| Objective:The aim of the research is to study the thyroid function and ultrasonic features of Hashimoto's thyroiditis patients with different TCM syndromes;according to the methods of supplementing Qi and nourishing Yin,promoting blood circulation and removing stasis,softening and strengthening and dispersing stasis,Qilong Sanjie prescription(QLSJ)was established,and the effect and mechanism of QLSJ in the treatment of experimental autoimmune thyroiditis(EAT)rats were studied.Methods:1.Study on the clinical characteristics of different TCM syndromes of Hashimoto's thyroiditis:73 cases of Hashimoto's thyroiditis were involved and divided into different syndrome by retrospective analysis.The thickness of lateral lobe and isthmic portion,echo characteristics of thyroid parenchyma,thyroid function,TPOAb and Tg Ab levels in serum were analyzed between different TCM syndromes.2.Based on the methods of replenishing qi and nourishing Yin,promoting blood circulation and removing stasis,softening and strengthening and dispersing knots,qlsj formula was established,which consists of Astragalus mongholicus Bunge(30g),ginseng(20g),Sparganii Rhizoma(15g),Curcumae Rhizoma(15g),Coix lacryma-jobi var.ma-yuen(Rom.Caill.)Stapf(15g),Ligustrum lucidum(20g),20g Polygonum multiflorum(20g),Glycyrrhiza glabra L.(6g),etc.3.Study on the effect of Qilong Sanjie prescription(QLSJ)in the treatment of EAT rats:88 female Lewis rats were divided into control group(NC)and model group and fed adaptively for one week.From the second week,the experimental autoimmune thyroiditis(EAT)rat model was established by feeding 0.064%high iodine water and subcutaneous injection of thyroglobulin.At the end of the 11th week,thyroid ultrasonography and the expression of TPOAb,Tg Ab,FT3 and FT4 in serum of all rats were used to determine the success of eat model.At the 12th week,the rats in the model group were divided into model+distilled water group(EAT+WATER),model+Qilong Sanjie low dose group(EAT+QLSJ-L),model+Qilong Sanjie medium dose group(EAT+QLSJ-M),model+Qilong Sanjie high dose group(EAT+QLSJ-H).From the 13th week,rats in EAT+WATER group were intragastrically given 2ml distilled water once a day for 12 weeks;rats in EAT+QLSJ-L group,EAT+QLSJ-M group and EAT+QLSJ-H group were treated with QLSJ at the doses of 0.15 g/Kg·d,0.30 g/Kg·d and 0.60 g/Kg·d once a day for 12 weeks;thyroid ultrasonography were performed and the expression of TPOAb,Tg Ab,FT3 and FT4 in serum were detected before killed.4.The thyroid tissues of rats were weighed and the pathological changes of thyroid tissues were observed by HE staining.5.Predict the mechanism of QLSJ in the treatment of autoimmune thyroiditis by network pharmacology.6.The expressions of CD4+IFN-?+(Th1),CD4+IL-4+(Th2),CD4+IL-17A+(Th17)and CD4+CD25+Foxp3+(Treg)in peripheral blood mononuclear cells(PBMC)in NC group?EAT+WATER group and EAT+QLSJ-H group were detected by flow cytometry.7.The expressions of serum TNF-?,IL-6,IL-17A,TGF-?1 and IL-10 in NC group,EAT+WATER group and EAT+QLSJ-H group were detected by ELISA.Results:1.Clinical data characteristics of patients with Hashimoto's thyroiditis:a total of 73patients with Hashimoto's thyroiditis were included.(1)the high incidence age of Hashimoto's thyroiditis was 40-70 years old,and the female prevalence rate was higher.(2)Hashimoto's thyroiditis is mainly divided into five types:Qi stagnation and phlegm stagnation type,phlegm stagnation and blood stasis type,liver depression and spleen deficiency type,Qi and yin deficiency type and spleen and kidney yang deficiency type.(3)The thickness of the left and right thyroid lobes and isthmus of the five syndrome types were different,the thicknesses of the phlegm stagnation and blood stasis type and Qi Yin deficiency type were the largest,while the thicknesses of the qi stagnation and phlegm obstruction type,liver stagnation and spleen deficiency type and spleen kidney yang deficiency type were smaller.(4)The ultrasonic echo of thyroid parenchyma of five types of syndromes were mainly diffuse hypoechoic.The patients with phlegm and blood stasis type were mainly diffuse hypoechoic with wireless hyperechoic.The patients with spleen and kidney yang deficiency type were mainly diffuse hypoechoic with wired hyperechoic.(5)There were differences in the type of thyroid function among the five syndrome types.The thyroid function in Qi stagnation and phlegm stagnation and blood stasis group was mainly normal,the thyroid function in Qi Yin deficiency group was mainly hyperthyroidism,the thyroid function in liver stagnation and spleen deficiency and spleen kidney yang deficiency group was mainly hypothyroidism.(6)There was no statistical difference in the titer of TPOAb and Tg Ab among the five TCM syndromes(P>0.05).2.Evaluation of EAT rat model:compared with NC group,the levels of serum FT3,FT4,Tg Ab and thyroid volume of each model group were increased before treatment(P<0.01)at the end of the 11th week,indicating that the EAT model was successfully established.There was no significant difference in serum levels of TPOAb level between the model groups and the NC group(P>0.05).3.The effect of QLSJ on the treatment of EATThe levels of serum TGAb and thyroid volume in EAT+QLSJ-M and EAT+QLSJ-H groups were lower than those before treatment.The levels of TGAb in EAT+QLSJ-M and EAT+QLSJ-H groups were not changed.There were no changes in serum FT3,FT4 and TPOAb before and after intragastric administration.QLSJ can reduce the relative weight of thyroid in a dose-dependent manner,and the effect of high dose on reducing thyroid index is more significant.In NC group,thyroid follicles were well distributed,and there was no inflammatory cell infiltration between the follicles.In EAT+WATER group,a large number of thyroid follicles were destroyed,the follicular space was enlarged,the shape of the follicles was irregular,and a large number of inflammatory cells were infiltrated in the follicular cavity and stroma,indicating the success of modeling.In EAT+QLSJ-L group,a large number of thyroid follicles were destroyed,and a large number of lymphocytes infiltrated into the follicular cavity and stroma;In EAT+QLSJ-M and EAT+QLSJ-H groups,the infiltration of thyroid lymphocytes was significantly reduced.4.Network pharmacology analysis results:QLSJ,a multi-component and multi-targettraditional Chinese decoction,may play a therapeutic role by regulating immunity in the treatment of EAT.5.The expression of Th1,Th2,Th17 and Treg lymphocyte subsets in rat PBMCs:the expression of Th1 in EAT+WATER group was significantly higher than that in NC group,while the expression of Treg was decreased;The level of Th1 in EAT+QLSJ-H group was significantly decreased,while the level of Treg was increased.6.The serum levels of TNF-??IL-6?IFN-??IL-17A were significantly higher than that of NC group(P<0.01),The expression of TGF-?1?IL-10 decreased significantly(P<0.01);after treatment,the expression of TNF-??IL-6?IFN-??IL-17A was significantly down regulated(P<0.01 or P<0.05);The expression of TGF-?1?IL-10 increased significantly(P<0.05)?Conclusion:1.The patients with phlegm and blood stasis,Qi and yin deficiency accounted for 32.9%and 21.9%respectively.The thickness of lateral lobe and isthmus of thyroid gland in these two types of patients was relatively large,which indicates that the methods of Supplementing Qi and nourishing Yin,activating blood circulation and removing stasis,softening hardness and dispersing knot are of great significance in the treatment of HT,especially in reducing the volume of thyroid gland and preventing further changes of pathogenesis.2.QLSJ decoction was established according to the methods of replenishing qi and nourishing Yin,activating blood circulation and removing stasis,softening hard and dispersing knot.QLSJ(0.15 g/Kg·d,0.30 g/Kg·d and 0.60 g/Kg·d)could reduce thyroid volume and relative weight in a dose-dependent manner.0.30 g/Kg·d and 0.60 g/Kg·d could significantly reduce serum Tg Ab level and reduce thyroid inflammatory cell infiltration in eat rats.3.QLSJ(0.60 g/Kg·d)can regulate the levels of Th1 and Treg lymphocyte subsets in rat spleen and PBMCs,and the levels of TNF-?,IL-6,IL-17A,TGF-?1,IL-10 in serum.It play an important role in immune regulation and anti-inflammatory.4.The methods of supplementing Qi and nourishing Yin,promoting blood circulation and removing stasis,softening and strengthening and dispersing stasis have therapeutic and immunomodulatory effects on eat rat model. |
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