| Graves disease(GD)is an autoimmune thyroid disease,characterized by goiter and hyperfunction,has been on the rise in recent years.Antithyroid drugs are the main treatment of GD at present,but they have the characteristics of high recurrence rate,and goiter is an important factor affecting the prognosis of the disease.The tutor believes that "phlegm coagulation in front of neck" is the core feature of GD,and"resolving phlegm and dispersing stagnation" is the basic treatment,which has a significant clinical effect.The series of research on "resolving phlegm and dispersing phlegm”has been funded by the National Natural Science Foundation of China for four times.This study was funded by the National Natural Science Foundation of China(NSFC)"The effect of resolving phlegm and dispersing stagnation method on the mechanism of immune tolerance switch and its modification in Graves disease mice"(No.81774293).Based on previous research,the key mechanism of resolving phlegm and dispersing stagnation method for immune tolerance in GD mice was explored.Chapter 1 Optimization of GD Mouse ModelObjective:To observe the time,rate and maintenance time of GD mouse model constructed by one-time immunoassay,so as to provide a carrier for the study.Methods:Adenovirus Ad-TSHR289 was constructed and BALB/c mice were divided into model group(GD)and normal group(NC).The GD group was injected with 2*109 Pfu virus at the first week of the experiment,and the NC group was given the same PBS solution.At the 4th,7th,13th and 18th weeks,mice in GD group and NC group were executed,and the contents of T4 and TRAb in blood were detected.The morphological changes of thyroid were observed and HE staining was observed.Results:(1)Thyroid function:At week 4,the positive rate of T3 and T4 in GD group was 4/6 and 3/6;at week 7,the positive rate of T3 and T4 in GD group was 6/6 and 5/6;at week 13,the positive rate of T3 and T4 in GD group was 6/6 and 5/6;at week 18,the positive rate of T3 and T4 in GD group was 6/6.The positive rate of TRAb was 6/6 in each period.(2)Thyroid morphology:At the 4th week,there was no obvious enlargement in the general thyroid morphology of NC group and GD group,but the thyroid coefficient(thyroid mass/body weight,TW/BW)of GD group was significantly higher than that of NC group(P<0.05).HE staining in GD group showed that thyroid follicular epithelial cells proliferated slightly,the surface of follicular cavity was smooth,and the follicular cavity was filled with glia.From the seventh week,goiter in GD group was obvious,the proportion of goiter was 5/6.TW/BW in GD group was significantly higher than that in NC group(P<0.001).Thyroid follicular epithelial cells in GD group proliferated obviously,protruded inward in many places,and intraluminal glia decreased.At the 13th and 18th weeks,goiter was obvious in GD group,and the proportion of goiter was 6/6.TW/BW in GD group was significantly higher than that in NC group(P<0.001).The proliferation of thyroid follicular epithelial cells in GD group was further aggravated,intraluminal glia decreased and local intraluminal fusion occurred.(3)From the 4th week,the serum T3 level in GD group increased gradually.At the 13th week,the serum T3 level was significantly higher than that at the 3rd week(P<0.05),and at the 18th week,the serum T3 level was significantly higher than that at the 3rd week(P<0.01).T4 content in GD group increased gradually from the 4th week to the 7th week,and reached the peak at the 7th week.The serum T4 content at the 7th,13th and 18th week was significantly different from that at the 4th week(P<0.05),and it tended to be stable from the 7th to 18th week.There was no significant difference in T4 content between the 7th,13th and 18th weeks(P>0.05).The content of TRAb in GD group increased gradually from the 4th week to the 7th week.The content of TRAb increased significantly(P<0.01).From the 7th week to the 13th week,the content of TRAb decreased(P<0.05).From the 13th to 18th week,the content of TRAb tended to be stable with no statistical significance(P>0.05).The trend of TW/BW in GD group was consistent with that of serum T4.(4)T3,T4,TRAb and TW/BW showed significant positive correlation.Conclusions:The GD mice model constructed by one immunization method can be identified at the 4th week and maintained until the 18th week.The rate of model formation is over 83.33%after 7th week.It can be used as a carrier for disease research.Chapter 2 The effect of resolving phlegm and dispersing stagnation on thyroid dysfunction and morphology in GD miceObjective:To observe the effect of Huatan Sanjie medicine on thyroid function and morphology in GD mice,to further evaluate its efficacy and to explore the mechanism of improving goiter.Methods:(1)Grouping and administration:The experiment was divided into four groups:normal control(NC)group,model(GD)group,methimazole(MMI)group and phlegm-resolving and resolving drugs(HTSJ)group.GD group,MMI group and HTSJ group were modeled according to the experimental method.Intervention:According to the equivalent dose of human and rat,MMI group mice were given 23 mg/kg/d of methimazole tablets,HTSJ group mice were given 7 g/kg/d of traditional Chinese medicine,suspended in pure water respectively.The mice were given 0.2 mL liquid every day.The NC group and GD group were given the same volume of pure water once a day.The drug was given for 6 weeks.(2)Radioimmunoassay was used to detect the contents of T4 and TRAb.(3)HE staining was used to observe the thyroid morphology of mice and scanning electron microscopy was used to observe the surface morphology of thyroid tissue.Results:(1)Thyroid function:Compared with NC group,T4 content in serum of GD group was significantly higher(P<0.001),while T4 content in MMI group and HTSJ group was significantly lower(P<0.001),indicating that drug intervention could improve thyroid hormone level.Compared with NC group,the serum TRAb level in GD group was significantly higher(P<0.001),but after drug intervention,the levels of TRAb in MMI group and HTSJ group were significantly lower than those in GD group(P<0.001).(2)HE staining and scanning electron microscopy:thyroid follicular epithelial cells in NC group were flat,arranged neatly,with smooth margin and filling in the follicular cavity.In GD group,thyroid follicular epithelial cells proliferated significantly,with rough margins,irregular convex follicular epithelial cells,insufficient material in follicular cavity and partial regional fusion.After 6 weeks of drug treatment the proliferation of follicular epithelial cells in MMI group was reduced,the convexity was reduced,the shape was changed greatly,and the follicular cavity was filled.The follicular epithelial cells in MMI group were still proliferated under 400-fold microscope.In HTSJ group,the proliferation of thyroid follicular epithelial cells was significantly reduced,no convexity was found,and the follicular cavity was filled.The morphological recovery of thyroid tissue in HTSJ group was better than that in MMI group.Scanning electron microscopy showed that the surface of thyroid follicular epithelial cells in NC group showed spherical structure without obvious microvilli.A large number of dense and slender microvilli were observed on the surface of GD group,suggesting that TSH stimulated endocytosis was active and the metabolic area of cell surface increased.After 6 weeks of drug intervention,the surface microvilli of thyroid follicular epithelial cells in MMI group and HTSJ group were significantly reduced,but the microvilli in MMI group were still slender,and the microvilli in HTSJ group were significantly shortened and sparse.These results suggest that after drug intervention,the endocytosis stimulated by TSH is weakened and the metabolic area of cell surface is reduced.Conclusions:Huatan Sanjie Drug can significantly reduce T4 and TRAb levels in GD mice,improve thyroid tissue morphology.Chapter 3:Effect of resolving phlegm and dispersing stagnation on oxidative stress in thyroid tissue of GD miceObjective:To observe the effect of Huatan Sanjie Drug on oxidative factors in thyroid tissue of GD mice,and to further explore its mechanism of improving goiter.Methods:The contents of T-SOD,MDA,GSH-px,GSH and T-AOC in thyroid tissue of mice were detected by Elisa kit.Results:Compared with NC group,the T-SOD content in thyroid tissue of GD group was significantly lower(P<0.001).After drug intervention,the T-SOD content of MMI group and HTSJ group was higher than that of GD group in varying degrees,but there was no statistical significance(P>0.05).Compared with NC group,the content of MDA in thyroid tissue of mice in GD group increased,but there was no statistical significance(P>0.05);after drug intervention,the content of MDA in MMI group was significantly lower than that in GD group(P<0.01),while that in HTSJ group decreased,but there was no statistical difference(P>0.05).Compared with NC group,the content of GSH-Px in thyroid tissue of mice in GD group decreased significantly(P<0.05);after drug intervention,the content of GSH-px in MMI group and HTSJ group increased,but there was statistical difference(P>0.05).Compared with NC group,the content of T-AOC in thyroid tissue of GD group was significantly lower(P<0.05).Compared with GD group,T-AOC content in MMI group and HTSJ group increased significantly(P<0.01;P<0.05).Compared with NC group,the GSH content in GD group was significantly lower(P<0.05),but after drug intervention,the GSH content in MMI group was significantly higher than that in GD group(P<0.05),and the GSH content in HTSJ group was significantly higher than that in GD group(P<0.01);compared with MMI group,the GSH content in HTSJ group was significantly higher(P<0.05).Conclusion:The drugs of resolving phlegm and dispersing stagnation regulate the balance of oxidation and antioxidation in thyroid tissue.Chapter 4 Effect of Resolving Phlegm and Dispersing Stasis Method on Immune Imbalance of Thl/Th2 Cells in GD MiceObjective:To observe the effect of resolving phlegm and dispersing stagnation on Thl/Th2 cell balance in GD mice,and to study the mechanism of regulating immune imbalance by resolving phlegm and dispersing stagnation.Methods:(1)The spleen coefficient of mice was determined.(2)After killing mice,the spleen was taken to prepare lymphocyte suspension.Antibodies such as CD3-percp,CD4-FITC,IL-4-PE and IFN-gamma-APC were added to the suspension and then detected by flow cytometry.(3)RT-PCR was used to detect the expression of Thl and Th2 cell-specific transcription factors T-bet and GATA-3.(4)The contents of IL-2,IFN-gamma,IL-4 and IL-6 were detected by Elisa.Results:(1)Spleen index of GD mice was significantly higher than that of NC mice(P<0.001).Spleen index of MMI and HTSJ mice was lower than that of GD mice(P<0.05;P<0.01).(2)Compared with NC group,the ratio of Thl cells to Th cells in GD group increased with statistical significance(P<0.05).MMI and HTSJ drugs decreased Thl expression to a certain extent,but there was no statistical difference(P>0.05).Compared with NC group,the expression of Th2 cells in GD mice decreased significantly(P<0.05).Compared with GD group,the expression of Th2 cells in MMI and HTSJ drugs increased,but both of them showed an upward trend.There was no statistical difference(P>0.05).Th1/Th2 ratio in GD group was significantly higher than that in NC group(P<0.001).After intervention with MMI and HTSJ,the ratio of Thl/Th2 in GD group was lower than that in model group to a certain extent,especially after intervention with phlegm-resolving and resolving drugs(P<0.05).(3)Compared with NC group,the expression of T-bet in spleen tissue of GD group was significantly increased(P<0.001).After drug intervention,compared with GD group,the expression of MMI and HTSJ group decreased significantly(P<0.01).Compared with NC group,the expression of GATA-3 in spleen tissue of GD group was significantly down-regulated(P<0.05).After drug intervention,the expression of GATA-3 in MMI and HTSJ group was higher than that in GD group.(4)IFN-gamma in serum of mice in GD group was significantly higher than that in NC group(P<0.001).After drug intervention,the levels of IFN-gamma in MMI group and HTSJ group were lower than those in GD group(P<0.01;P<0.05).Compared with NC group,the level of IL-2 in serum of mice in GD group increased significantly(P<0.01).Compared with GD group,the level of IL-2 in MMI group decreased significantly(P<0.05),while that in HTSJ group decreased significantly(P>0.05).Compared with NC group,the level of IL-4 in serum of mice in GD group decreased significantly(P<0.01);compared with GD group,the level of IL-4 in MMI group and HTSJ group increased,but there was no statistical significance(P>0.05).Compared with NC group,the level of IL-6 in serum of GD group increased significantly(P<0.001);after treatment with MMI and HTSJ,the level of IL-6 decreased significantly(P<0.01).Conclusions:Huatan Sanjie Drug can regulate the gene expression of Thl/Th2 specific transcription factors T-bet and GATA-3,regulate the level of Thl/Th2 secreting cytokines and restore the balance of Th1/Th2 cell expression.Chapter 5 The effect of resolving phlegm and dispersing stagnation on immune tolerance disorder in GD miceObjective:To study the effect of Huatan Sanjie on Treg cell expression,immunosuppression and cytokines secreted by Treg cells in GD mice,and to study the effect of Huatan Sanjie on immune tolerance of GD mice.Methods:(1)The expression of Treg cells was detected by flow cytometry.(2)CD4+CD25+cells and CD4+CD25-cells were co-cultured after magnetic beads were sorted.CCK-8 solution was added to detect the immunosuppressive rate by enzyme label.(3)The contents of IL-10 and TGF-beta were detected by Elisa kit.Results:(1)Compared with NC group,Treg cell content in GD group decreased significantly(P<0.001).Compared with GD group,there was significant difference between MMI group and HTSJ group(P<0.05).(2)When CD4+CD25-was cultured alone,i.e.20000:0,the OD values of each group were basically the same,but when it was co-cultured with CD4+CD25+cells(20000:20000;20000:10000),OD values of each group decreased in varying degrees;when CD4+CD25-and CD4+CD25+were cultured in the number of cells at 20000:20000,the inhibition rate of GD group was significantly lower than that of NC group(P<0.05),but after drug intervention,the inhibition rate of MMI group was significantly lower than that of NC group(P<0.05).Compared with HTSJ group,the immunosuppressive rate in HTSJ group was higher than that in GD group(P<0.05);(3)IL-10 in serum of GD group was significantly lower than that in NC group(P<0.001).After drug intervention,the immunosuppressive rate in MMI group and HTSJ group was higher than that in GD group(P<0.001).Compared with NC group,the serum level of TGF-beta in GD group decreased significantly(P<0.01).Compared with GD group,the serum level of TGF-beta in HTSJ group increased significantly(P<0.05),while that in MMI group decreased significantly(P>0.05).Conclusion:The method of resolving phlegm and dispersing stagnation can maintain the stability of immune tolerance in GD mice.Chapter 6 Effect of resolving phlegm and dispersing stagnation on upstream regulation of immune tolerance disorder in GD miceObjective:To observe the effect of Huatan Sanjie on DNA methylation and Hippo signaling pathway of Foxp3 in spleen of GD mice,and to study the upstream regulation mechanism of Huatan Sanjie on immune tolerance abnormality of GD mice.Methods:(1)CD4+CD25+Treg cells were selected by immunomagnetic bead sorting method.DNA extraction,detection,sulfite treatment,purification and amplification were performed.(2)RT-PCR was used to detect the expression of Hippo signaling pathways Mstl,YAP1 and TAZ in spleen tissues.Results:(1)Compared with NC mice,DNA methylation of Foxp3 in GD mice increased significantly at CpG2,CpG3,CpG4 and CpG6 loci(P<0.05).Compared with GD group,there was no significant change in DNA methylation degree between MMI group and HTSJ group(P>0.05).At CpG6 site,MMI group and HTSJ group had a decreasing trend compared with GD group.There was no significant difference in CpGl,CpG5 and CpG7 loci among groups(P>0.05).(2)Compared with NC group,Mstl expression was down-regulated and YAP and TAZ expression was up-regulated in GD group(P<0.05).Compared with GD group,the relative expression of Mstl was up-regulated in MMI group and HTSJ group,and the expression of YAP and TAZ was down-regulated(P<0.05),The expression of TAZ in spleen of mice in GD group was significantly higher than that in NC group.After drug intervention,the expression of TAZ in spleen of mice in GD group was improved in varying degrees(P<0.01).Conclusion:DNA methylation of Foxp3 may be one of the pathogenic factors of GD mice,but the drugs of resolving phlegm and dispersing stagnation have no obvious effect on its regulation.Phlegm-resolving and resolving drugs can affect the expression of key factors in Hippo signaling pathway,which may be one of the ways of regulating Foxp3 expression.Summary:Huatan Sanjie Drug can regulate the expression of key factors of Hippo signaling pathway(Mstl,YAP,TAZ),increase the expression of Foxp3,enhance Treg secretion and immunosuppressive effect,restore Th1/Th2 balance,improve overall immune disorder,and regulate the balance of antioxidation and oxidation in thyroid tissue,so as to play a therapeutic role in GD mice. |
PDF Full Text Request