| Purpose:By combinating zoopery and clinical trial,subjective and objective evidence,also with theoretical analysis,we observed the thyroid and systemic effects of Chaihu Ruanjian Xiaoying granules on subclinical HT patients with thyroid nodules and Liver stagnation and spleen deficiency syndrome.Furthermore,the animal experiments were conducted to explore the therapeutic mechanism of Chaihu Ruanjian Xiaoying granules to balance Th1/Th2 and related T lymphocyte subsets and inflammatory factors by T-bet/GATA3 axis in regulating immunological inflammatory response.In addition,we explored the promoting effect of the pathogenesis of liver depression and spleen deficiency on thyroid nodules occurrence in subclinical HT patients,as well as the influence of thyroid changes on immune homeostasis,based on the theory"dimple corresponding".We proposed that the thyroid treated with the whole body-softening hardness and dispersing mass,soothing liver and invigorating spleen could eliminate the negative influence between the thyroid and the whole body of HT patients.To provide a solid theoretical basis and scientific,safe,effective drugs for clinical diagnosis and treatment of HT.Material and method:Clinical trial:The cases were selected from the patients with Hashimoto disease accompanied by thyroid nodules in the outpatient department of our hospital,who dialectically belonged to the syndrome of liver depression and spleen deficiency.The 54patients with thyroid nodules indicated by thyroid color doppler ultrasound,laboratory examination showed that thyroid anti-autoantibody increased and thyroid function were normal,were given Chaihu Ruanjian Xiaoying granules combined with lifestyle intervention for 3 months.Chaihu Ruanjian Xiaoying granules were given 3 times a day and 2 bags each time,fully brewed with boiling water and taken orally.Baseline data,liver and kidney function and safety indicators,TCM symptom score,five items of thyroid function,degree of goiter,maximum diameter of the largest thyroid nodule were observed and recorded at the beginning and end stage of the treatment course.Th1,Th2,Th17 and Treg,CXCL10,CXCR3,IFN-γand TNF-αof serum were detected by ELISA method.The correlation between the observations and the curative effect of thyroid nodules accomplished with HT was investigated.The indicators were used to evaluate the efficacy and preliminary regulatory mechanism of Chaihu Ruanjian Xiaoying granules.Animal experiment:48 SPF female SD rats weighing between 182-214g were selected for the experiment,adaptively fed for 7 days firstly,and then randomly divided into normal group(12 rats)and model group(36 rats),and fed by natural feeding method for normal group.At the same time,the model group was made model by combining with"heterologous thyroid antigen induction+high-iodine water combined with diet disorder+continuous restraint stress+swimming fatigue",and the compound modeling method was used to replicate the EAT model similar to clinical syndrome with liver depression and spleen deficiency.At the end of the 8th week,the disease models were evaluated.Then,the successful disease models were divided into model group,Jinshuibao tablet group and Chaihu Ruanjian Xiaoying granule group for further treatment of 8 weeks:the model group was given10ml/kg intragastric distilled water daily according to the weight as the normal group;Jinshuibao tablet group was given 0.45g/kg/d intragastric Jinshuibao tablet solvent;Chaihu Ruanjian Xiaoying granule group was given 12.86g/kg/d intragastric Chaihu Ruanjian Xiaoying granule solvent.After treatment,the rats in each group were treated with:1.determining the thyroid antibodies by ELISA method,TGAb,TPOAb as effect indexes.2.observing the pathological damage of thyroid tissue section under the light microscope.3.determining the expression of T-bet,GATA3,Foxp3 and RORγt in thyroid tissues by WB method.4.determining the expression of Th1,Th2,Th17,Treg,IL-1,2,4,6,10,12,IFN-γand TNF-αby ELISA method.5.determining the expression of CD3~+,CD4~+,CD3~+CD4~+,CD4~+CD28~+and CD4~+CD28~-in thymocytes by flow cytometry.Theoretical study:To explore the promoting effect of the pathogenesis of liver depression and spleen deficiency on thyroid nodule occurrence in subclinical stage of HT,and the influence of thyroid changes on immune homeostasis.at the same time,we point out the thought for treatment"dimple corresponding"-softening hardness and dispersing mass,soothing liver and invigorating spleen.Results:Clinical trial:(1)Efficacy of TCM:the TCM score before treatment was 10.65±2.96,and TCM score after treatment was 4.35±1.98,the contrast showed significant difference(P<0.01),the total efficiency was 94.44%;It showed significant difference in each single TCM score before and after treatment(P<0.01).It is suggested that Chaihu Ruanjian Xiaoying granules could significantly improve the symptoms of liver depression and spleen deficiency.(2)Comparison of FT3,FT4 and TSH:there were no significant differences in the three items of thyroid function between HT patients before and after treatment(P>0.05).(3)Efficacy of TGAb and TPOAb titer reduction:After treatment,TGAB and TPOAB titer declined significantly,and the total was 77.78%.(4)Efficacy of thyroid nodules:after treatment,the maximum diameter of the largest thyroid nodule accomplished with HT was significantly reduced compared with that before treatment(P<0.01),and the total efficiency was 81.48%.It is suggested that Chaihu Ruanjian Xiaoying granules could shrink the nodules accomplished with HT.(5)Curative effect of goitre:after treatment,7 patients had no goitre,and no case withⅢdegree goitre,the curative effect of goitre was significant(P<0.01).It is suggested that Chaihu Ruanjian Xiaoying granules could alleviate goitre.(6)Elisa results of serum:after treatment,Th1,Th17,TNF-α,IFN-γ,CXCL10,CXCR3,Th1/Th2,Th17/Treg were significantly declined(P<0.01),while Th2 and Treg were significantly increased(P<0.01).These results suggested that Chaihu Ruanjian Xiaoying granules could balance T lymphocyte differentiation and reduce inflammatory response effectively.(7)Correlation analysis between thyroid nodule and different indicators before treatment:the maximum diameter of the largest thyroid nodule was significantly correlated with Th1,CXCL10,Th1/Treg,TGAb and TCM score of 54 patients before treatment(P<0.05),and the correlations were positive(R>0).Other indicators showed no significant correlation with the maximum diameter of the largest thyroid nodule before treatment(P>0.05).(8)Comparison of indicators between different therapeutic groups of thyroid nodule:there were significant differences in Th1,CXCL10,TGAb and TCM score between the effective and ineffective groups(P<0.05).Th1,CXCL10,TGAb and TCM score in the ineffective group were higher than those in the effective group.There were no significant differences among the other indicators(P>0.05).Binary Logistic regression results showed that Th1 and TGAb levels were significantly positively correlated with of thyroid nodule(P<0.05),and they were risk factors of treatment failure(OR>1).That is,the higher serum Th1 and TGAb levels may result in the higher probability of treatment failure.While CXCL10 and TCM score showed no significant correlation with curative effect(P>0.05).(9)Safety:There were no significant abnormal changes in blood and urine routine,liver and kidney functions and ECG indicators before and after treatment,and no patients complained of adverse reactions during the course of treatment.Animal experiment:(1)General state:The fur was smooth and glossy of the rats in the normal group.The rats in normal group were good activity,they were in good sprits,and ate normally,while with no abnormal bowel movement.After combined induction,the rats in the modeling groupstruggled intensely in the early stage,and showed obvious resistance in the bound barrel,when they came out of the barrel,the obvious resistance wasn’t disappear,they presentedconfrontation state continuously.In the late stage,the changes occurred obviously,the struggle response of the rats was weakened significantly,and their fur was withered,yellow and brittle,their activity was reduced,they were in poor sprits,and ate less,the defecation showed loose stools.After treatment with Jinshuibao tablet and Chaihu Ruanjian Xiaoying granules,theappearance and behavior of the rats were significantly improved.(2)Five items of thyroid function:compared with the normal group,the titers of serum TGAb and TPOAb in the modeling rats were increased,and more than doubled that in the normalgroup,which showed significant differences(P<0.01),and TSH in the modeling rats was reduced(P<0.01),while there were no significant differences in FT3 and FT4 between the two groups(P>0.05).The result indicated that the subclinical hyperthyroidism modeling was successful completed.After the modeling,the test results of the modeling rats showed that the titers of the antibodies in 2 of the modeling group were less than doubled mean of the normal group,thus the 2 rats were eliminated.After intervention,FT3 and FT4 in the 4 groups were no significant differences.Compared with the normal group,the titers of the antibodies inmodel group were more than doubled that in the normal group,and TSH was reduced,which significantly increased(P<0.01).Compared with the model group,the titer of antibodies in Jinshuibao tablet and Chaihu Ruanjian Xiaoying granule groups was significantly decreased,and TSH was increased(P<0.01),while the titers of Chaihu Ruanjian Xiaoying granulegroup were improved more obviously than that in Jinshuibao tablet group(P<0.01).(3)The corresponding indicators of liver depression and spleen deficiency:compared with the normal group,the total length of 5min motion route,the number of punctures in the central area,retention time,body weight and food intake of the model group were significantly less than those of the normal group(P<0.05).The fecal moisture content in this group wassignificantly higher than that in the normal group(P<0.01).(4)Two rats classified as unmodeled were eliminated,and the modeling rate reached 94.44%.Then 34 model rats were randomly divided into the model group(12),the Chaihu Ruanjian Xiaoying Granule group(11)and the Jinshuibao tablet group(11).During the intervention,one rat in the model group struggled during intragastrical administration,resulted in death of the drug solution into the lung,thus the final model group included 11 experimentalspecimens.(5)Thyroid weight index:compared with the normal group,the thyroid weight index of themodel group was increased,and the difference was significant(P<0.01).The result indicated the inflammatory hyperplasia occured in the thyroids of the rats in the model group.Afterintervention,inflammatory hyperplasia was in remission in both Jinshuibao tablet and Chaihu Ruanjian Xiaoying Granule groups(P<0.01),and the improvement was more obvious inChaihu Ruanjian Xiaoying Granule group(P<0.01).(6)Pathology of thyroid:in the normal group,the configurations of thyroid were not damaged,the tissue of thyroid was uniform and regular,the follicle cavity and epithelial cells had regular configuration and morphology,and there was no obvious abnormality and inflammatory infiltration in the follicle cavity.In the model group,compared with the normal group,the configurations of thyroid were incomplete,the tissue of thyroid was destroyed,the morphology of follicle was irregular,the glial components in the follicular lumen were reduced,and a large number of inflammatory infiltrates appeared inside and outside the follicular lumen.Compared with the model group,the configurations of thyroid of jinshuibao tablet group were relatively intact,the destruction of thyroid tissue and the morphological changes of follicles were not obvious,the nuclear infiltration inside and outside the follicle cavity was relatively decreased,and the inflammatory infiltration was relatively attenuated.Compared with the Jinshuibao tablet group,the configurations of thyroid and the destruction of thyroid tissue of the Chaihu Ruanjian Xiaoying granule group were similar to that of the Jinshuibao tablet group,both of which were better than that of the model group.The nuclear infiltration inside and outside the follicle cavity was attenuated compared with that of the Jinshuibao tablet group,and the inflammatory infiltration was attenuated as well.(7)Expression of thymocyte:compared with the normal group,the expression of CD3~+,CD4~+,CD3~+CD4~+and CD4~+CD28~-cells in the thymus of rats in the other 3 groups was increased,while the expression of CD4~+CD28~+cells was decreased,with significant difference(P<0.01).Compared with the model group,the expression of CD3~+,CD3~+CD4~+and CD4~+CD28~-cells in Chaihu Ruanjian Xiaoying granule and Jinshuibao groups was decreased,and the expression of CD4~+CD28~+cells was increased,the difference was significant(P<0.01),the expression of CD4~+cells in Chaihu Ruanjian Xiaoying granule group was decreased(P<0.05),and the expression of CD4~+cells in Jinshuibao tablet group was decreased,but with no significant difference.Compared with jinshuibao tablet group,the expression of CD3~+,CD3~+CD4~+and CD4~+CD28~-cells in Chaihu Ruanjian Xiaoying granule group was decreased,and the expression of CD4~+CD28~+cells was increased,with significant difference(P<0.01).The expression of CD4~+cells in Chaihu Ruanjian Xiaoying granule group was decreased,but with no significant difference compared with that in Jinshuibao tablet group.It is suggested that Chaihu Ruanjian Xiaoying granules could decrease the expression of CD3~+,CD4~+,CD3~+CD4~+,CD4~+CD28~-and increase the expression of CD4~+CD28~+,and the effect of Chaihu Ruanjian Xiaoying granules is better than that of Jinshuibao tablets.(8)Expression of protein in thyroid:compared with the normal group,the expression of GATA3 and Foxp3 in model and Jinshuibao tablet groups were significantly decreased,while the expression of T-bet and RORγT was significantly increased(P<0.01),the expression of RORγT in Chaihu Ruanjian Xiaoying granule group was increased(P<0.01),while the expression of T-bet,GATA3 and Foxp3 was not significantly different from that in normal group.Compared with the model group,the expression of GATA3 and Foxp3 in Jinshuibao tablet group and Chaihu Ruanjian Xiaoying granule groups was significantly increased,while the expression of T-BET and RORγT were significantly decreased(P<0.01).Compared with Jinshuibao tablet group,the expression of GATA3 and Foxp3 in Chaihu Ruanjian Xiaoying Granule group was significantly increased,while the expression of T-bet and RORγT was significantly decreased(P<0.01).It is suggested that Chaihu Ruanjian Xiaoying granules could down-regulate the expression of T-bet and RORγT and up-regulate the expression of GATA3 and Foxp3,while the effect is better than that of Jinshuibao tablets.(9)Elisa results of serum:compared with the normal group,the expression of Th1,Th17,Th1/Th2,Th17/Treg,IL-1,2,6,12,IFN-γand TNF-αin model group was significantly increased,while the expression of Th2,Treg,IL-4 and 10 was significantly decreased(P<0.01).Compared with the model group,the expression of Th2,Treg,IL-4 and 10 was significantly increased in Jinshuibao tablet and Chaihu Ruanjian Xiaoying Granule groups,while the expression of Th1,Th17,Th1/Th2,Th17/Treg,IL-1,2,6,12,IFN-γand TNF-αwas significantly decreased(P<0.01).Compared with Jinshuibao tablet group,the expression of Th2,Treg,IL-4 and 10 was significantly increased in Chaihu Ruanjian Xiaoying Granule group,while the expression of Th1,Th17,Th1/Th2,Th17/Treg,IL-1,2,6,12,IFN-γand TNF-αwas significantly decreased(P<0.01).It is suggested that Chaihu Ruanjian Xiaoying granules could regulate the balance of T lymphocyte subsets and related cytokines,while the effect is better than that of Jinshuibao tablets.Theoretical study:(1)The essential meaning of the concept"dimple corresponding"and its extended ideological core of dialectical unity of TCM.(2)To explore the pathogenesis of subclinical HT based on the theory"dimple cor-responding".(3)The theoretical core of treating Hashimoto’s disease based on"dimple corresponding"smoothing liver and invigorating spleen,softening hardness and dispersing mass.Conclusion:1.Chaihu Ruanjian Xiaoying granules could balance T lymphocyte subsets and regulate related cytokines,improve the clinical symptoms of Hashimoto’s thyroiditis with liver depression and spleen deficiency significantly,as well as decrease the titer of thyroid-related antibodies and reduce the TCM syndrome score,shrink the nodules accomplished with HT,and alleviate goitre,with safety and effectiveness.2.By increasing the expression of GATA3 and Foxp3,reducing the expression of T-BET and RORγT,balancing the T lymphocyte subsets and regulating related cytokines,Chaihu Ruanjian Xiaoying granules could regulate the immune response and reduce the inflammatory response,improve the symptoms of rats with liver depression and spleen deficiency,as well as alleviate the inflammatory swelling and pathological morphological changes of thyroid.3.Liver depression and spleen deficiency(the disorder of"Ben")is the main cause of sub-clinical HT,resulting in"dimple"disease,at the same time,the injury of thyroid(dimple)could deteriorate the disorder of"Ben".Therefore,we proposed that regulating"dimple"together with"Ben"corresponding is an effective way to treat Hashimoto’s disease. |
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