Correlation Between TCM Syndromes And Cellular Immunity In Graves’ Disease And Effect Of Chinese Medicine Of Reducing Phlegm And Resolving Masses On T Cell Subsets In The GD Mouse Model

BackgoundGraves’disease (GD) is an autoimmune disease, accounting for about90%of hyperthyroidism, its incidence has increased year by year, which is significantly high among women. GD has high metabolic syndrome and thyroid enlargement as the main feature, there are also problems about high recurrence rate, long-term medication, side effects, unclear drug withdrawal syndrome, which seriously affected the normal life of GD patients.It is generally considered that external environment, the genetic and immune interactions determine the incidence of GD. In recent years, immunological mechanisms of GD is attracted more and more attention and to study the relationship between GD and T cell subsets with regulatory factors become hot. T cells and its regulatory factors involved in the immune response and produce thyroid stimulating hormone receptor antibody (TRAb) and other auto-antibodies, resulting in the occurrence and development of GD. Clinical treatment for Graves’disease mainly contains anti-thyroid drug (ATD) treatment,131I treatment and surgical treatment. The anti-thyroid drug therapy is the most commonly used method, but there’s much side effect, and GD is easy to relapse. However, Chinese medicine treatment of Graves disease has a unique advantage, can reduce the thyroid drug dosage and reduce thyroid drug side effects. Traditional Chinese medicine believes that phlegm is not only an important cause of GD, through the thyroid enlargement, but also the basis for GD syndrome. Combined with other causes, they can form different types of syndrome. Therefore the method of Reducing Phlegm and Resolving Masses is essential to the Chinese medicine treatment of GD, can improve thyroid enlargement in patients with GD.In earlier stage, the experiment studied the mechanism of Chinese medicine of Reducing Phlegm and Resolving Masses in the treatment of GD, indicating Chinese medicine of Reducing Phlegm and Resolving Masses can inhibit thyroid cell proliferation, promote its apoptosis, affect the expression of Bax. Bcl-2and finally improve the dynamic balance between proliferation/apoptosis in thyroid cells. However, there is no exploration for the correlation between T cell-mediated cellular immunity and TCM syndrome, and not much study on the intervention mechanism of Chinese medicine of Reducing Phlegm and Resolving Masses on GD cellular immunity, which severely limited the widely application of traditional Chinese medicine. Therefore, Graves’ disease is the core in the whole study, and in clinical study we take the TCM syndromes as the foothold, the cellular immunity as the breakthrough point; in animal experiment we established the GD model with Ad-TSHR289virus to immune mice as the carrier, Chinese medicine of Reducing Phlegm and Resolving Masses as intervention measure, T cell subsets as object, to explore the correlation between GD and TCM syndrome, and the impact mechanism of Chinese medicine of Reducing Phlegm and Resolving Masses on the T cell subsets, which had certain clinical significance and application value.Objective1.To investigate the correlation in patients with GD between syndrome and cellular immunity.2.To investigate the effect of traditional Chinese medicine of Reducing Phlegm and Resolving Masses on T cell subsets in the spleen of Graves mouse model.Method1.Collecting30patients with Graves’disease, including16cases of thyroid dysfunction,14cases normal thyroid function, and20cases of healthy adults as normal control group from the outpatient department of Endocrinology in the Guang’anmen Hospital of China Academy of Chinese Medical Sciences. Compare the differences between groups of thyroid function, symptoms and signs, immunological indexes (the ratio of CD4,CD8,Treg,Th17,the Foxp3, RORyt expression of mRNA,IL-10,IL-17). Analyze the correlation between the immunological indexes, between symptoms and the indexes of immunology by Multiple Linear Regression.2. The PCR fusion gene was plasmid and vector by Gateway Technology, adenovirus packaging used in this experiment, successfully construct of Ad-TSHR289recombinant adenovirus. Preparation of Graves disease animal model by adenovirus immunity of mice, select mice model intervention delivery, detect their T4, TRAb and T cell subsets (CD4,CD8, Treg, Th17cells ratio) of the spleen, analyse the effect of Chinese medicine of Reducing Phlegm and Resolving Masses on T cell subsets.Result1. The characteristics of TCM Syndromes of GD patientsAssociated with the symptoms and signs of TCM of the collected30GD patients, summarized qi stagnation and phlegm syndrome in13cases (43.3%), heat in the heart and liver syndrome in10cases (33.3%), yin deficiency inner heat syndrome in7cases (23.3%).2. The immunological indexes in each group2.1CD4,CD8, Treg,Th17cell ratio:Compare the CD4,CD8,Treg cell ratio of the normal group, the GD normal thyroid function group and GD abnormal thyroid function group, P>0.05, there’s no statistical difference. Compare the percentage of Th17cells, the GD normal thyroid function group (3.1029±0.48392) and GD abnormal thyroid function group (3.5663±0.31239) were higher than that of the normal group(2.247±0.30051), P<0.01, GD abnormal thyroid function group was higher than that in GD normal thyroid function group, P<0.05, with significant difference.2.2Foxp3, RORyt mRNA expression levels:Compare the Foxp3, RORyt mRNA expression levels of the normal group, the GD normal thyroid function group and GD abnormal thyroid function group, P>0.05, there’s no statistical difference.2.3IL-10, IL-17levels:Compare the IL-10, IL-17of the normal group, the GD normal thyroid function group and GD abnormal thyroid function group, P>0.05, there’s no statistical difference.3. The correlation between TCM Syndromes and the index of Immunology in GD patientsThe qi stagnation and phlegm syndrome of GD patients and the immunol indexes of CD4,CD8, Treg, Th17, Foxp3, RORyt, IL-10, IL-17showed no obvious correlation, P>0.05, there’s no statistical difference; the heat in the heart and liver syndrome had negative correlation with CD8cell ratio, β=-0.509, P=0.008<0.05, with statistical difference, but there’s no correlation between the heart and liver syndrome and CD4, Treg, Th17, Foxp3, RORγ t, IL-10, IL-17, P>0.05, there’s no statistical difference; the yin deficiency inner heat syndrome of GD patients had positive correlation with CDg cell ratio, β=0.440, P=0.039<0.05, with statistical difference, but there’s no correlation between the yin deficiency inner heat syndrome and CD4, Treg, Th17, Foxp3, RORγt, IL-10, IL-17, P>0.05, ther’s no statistical difference.4. Animal experimental model rate reached61.7%.5. Effect of Chinese medicine of Reducing Phlegm and Resolving Masses on T4, TRAb levelsAfter drug intervention, the T4level of large dose Chinese medicine group (95.72±20.86), western medicine group (99.12±19.39) were lower than that of model group (145.18±20.53), P<0.01,the difference was statistically significant; Chinese medicine small dose group (124.21±19.61) was lower than that of model group,P<0.05, there’s statistical difference.After drug intervention, the large dose Chinese medicine group (15.69±7.43), western medicine group (18.25±8.30) TRAb were lower than that of model group (45.37±15.31), P<0.01, the difference was statistically significant; Chinese medicine small dose group (32.11±10.81) was lower than that of model group, P<0.05, there’s statistical difference.6. Effect of Chinese Medicine of Reducing Phlegm and Resolving Masses on CD4, CD8, Treg, Th17cell ratioAfter drug intervention, compare the CD4,CD8, Treg, Th17cell ratio in the spleen of mouse model of each group, P>0.05, there’s no statistical difference.Conclusion1. Analysing the correlations between TCM syndromes and cellular immunology, it indicated that the heat in the heart and liver syndrome had negative correlation with CD8cell ratio, the yin deficiency inner heat syndrome had positve correlation with CD8cell ratio, but there’s no correlation among the both syndrome and CD4, Treg, Th17, Foxp3, RORγt, IL-10, IL-17.The qi stagnation and phlegm syndrome of GD patients and the immunol indexes mentioned above showed no obvious correlation.2. Immuning BALB/c mice with Ad-TSHR289recombinant adenovirus could successfully establish mouse model of GD, which had clinical feature similarity, the model rate reached61.7%3. Reducing Phlegm and Resolving Masses could effectively reduce the levels of thyroid hormones and TRAb, it might inhibit thyroid hormone synthesis or secretion.4.Reducing Phlegm and Resolving Masses had no obvious effect on T cell subsets, but may indirectly regulate cellular immune, which need further study.