Study On The Immune Regulation Of Goiter And Hyperfunction In Mice With Graves Disease By Huayu Sanjie Method

Graves’ disease is an autoimmune thyroid disease.In China and Western countries,the prevalence of GD is approximately 2-3.0%and 0.5-2%,respectively.In general,the incidence of GD is increasing steadily.Goiter and excessive function of thyroid are the features of GD.GD is related to immune disorder.Our professor believes goiter is tangible and named phlegm turbidity.Excessive function of thyroid is called hyperactivity of fire.The basic pathogenesis of GD is the accumulation of phlegm-fire on neck,so the principle of treatment on GD is Reducing Phlegm and Resolving Masses,Removing heat and eliminating goiter.A prescription,which is composed mainly of Prunella vulgaris L.,Scrophularia ningpoensis Hemsl and Bolbostemma paniculatum(Maxim.)Franquet,is made to treat GD.The prescription has been used in clinical for over 20 years and has a good effect on GD.Our research has been supported by National Natural Science Foundation of China three times.And this study came from Effect of Reducing Phlegm and Resolving Masses on events of thyroid hormone synthesis downstream in GD mouse model,which is the project of National Natural Science Foundation(Grant No.81373593).Three aspects which contained goiter,Excessive function of thyroid and immune disorder were carried out.We try to find the relationship among them and explore the treatment characteristics of Reducing Phlegm and Resolving Masses.ObjectiveTo study the effect of Reducing Phlegm and Resolving Masses on goiter and excessive function of thyroid.And try to explore its mechanism from immune disorder,proliferation and apoptosis,downstream events of thyroid hormone systhesis..Methods1.Preparation of GD mice model and grouping:All the female BABL/c mice were divided into 3 groups(Normal,Ad-Control and Ad-TSHR289).Mice in Ad-TSHR289 were injected intramuscularly(i.m.)with phosphate-buffered saline(PBS)containing Ad-TSHR289.Mice were i.m.injected three times at 3-week intervals(in the 1st,4th and 7th week).The activity of the recombinant adenovirus were tested every time before injection.Mice in Normal and Ad-Control were immunized with PBS and PBS containing Ad-Control respectively by the same method.3 weeks after the last injection(in the 10th week),the level of TRAb and T4 in mice were tested.Only the mice model forming rate is higher than 70%can the mice be used in follow-up experiment.After inducing the GD model successfully,mice in Normal and Ad-Control were kept.And the left mice in Ad-TSHR289 were randomly divided into 3 groups and oral gavaged once a day for 4 weeks with distilled water as GD group(GD),methimazole dissolved in water as methimazole group(MMI),or the TCM prescription suspended in water as HTS J group(HTSJ).The dosage of drugs was calculated according to the dose conversion between human and animal.Mice from Normal and Ad-Control were also oral gavaged with distilled water.After treatment,All the mice were executed and their blood,spleens,and thyroid tissues were obtained for further tests.2.Detection of thyroid secretion in GD mouse model:the serum levels of T4 and TRAb were detected by radioimmunoassay,and the effect of Reducing Phlegm and Resolving Masses on thyroid function of GD mice was observed.3.Observation of thyroid morphology and histology in GD mouse model:The body and thyroid weight of mice were weighted and thyroid index(thyroid weight/body weight)was calculated.Mice thyroid morphological changes were observed by stereomicroscope.Mice thyroid histological changes were observed by light microscopy after HE staining.Mice thyroid ultrastructural changes were observed by transmission electron microscope.The effect of Reducing Phlegm and Resolving Masses on thyroid morphology and histology in GD mice was observed.4.Detection of molecules of thyroid hormone synthesis downstream in GD mouse model:The mRNA expression of NIS,TPO and Tg in mice were detected by RT-PCR.The protein expressions of NIS,TPO and Tg in mice were detected by Western Blot.The effect of Reducing Phlegm and Resolving Masses on thyroid hormone synthesis in GD mice was observed.5.Detection of the thyrocyte proliferation and apoptosis in GD mouse model:The protein expressions of PCNA,Fas,FasL,Bcl-2 and bax in mice were detected by immunohistochemistry.The effect of Reducing Phlegm and Resolving Masses on thyrocyte proliferation and apoptosis in GD mice was observed.6.Detection of the Treg/Th17 cells of spleen lymphocytes in GD mouse model:The percentage of CD4+CD25+Foxp3+ Treg cells and CD4+IL-17+ Th17 cells in spleen lymphocytes of mice were detected by four colour flow cytometry.The expression of Foxp3 mRNA and IL-17 mRNA were detected by RT-PCR.The effect of Reducing Phlegm and Resolving Masses on immune disorders in GD mice was observed.Result1.Induced the GD mouse model successfully:The serum T4 of Ad-TSHR289 is 230.16 ±41.01ng/ml,which is 152.90%higher than it in Normal(P=0.000).The serum TRAb of Ad-TSHR289 is 12.34 ± 3.51u/l,which is 1423.46%higher than it in Normal(P=0.000).All the serum T4 and TRAb in Ad-TSHR289 were higher than the upper normal limits of Normal.And the model forming rate in Ad-TSHR289 is 100%.2.Effect of Reducing Phlegm and Resolving Masses on thyroid hormone secretion in GD mouse model:The serum T4 of HTSJ is 181.46 ± 47.69ng/ml,which is 18.73%decreased compared with GD(P=0.021).The serum TRAb of HTSJ is 10.55±2.37 u/1,which is 35.47%decreased compared with GD(P=0.000).3.Effect of Reducing Phlegm and Resolving Masses on thyroid morphology and histology in GD mouse model:The thyroid index of HTSJ is(3.189± 1.220)×10-4,which is 41.69%decreased compared with GD(P=0.000).The congestion of thyroid in HTSJ has been improved compared with GD.The protrusions on the follicular lumen disappeared and became smooth.The hypertrophy and hypercellular of the thyroid follicular epithelia in HTSJ had been improved significantly.The shape of the thyroid follicular epithelia changed from columnar to flat.And the colloid in the follicular lumen was decreased.The number of mitochondria decreased and the irregular swelling reduced in HTSJ,and the extension of rough endoplasmic reticulum dilataltion was also reduced.The structure of mitochondria cristae became clear and the number of lipid droplets decreased compared with GD.4.Effect of Reducing Phlegm and Resolving Masses on molecules of thyroid hormone synthesis downstream in GD mouse model:The expression of NIS protein and NIS mRNA in thyrocytes of HTSJ were 78.80%and 45.97%decreased respectively compared with GD(P=0.003,P=0.000).The expression of TPO protein and TPO mRNA in thyrocytes of HTSJ were 72.85%and 66.24%decreased respectively compared with GD(P=0.000,P=0.000).The expression of Tg protein and Tg mRNA in thyrocytes of HTSJ were 81.06%and 62.79%decreased respectively compared with GD(P=0.000,P=0.000).5.Effect of Reducing Phlegm and Resolving Masses on the thyrocytes proliferation and apoptosis in GD mouse model:The number of PCNA positive thyroid follicular epithelia in HTSJ decreased compared with GD.The number of Fas positive,FasL positive,and Bax positive thyroid follicular epithelia in HTSJ increased compared with GD.The number of Bcl-2 positive thyroid follicular epithelia in HTSJ didn’t changed compared with GD.6.Effect of Reducing Phlegm and Resolving Masses on the immune function in GD mouse model:The percentage of CD4+CD25+Foxp3+ Treg cells in spleen lymphocytes of HTSJ increased 73.62%compared with GD(P=0.037).The expression of Foxp3 mRNA in spleen lymphocytes of HTSJ increased 186.67%compared with GD(P=0.010),and the expression of IL-17 mRNA were 71.27%decreased compared with GD(P=0.003).Conclusion1.Reducing Phlegm and Resolving Masses can decrease the excessive thyroid function and improve goiter.2.The possible mechanism of Reducing Phlegm and Resolving Masses on GD mice:Reducing Phlegm and Resolving Masses can regulate the immune disorder by up-regulating the percentage of CD4+CD25+Foxp3+ Treg cells in spleen lymphocytes,increasing the expression of Foxp3 mRNA,reducing the expression of IL-17 mRNA.These regulation can reduce the production of TRAb,which can ①decrease the expression of NIS,TPO and Tg,leading the reduction of the synthesis of thyroid hormone in GD mice,②inhibit the proliferation and accelerate the apoptosis of thyroid follicular epithelia,leading to improve goiter.Reducing Phlegm and Resolving Masses treats GD by the comprehensive regulation of three aspects which contained the goiter,excessive thyroid function and immune disorder.