| Purpose:This study includes three parts:clinical study,animal experiment and theoretical discussion.Clinical research is a part of a multicenter,randomized controlled study on the subclinical treatment of chronic lymphocytic thyroiditis with modified Xiaoyaosan,a traditional Chinese medicine compound with the effect of soothing the liver and strengthening the spleen developed by Professor Zhang Lan,Objective to evaluate the efficacy of Xiaoyaosan in the treatment of subclinical Hashimoto's thyroiditis,and to establish eat model of liver depression and spleen deficiency through animal experiments.To explore the mechanism of Xiaoyaosan from the perspective of mi R326 regulating Th17 cell differentiation.In the theoretical part,based on the concept of unity of spirit and form,it is proposed that disharmony of spirit and form,that is,liver depression and spleen deficiency,is the core of pathogenesis of subclinical stage of Hashimoto's thyroiditis.To provide scientific basis and therapeutic drugs for the subclinical treatment of Hashimoto's disease,and enrich the guiding role of TCM disease prevention theory in clinical application.method:1 clinical research:the clinical research of this subject adopts the random,positive drug control method,according to the subclinical stage of HT,Hashimoto's thyroiditis patients with liver depression and spleen deficiency and kidney deficiency syndrome,they are randomly divided into the treatment group and the control group,The treatment group was given Xiaoyaosan Jiawei granules,two bags(about 15g each),three times a day;The control group was given Jinshuibao tablets,4 tablets at a time,3 times a day.The course of treatment in both groups was 12 weeks.First of all,through the two groups of clinical trial data,the effect of Modified Xiaoyao Powder on the decrease rate of thyroid autoantibody titer and TCM symptom index of HT patients in subclinical stage was observed,and the curative effect of Modified Xiaoyao Powder on Hashimoto's thyroiditis of liver depression and spleen deficiency type was evaluated;Secondly,the multiple regression relationship between TGAb titer(dependent variable)of 110 patients before treatment and age,BMI,T cell subsets and related cytokines,blood cell cluster differentiation antigen and total score of TCM symptoms was studied,and the correlation was analyzed;Finally,according to the efficacy criteria of thyroid double antibody titer reduction rate,the patients with TGAb or TPOAb titer reduction rate?30%were regarded as effective cases and were classified into effective case group,while the patients with TGAb or TPOAb titer reduction rate<30%were regarded as invalid cases and were classified into invalid case group.The differences of each index between the two groups before treatment were compared.In order to further test the correlation between the different observation indexes and whether they are effective or not,and seek the relationship between them,we choose binary logistic regression model,take whether they are effective or not as the dependent variable(invalid assignment is 1,effective is 0),and take the different observation indexes as the independent variable for binary logistic regression analysis.Logistic regression analysis was used to analyze the factors influencing the curative effect of Xiaoyao Powder in the treatment of HT with liver depression and spleen deficiency.2 animal experiment:the rat model of eat was induced by high iodine induction combined with heterologous thyroid antigen immunization with SPF Rats as carrier.On the basis of eat rat model,the rat model of liver depression and spleen deficiency syndrome of traditional Chinese medicine was reproduced by the method of chronic restraint stress+excessive fatigue+diet disorder.The rat model of liver depression and spleen deficiency syndrome of eat was established,Compared with normal SPF Rats(normal group),the changes of thyroid tissue in each group were observed by HE staining.The levels of FT3,FT4,TSH,TGAb and TPOAb in peripheral blood were measured by enzyme-linked immunosorbent assay.The levels of mir326,IL-4 m RNA,IL-17 m RNA and IFN in thyroid tissue were detected by RT-PCR-?Western blot was used to detect the expression level of Ets-1 protein in thyroid tissue,and flow cytometry was used to detect CD4~+IFN in spleen tissue-?The secretion of T cells,CD4~+IL-4 T cells and CD4~+IL-17 T cells.All the experimental data were statistically processed by spss.22 software.Theoretical discussion:according to the pathogenesis core of liver depression and spleen deficiency or kidney deficiency in subclinical stage of HT,this paper tries to expound the theoretical connotation of treating subclinical stage of Hashimoto's disease from the perspective of"unity of spirit and form"and modern medicine,so as to provide new ideas for clinical prevention and treatment of HT.Results:1 clinical trial results:(1)Before treatment,there was no significant difference in gender,age,BMI and other indicators between the two groups(P>0.05),that is,the baseline data of the two groups were consistent and comparable.(2)Comparison of FT3,FT4 and TSH in peripheral blood before and after drug treatment between the treatment group and the control group:there was no significant difference in serum FT3,FT4 and TSH between the two groups after treatment(P>0.05);There was no significant difference in serum TGAb and TPOAb between the two groups(P>0.05).(3)Comparison of TGAb,TPOAb in peripheral blood of treatment group and control group before and after drug treatment and the decrease range:in the two groups,the serum TGAb and TPOAb decreased significantly after treatment(P<0.01);Compared with the control group,serum TGAb and TPOAb in the treatment group decreased significantly(P<0.01).Compared with the control group,the decrease of serum TGAb in the treatment group was significant(P<0.01),while the decrease of serum TPOAb in the treatment group was equivalent to that in the control group(P>0.05).(4)Statistical comparison of single TCM symptom score and total TCM symptom score between the treatment group and the control group before and after drug treatment:there was no significant difference in single TCM symptom score and total TCM symptom score between the two groups before treatment(P>0.05),which was comparable.After treatment,the clinical symptoms such as goiter,fatigue,chest fullness,restlessness,loose stool,tongue and pulse,and the total score of TCM symptoms in the treatment group were significantly improved compared with those before treatment(P<0.01);In the control group,the single symptoms were improved except for general fatigue,and the difference was statistically significant(P<0.01 or P<0.05).Compared with the control group,the improvement degree of single TCM symptom score and total TCM symptom score of the treatment group were significantly improved(P<0.01),which had statistical significance.(5)Comparison of TGAb,TPOAb titer decline rate and curative effect judgment results:the total effective rate of the treatment group was 74.55%,and that of the control group was52.73%,and the total effective rate of the treatment group was significantly higher than that of the control group(P<0.05).(6)The comparison of curative effect evaluation results of single TCM symptom between the two groups can be seen from table 3-2.The curative effect of the treatment group in improving"loose stool"and"tongue image"of liver depression and spleen deficiency is different from that of the control group,and the difference is statistically significant(P<0.05);The effective rates of improving the symptoms of goiter,fatigue,chest fullness,restlessness and pulse condition were 81.82%,89.09%,78.18%,87.27%and 85.45%respectively,which were significantly higher than 65.22%,52.17%,50.00%,64.71%,64.00%and 71.43%in the control group(P<0.01).(7)Comparison of comprehensive curative effect evaluation results of TCM symptoms between the two groups:table 3-3 shows that the overall effective rate of the treatment group is 93.36%,and that of the control group is 30.91%.By chi square test,the total effective rate of TCM symptom score of the treatment group is significantly higher than that of the control group(P<0.01).It is suggested that Modified Xiaoyao Powder is better than Jinshuibao tablet in improving TCM symptoms.(8)Multiple linear regression analysis of serum TGAb before treatment:there was correlation between serum TGAb titer and Th17,CD4~+T cell percentage,CD16~+CD56~+NK cell percentage,CD19~+B cell percentage,TCM symptom score(P<0.05)There was a positive correlation between the percentage of CD19~+B cells and TGAb before treatment(r>0,P<0.05),and a negative correlation between the percentage of CD16~+CD56~+NK cells and TGAb before treatment(r<0,P<0.05).(9)Comparison of indicators before treatment among different therapeutic groups of 55patients in the treatment group:comparison of indicators among different therapeutic groups of 55 patients in the treatment group,there were significant differences in Th17,CD4~+T cell percentage,CD16~+CD56~+NK cell percentage and TCM symptom score between the two groups of effective cases and ineffective cases(P<0.05)The TCM symptom score was significantly higher than that in the effective group,and the percentage of CD16~+CD56~+NK cells in the ineffective group was significantly lower than that in the effective group.There was no significant difference in other indexes between the two groups(P>0.05).Logistic regression results showed that there was a significant positive correlation between the serum Th17 expression level and no curative effect in the treatment group(P<0.05);There was a negative correlation between the percentage of CD16~+CD56~+NK cells and the efficacy(P<0.05).Both of them were independent risk factors(or>1).The higher the serum Th17 level and the lower the percentage of CD16~+CD56~+NK cells,the higher the probability of failure after treatment.There was no significant correlation between the percentage of CD19~+B cells and the score of medical symptoms and the curative effect(P>0.05).(10)Adverse reaction events:in the clinical observation of the treatment group and the control group,ECG,blood and urine routine,alanine aminotransferase,aspartate aminotransferase,serum creatinine and other liver and kidney function indexes were not abnormal before and after treatment,and there were no adverse events in the two groups.2 animal experiment results(1)Successful establishment of eat model:after 8 weeks of modeling,the expression level of TSH in rat serum was significantly lower than that in normal group(P<0.01),indicating that rats were in subclinical hyperthyroidism stage;The expression levels of TGAb and TPOAb were significantly increased,and the increased level was 2 times higher than that of the normal group,which indicated that eat model was successful.The thyroid autoantibodies in 3of 36 rats were less than 2 times of the normal group,so they were eliminated.There was no significant difference in the expression of FT3 and FT4 between the model group and the normal group(2)The spleen deficiency model rats were successfully constructed:compared with the normal group,the growth rate of body weight in the model group was significantly decreased(P<0.01).The food intake decreased significantly(P<0.01)and the fecal moisture content increased significantly(P<0.01),suggesting that the rats had spleen deficiency symptoms.(3)Successful construction of liver depression model rats:after 8 weeks of modeling,compared with the normal group,there were significant differences in the total movement distance and the number of central punctures of the model rats in 5 minutes(P<0.01);There was no significant difference in the total distance and residence time of the central region between the model group and the normal group(P>0.05),but the total distance of the central region in the model group was lower than that in the normal group,and the residence time was longer than that in the normal group.(4)The above results showed that 33 rats successfully established eat model of liver depression and spleen deficiency,and the modeling rate was 91.67%.Furthermore,33 eat rats were randomly divided into model group,Xiaoyaosan group and Jinshuibao group.Five rats died in the drug intervention stage,including 3 rats in the modified Xiaoyaosan group and 2rats in the Jinshuibao tablet group.Autopsy of the dead rats showed that there was no obvious abnormality in other tissues except for residual drugs in the lungs.After 8 weeks,the follow-up study was carried out.(5)The general behavior of rats in each group was observed.The normal group had normal food and drinking water,bright hair color,good mental state,sensitive action,deep stool color,plenty of granules and no abnormal appearance and behavior;The rats in the model group were irritable,irritable,struggling to resist and accompanied by hissing before the model was established.After the shackles were released,they quickly avoided and their back hair was erect.In the model group,the response to external stimulation was slow,the duration of struggle was significantly reduced,the rats liked to gather together,and accompanied with symptoms such as laziness,squinting,tiredness,dull hair color,less food,light and soft stool color,smaller particles,and even loose stools.After intragastric administration,the general condition of rats in Xiaoyaosan modified group and Jinshuibao group was significantly better than that in model group,such as eating condition,mood,hair color and a series of appearance behaviors.(6)After 8 weeks of treatment,there was no significant difference in the expression levels of FT3 and FT4 in serum among the groups.Compared with the normal group,the expression of serum TSH in the model group was significantly decreased(P<0.01).After drug treatment,the expression level of serum TSH in the Xiaoyaosan group was significantly increased,which was statistically significant compared with the model group,but there was no significant change in the expression level of serum TSH in the Jinshuibao group,The expression of serum TSH in Xiaoyaosan group was significantly higher than that in Jinshuibao group,with statistical significance.Compared with the normal group,the expression levels of serum TGAb and TPOAb in the model group were significantly increased(P<0.01);After treatment with Xiaoyaosan or Jinshuibao,the expression levels of serum TGAb and TPOAb were significantly decreased compared with the model group(P<0.01),and the expression levels of serum TGAb and TPOAb in Xiaoyaosan group were not statistically significant compared with Jinshuibao group,but there was a downward trend.The above results showed that modified Xiaoyaosan could significantly increase the expression level of TSH in rats,and significantly reduce the levels of TGAb and TPOAb,and the therapeutic effect was better than Jinshuibao tablets.(7)After 8 weeks of drug treatment,the expression of mir326,IL-4,IL-17 and IFN-?in thyroid tissue of rats in each group were detected The expression of mir326 and IL-17 m RNA in the model group were significantly higher than those in the normal group(P<0.01).The expression of mir326 and IL-17 m RNA in Xiaoyaosan group were significantly lower than those in Jinshuibao group.The levels of IL-4 and IFN-?in thyroid tissue of rats in each group were higher than those in the control group.There was no significant difference in m RNA expression.The above results showed that modified Xiaoyaosan could significantly reduce the expression of mir326 and IL-17 m RNA,and the efficacy of modified Xiaoyaosan was significantly better than that of Jinshuibao tablets.(8)T lymphocyte abnormality is one of the causes of autoimmune diseases.Autoimmune effector T cells include Th1,Th2 and Th17 cells,which secrete cytokines IFN-?,IL-4 and IL-17.In order to determine whether modified Xiaoyaosan can affect effector T cells,we measured the proportion of Th1,Th2 and Th17 cells in rat spleen.CD4~+IFN-~+T cells,CD4~+IL-4~+T cells and CD4~+IL-7~+T cells represent Th1 cells,Th2 cells and Th17 cells,respectively.The proportion of Th1 and Th17 cells in spleen tissue of model group was significantly higher than that of normal group,and the difference was statistically significant(P<0.01),which was also consistent with the increase of IL-17 m RNA level,while IFN-?There was no difference in m RNA level among the groups.There was no difference in the proportion of Th2 cells among the groups,which was consistent with the expression level of IL-4 m RNA among the groups.These results suggest that Th17 cells may be involved in the pathogenesis of Hashimoto's thyroiditis.After treatment with Jinshuibao,the proportion of Th1 cells decreased,but there was no statistical significance.However,the proportion of Th1and Th17 cells in spleen tissue of rats treated with modified Xiaoyaosan decreased significantly(P<0.01),and the proportion of Th17 cells in Xiaoyaosan group was significantly lower than that in Jinshuibao group(P<0.01).The above results show that Modified Xiaoyao Powder can treat Hashimoto's thyroiditis by regulating Th17 cells,and its regulatory effect on Th17 cells is significantly better than that of Jinshuibao tablets.(9)Western blot was used to detect the expression of Ets-1 protein in the thyroid of the four groups.Compared with the normal group,the expression of Ets-1 protein in the model group was significantly down regulated(P<0.01).Compared with Jinshuibao group,the expression of Ets-1 protein in Xiaoyaosan group was significantly increased(P<0.01).These results indicate that modified Xiaoyaosan can up regulate the expression level of Ets-1,the downstream target protein of mi R326.(10)In order to detect the effect of modified Xiaoyaosan on thyroid pathological morphology of eat rats with liver depression and spleen deficiency,thyroid tissue sections of four groups of rats were stained with he.In the normal group,the thyroid structure was intact,the size and shape of follicles were regular,and there was no obvious lymphatic infiltration;In the model group,the thyroid tissue structure was incomplete,a large number of follicles atrophied,and a large number of lymphocytes infiltrated into the follicular cavity and interstitial tissue;In the modified Xiaoyaosan group,the follicular cavity of thyroid tissue was enlarged,part of the follicular structure was destroyed,and a small amount of lymphocytes infiltrated in and around the follicular cavity,which was lighter than that in the model group;The histological structure of thyroid gland in Jinshuibao tablet group was incomplete,the pathological morphology was similar to that in Xiaoyaosan group,and the lymphocyte infiltration degree in Jinshuibao tablet group was more severe than that in Xiaoyaosan group.These results indicate that modified Xiaoyaosan can reduce the inflammatory reaction of thyroid tissue in eat rats with liver depression and spleen deficiency,and has protective effect on thyroid tissue.3.Theoretical discussion(1)The origin and development of the meaning of"Shen-xing";(2)The relationship between disharmony of body and spirit and stagnation of liver qi and deficiency of spleen;(3)The theoretical connotation of treating Hashimoto's disease from the perspective of liver,spleen and kidney.Conclusion:1 Modified Xiaoyao Powder can effectively reduce the titer of thyroid related autoimmune antibody in HT patients,reduce the TCM symptom score of HT patients with liver depression and spleen deficiency,and has significant effect on TCM syndrome.The higher the serum TGAb titer of 110 patients before treatment,the higher the expression level of Th17 cells,the percentage of CD4~+T cells,the percentage of CD19~+B cells,and the lower the percentage of CD16~+CD56~+NK cells.The expression level of Th17 in peripheral blood and the percentage of CD16~+CD56~+NK cells are independent predictors of the therapeutic effect of Xiaoyaosan on HT patients with liver depression and spleen deficiency.The results showed that Xiaoyaosan had no effect on the blood and urine routine,liver and kidney function and ECG indexes of HT patients,and there were no obvious abnormal reactions and serious adverse events after taking the medicine,which indicated that Xiaoyaosan,a Chinese medicine compound used in early clinical differentiation and intervention of HT,had good effectiveness and safety.Xiaoyaosan Jiawei powder can inhibit the differentiation of Th17 cells,reduce the expression of IL-17 m RNA,reduce the inflammatory reaction,and ultimately have a protective effect on thyroid tissue by reducing the expression of mir326 and up regulating the expression of Ets-1in thyroid tissue of eat rats with liver depression and spleen deficiency.3"spirit"disorder is involved in the pathogenesis of HT,and may be the potential cause of liver qi stagnation in HT patients.Deficiency of spleen and kidney leads to"shape"injury,which may be the basis of HT.Finally,from the perspective of soothing the liver,strengthening the spleen and tonifying the kidney,this paper interprets the theoretical connotation of"the unity of spirit and form". |
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