| Purpose:This study is based on Professor Zhang LAN’s years of clinical experience to develop a Chinese herbal compound with the effect of soothing the liver and strengthening the spleen--Ruanjian Xiaoying Granules,observe its intervention effect on thyroid autoantibody,thyroid morphology,structure and function of rats eating liver depression and spleen deficiency model,verify the efficacy of Ruanjian Xiaoying granules,and explain the pathogenesis of Hashimoto’s syndrome of liver depression and spleen deficiency with thyroiditis.By observing the effect of Ruanjian Xiaoying Granules on the expression of SOCS1,SOCS3,p-JAK2/JAK2 and p-STAT3/STAT3 proteins in Socs1/3 /JAK-STAT signaling pathway in thyroid tissue of rats with liver depression and spleen deficiency eating model.It is intended to explore the therapeutic mechanism of Ruanjian Xiaoying granules in the treatment of Hashimoto’s thyroiditis from the aspect of immune regulation.Combined with the characteristics of the disease and syndrome of HT,based on the theory of the five elements of traditional Chinese medicine,the relationship between the liver and the spleen and the pathogenesis of the traditional Chinese medicine caused by the dysfunction of the bridge disease were elaborated,providing the theoretical basis for the prevention and treatment of the syndrome of liver depression and spleen deficiency of HT with Ruanjian Xiaoying granules.Material and method:SD grade rats were selected as experimental subjects.40 rats in normal control group(10 rats)and modeling group(30 rats)were divided into model control group(10 rats),Tripterygium wilfordii polyglycoside tablet group(10 rats)and Ruanjian Xiaoying granule group(10 rats)according to random number allocation method.Except for the normal control group,the other rats were fed with high iodine drinking water and subcutaneous thyroglobulin injection to establish the EAT rat model.The EAT rat model was obtained by restraint stress method,excessive fatigue method and diet disjoint method,and the EAT model of liver depression and spleen deficiency was obtained for 8 weeks.(Thyroid peroxidase antibody,thyroid peroxidase antibody,thyroid peroxidase antibody,thyroid peroxidase antibody,thyroid peroxidase antibody,TPOAb)and thyroglobulin antibody(TGAb)levels(the levels of TGAb and TPOAb in the model group are twice or more than those in the normal group)were used to evaluate whether EAT model was successful.The success of the model of liver depression and spleen deficiency was determined by the experiment of rat behavior,sugar and water preference and the growth rate of body weight.Normal control group and model control group were routinely given 2m L distilled water by intragastric administration,Tripterygium wilfordii polyglycoside tablets group was given Tripterygium wilfordii polyglycoside tablets 9.45mg/kg,and Ruanjianxiaoying granules group was given Ruanjianxiaoying granules 16.17g/kg by intragastric administration every day.The intervention time of each group was 8 weeks.After 8weeks of treatment,the rats were killed.Blood was collected from abdominal aorta and thyroid tissue was reserved for examination(the amount of blood collected and the preservation method of thyroid tissue should be explained).Observation indexes and methods: The general condition and HE staining results of thyroid tissue of each group were observed,and the ultrastructure of thyroid tissue was observed under transmission electron microscope.Serum TGAb,TPOAb,Free Triiodothyronine(FT3),Free Thyroxine(FT4)and thyroid stimulating hormone(thyroid)were detected by enzyme-linked immunosorbent assay(ELISA)stimulating hormone(TSH)and Thyrotropin-releasing hormone(TRH)levels,The expression of the SOCS1/3-JAK-STATS related proteins was detected by Western blotting.The protein expressions of TLR3 and SOCS1/3 were detected by immunohistochemistry.Results:1.Comparison of the general conditions of rats in the modeling group and the normal group: no rats died during the experiment,and no abnormalities were observed in the appearance,daily behavior and activities of rats in the normal group after 8 weeks of modeling.All the modeling rats showed irritability in the early stage,strong resistance and struggle during restraint,and rapid avoidance and erect back hair after release of restraint.In the later stage of modeling,the rats gradually showed depressive symptoms from irritability,decreased activity,weakened response to restraint,easy loss of back hair gradually rough,dark yellow skin,lethargy,lethargy,slow growth of body mass,and occasionally loose stools.Difficulty eating,extreme inability to move or even move,inability to hold onto the cage.Compared with the normal group,the sugar water consumption rate and body weight growth rate of rats in the modeling group were significantly decreased(P < 0.05).It is suggested that the rats conform to the symptoms of liver depression and spleen deficiency.2.HE staining results of pathology of rats in each group: thyroid tissue sections were stained by HE.The thyroid structure was normal in the normal group,while the thyroid tissue was destroyed in the model group,with a large number of lymphocytes infiltrating in the follicular cavity and interstitial tissue,and serious interstitial fibrosis.Compared with model group,lymphocyte infiltration in Tripterygium wilfordii group was slightly less,and the degree of interstitial fibrosis was less.The morphological characteristics of Xiaoying granule group were basically the same as that of Tripterygium wilfordii group,but the number of lymphocyte infiltration was slightly less than that of tripterygium wilfordii group,and no interstitial fibrosis was formed.3.Comparison of TPOAb and TGAb levels among all groups: After 8 weeks of intervention,compared with normal group,TGAb and TPOAb levels in model group,Tripterygium Wilfordii group and Ruanjian Xiaoying granule group were increased,and the difference was statistically significant(P<0.05);Compared with model control group,serum TGAb and TPOAb levels in Ruanjianxiaoying granule group and Tripterygium wilfordii polyglycoside tablet group were decreased,and the difference was statistically significant(P<0.05).Compared with tripterygium wilfordii polyglycoside tablet group,serum TGAb and TPOAb levels in Ruanjianxiaoying granule group were lower(P<0.05).These results indicated that the two medication methods could reduce the levels of TGAb and TPOAb,but the decrease of Ruanjian Xiaoying granules was higher than that of tripterygium wilfordii group.4.Thyroid function analysis of rats in each group: Compared with normal group,serum FT3,FT4 and TRH levels of rats in model group,Tripterygium wilfordii group and Ruanjian Xiaoying group were decreased(P<0.05),while TSH level was increased(P<0.05),suggesting abnormal thyroid function in each group.Compared with model group,serum FT3,FT4 and TRH levels of rats in treatment group were increased(P<0.05),while TSH level was decreased(P<0.05).Compared with tripterygium wilfordii group,serum FT3,FT4 and TRH levels of rats in Ruanjian Xiaoying group were increased(P<0.05),while TSH levels were decreased(P<0.05).Thyroid function was improved after intervention of Tripterygium wilfordii and Ruanjian Xiaoying,and the effect of Ruanjian Xiaoying granules was better than that of tripterygium wilfordii group.5.Comparison of inflammatory factors in each group: After 8 weeks of intervention,the levels of IL-6 and TNF-α in the model group were increased compared with the normal group,with statistical significance(P < 0.05);Compared with model control group,serum levels of IL-6 and TNF-α in Ruanjian Xiaoying granule group and Tripterygium wilfordii polyglycoside tablet group were decreased,with statistical significance(P<0.05);Compared with tripterygium wilfordii polyglycoside tablet group,serum levels of IL-6 and TNF-α in Ruanjian Xiaoying granule group were decreased,and the difference was statistically significant(P<0.05).These results suggested that the two drugs could regulate IL-6 and TNF-α cytokines,but the down-regulation range of Ruanjian Xiaoying granules was higher than that of tripterygium wilfordii group.6.The protein expressions of SOCS1,SOCS3,p-JAK2/JAK2 and p-STAT3/STAT3 in thyroid tissues of rats in each group were detected by Western blotting.Compared with normal group,SOCS1 and SOCS3 expressions were decreased and p-Jak2 /JAK2 and p-Stat3 /STAT3 protein expressions were increased in model group,with statistical significance(P < 0.05).Compared with model control group,SOCS1 and SOCS3 were increased in Ruanjian Xiaoying granule group and Tripterygium wilforia polyglycoside tablet group,while protein expressions of p-JAK2/JAK2 and p-Stat3 /STAT3 were decreased,with statistical significance(P<0.05).Compared with tripterygium glycoside tablets group,SOCS1 and SOCS3 expressions were increased and p-JAK2/JAK2 and p-STAT3 /STAT3 protein expressions were decreased in Ruanjian Xiaoying granules group,with statistical significance(P<0.05).These results suggested that both tripterygium wilfordii and Ruanjian Xiaoying granules could improve the symptoms and thyroid function of rats with liver-qi and spleen-deficiency model,which may be related to the regulation of SOCS1/3-JAK-STAT signaling pathway,and the effect of Ruanjian Xiaoying granules was better than that of tripterygium wilfordii group.7.Compared with the normal group,the expressions of TLR3 and SOCS1/3 in the model group were increased,with statistical significance(P < 0.05).Compared with model control group,the expressions of TLR3 and SOCS1/3 in Ruanjian Xiaoying granule group and Tripterygium wilfordii polyglycoside tablet group were decreased,and the difference was statistically significant(P<0.05).Compared with tripterygium wilfordii polyglycoside tablets group,the expressions of TLR3 and SOCS1/3 in Ruanjian Xiaoying granules group were decreased,and the difference was statistically significant(P<0.05).These results indicated that both Ruanjianxiaoying granules and Tripterygium wilfordii polyglycoside tablets could inhibit the expression of TLR3 and SOCS1/3,and the effect of Ruanjianxiaoying granules was better than that of tripterygium wilfordii group.Conclusion:1.The rat model of eating liver depression and spleen deficiency was successfully established through the method of "chronic restraint stress + diet disjoint+ excessive exercise" for 8 weeks by using high iodine water feeding combined with subcutaneous thyroglobulin injection.It laid the foundation for the future study of therapeutic mechanism.2.Ruanjian Xiaoying granules can reduce thyroid autoantibody,reduce thyroid histopathological injury,improve thyroid function,and play a therapeutic role in eating liver depression and spleen deficiency model rats.3.Ruanjian Xiaoying granules can regulate the expression of SOCS1,SOCS3,p-JAK2/JAK2 and p-STAT3/STAT3 proteins in socs1/3 /JAK-STAT signaling pathway,and down-regulate the path-related cytokines such as IL-6 and TNF-α,suggesting that Ruanjian Xiaoying granules can regulate the signaling pathway.Reduce thyroid tissue damage by inhibiting inflammatory response.The JAK/STAT signaling pathway is expected to provide a new target for the treatment of HT and a new intervention method for clinical treatment.4.Ruanjian Xiaoying granules can inhibit the expression of TLR3,SOCS1 and SOCS3,suggesting that they may activate the innate immune response by regulating the expression level of Socs1/3-TLR3 in the early stage of the disease.Thus,it plays a protective role in thyroid tissue. |
PDF Full Text Request