mediated inhibition of the mRNA expression of these proliferation-associated proteins was also dose-dependent.9. In flow cytometry of spleen cells, there was no difference of CD3+, CD4+, CD8+and CD4+/CD8+among the three groups:Norm, GD and GD+High-dose Dio (P>0.05). Compare to the Norm group, the percentage of Treg cells (CD4+CD25+Foxp3+T cells) in the GD group was reduced (P<0.05). However, when High-dose Dio treatment was administered for24days, the reduced percentage of Treg cells was restored (P<0.05).Conclusions:1. Dio relieved goiter in the mouse model of GD induced through Ad-TSHR-289immunization. During Dio treatment, no obvious side effect was found on body weight and hepatorenal function. 2. This mechanism of the effect of Dio was related to the inhibition of thyrocyte proliferation, involving the suppression of IGF-1, NF-κB, cyclin D1and PCNA expression.3. Dio was able to restore the decline of the Regulatory T cells in the mouse model of GD induced through Ad-TSHR-289immunization. |
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