| BackgroundHashimoto’s Thyroiditis(HT,also known as Hashimoto’s disease),also known as chronic lymphocytic thyroiditis(CLT),is a common type of autoimmune thyroiditis(AIT).In recent years,with the increase of people’s iodine intake and the change of environmental factors such as the the pace of life has increased,the incidence of HT has gradually increased.Most HT patients lack clinical manifestations in the early stage and are at risk of developing permanent hypothyroidism,thyroid nodules or even thyroid cancer,which seriously affects their quality of life and long-term prognosis.The pathogenesis of HT is complex.At present,there is no specific treatment in clinic.Thyroid hormone replacement therapy is mainly adopted according to the patients’ thyroid function.Most people with normal thyroid function and only related antibodies are still in the passive observation stage.Traditional Chinese medicine(TCM)has accumulated some experience in treating HT.Moreover,TCM is effective in syndrome differentiation,relieving symptoms,regulating immunity and improving thyroid function,which is worthy of further study.However,at present,TCM syndrome differentiation of HT lacks unity,and there is no suitable algorithm in the field to explore the associated rules between HT syndromes and clinical characteristics.In addition,the mechanism of effective TCM prescription for HT is not clear.Therefore,this study relies on the National Natural Science Foundation(No.82074412)to explore the syndrome diagnosis model of HT,and to study the curative effect and mechanism of TCM in treating HT through animal experiments,so as to provide reference and scientific basis for the diagnosis and treatment of HT in TCM.Purposes1.The XGBoost algorithm was employed to construct the HT syndrome diagnosis model and explore the characteristic variables of syndrome classification.2.Network pharmacology was employed to explore the effective components,targets and mechanisms of effective TCM prescription Xiapuxiaoying recipe(XPXY recipe)in treating HT.3.Animal experiments were employed to observe the effect and mechanism of the XPXY recipe in treating HT.Methods1.141 HT patients in the case group and 236 non-HT people in the control group were included.The information such as indexes of thyroid function,thyroid ultrasonic examination,symptoms and tongue pulse were collected,and the TCM syndrome diagnosis model of HT was constructed by XGBoost algorithm,and the performance of the model was evaluated by accuracy,variable importance ranking and confusion matrix.2.The effective components of XPXY recipe were obtained by TCMSP,and the information was standardized by Uniprot protein database,and the HT disease-related genes were collected by GeneCards,OMIM,DrugBank and PharmGkb databases.Taking the drug-disease intersection gene,the protein interaction network was constructed by STRING,and the potential targets of XPXY recipe for HT were screened according to topological parameters.The compound-target network was constructed by Cytoscape 3.7.2.GO function and KEGG pathway enrichment were analyzed by DAVID to obtain potential pathways.3.54 female SPF SD rats aged 6-8 weeks were randomly divided into normal group(9)and model group(45).The model group was immunized with porcine thyroglobulin mixed with Freund’s adjuvant and fed with high iodine.After model establishment,they were divided into model group,high-dose group of XPXY recipe(17.24g/Kg/d),middle-dose group of XPXY recipe(8.62g/Kg/d),low-dose group of XPXY recipe(4.31g/Kg/d)and tripterygium wilfordii polyglycoside group(6.35mg/Kg/d)by gavage.After 8-week intervention,the animals were killed and samples were kept.The effects of XPXY recipe on the morphology,structure and function of thyroid gland in rats were observed by means of serum thyroid function test,HE staining and transmission electron microscope.RT-PCR,Western Blot,chemical fluorescence and ELISA were used to explore whether XPXY recipe affected the thyroid gland by regulating the NOX4/ROS/NF-κB pathway mediated by miR-423-5p in rats.Results1.① Analysis of the clinical features of the case group and the control group shows that there are differences in gender,levels of TPOAb,TGAb,TSH,FT4,TRAb,left thyroid lobe width,left thyroid lobe thickness,right thyroid lobe thickness,thyroid isthmus thickness and neck lymph node size between the two groups(P<0.05);In the case group,the most frequent subjective symptoms are fatigue,dry mouth,insomnia and dreaminess,chills of limbs and loose stool,and the most frequent single diagnosis contents of tongue and pulse information are pale red,normal tongue body,thin tongue coating,normal sublingual vein and pulse string;Comparing the clinical characteristics of patients with liver depression and spleen deficiency syndrome,qi stagnation and phlegm obstruction syndrome,heart-liver fire flourishing syndrome,phlegm-blood stasis syndrome,qi-yin deficiency syndrome and spleen-kidney yang deficiency of syndrome in the case group,the above-mentioned content indicated that TSH level is distinguishable among different syndrome types(P<0.05),but there is no correlation between the state of thyroid function(normal thyroid function,clinical hypothyroidism,subclinical hyperthyroidism,subclinical hyperthyroidism)and syndrome types(P<0.05)②XGBoost algorithm was used for feature selection,and the output of variable importance ranking of syndrome classification were TPOAb,lack of breath and laziness,TGAb,edema of lower limbs,pale complexion,thirst,abdominal distension,loose stool,soreness of waist and knees,foreign body sensation in the throat,dizziness,excessive phlegm,constipation,left thyroid lobe width,emaciation,the deterioration and remission of the disease with mood,chills and cold limbs,and fatigue.The accuracy rate of HT syndrome diagnosis model established by XGBoost algorithm is 0.99,the accuracy rate of non-HT diagnosis is 1.00,the recall rate is 1.00,and the F1 value is 1.00.The accuracy rate,recall rate and F1 value of the diagnosis of HT with liver stagnation and spleen deficiency syndrome were 1.00,1.00 and 1.00 respectively.The accuracy rate=1.00,the recall rate=0.83,and the F1 value=0.91 in the diagnosis of HT syndrome of qi stagnation and phlegm stagnation.The accuracy rate=0.97,the recall rate=1.00,and the F1 value=0.98 in the diagnosis of HT with hyperactivity of heart and liver fire.The accuracy rate,recall rate and F1 value of the diagnosis of HT with phlegm and blood stasis syndrome were 1.00,1.00 and 1.00 respectively.The diagnostic accuracy of HT deficiency of both Qi and Yin is 0.98,the recall rate is 1.00,and the F1 value is 0.99.The accuracy rate,recall rate,and F1 value of the diagnosis of deficiency of spleen and kidney yang in HT were 1.00,1.00 and 1.00,respectively.The overall model has high accuracy and good performance,and there is a strong causal logic between the clinical characteristics included in the study and the final syndrome diagnosis.2.①The core effective components of XPXY recipe in treating HT include β-sitosterol,baicalin,baicalein,quercitrin,isorhamnetin etc.,which can play the roles of antioxidation,anti-inflammatory and immunity.② The main target of XPXY recipe in treating HT may be involved in immunoregulation,Pyroptosis and inflammatory reaction by regulating PTGS2,ESR1,ESR2,CASP8,STAT3,BCL2 and IL1B.③ At present,XPXY recipe mainly acts on Th17 cell diffrentiation pathway,IL-17 signaling pathway,Toll-like receptor signaling pathway,etc.,affecting biological processes such as reactive oxygen species metabolism and lipopolysaccharide reaction,involving cell components such as membrane rafts and membrane micro-regions,and affecting molecular functions such as cytokine receptor binding and heme binding.3.① Experimental autoimmune thyroiditis rats(EAT)model fed with pig thyroglobulin mixed immune adjuvant and high iodine were successfully established,and the serum levels of TPO-Ab,TG-Ab,TSH,FT3 and FT4 in the model group were significantly higher than those in the normal group(P<0.01);Compared with the model group,the serum levels of TPO-Ab,TG-Ab,TSH,FT3 and FT4 in the high-dose group,the middle-dose group and the tripterygium wilfordii polyglycoside group were all decreased(P<0.05).The levels of serum FT3,TPO-Ab,TG-Ab,TSH and FT4 in the low-dose group decreased(P<0.05),but there was no significant difference between them and the model group.In terms of pathological morphology HE staining showed that thyroid follicles in model group were destroyed,more lymphocytes infiltrated in glands,and more fibrous hyperplasia and necrotic cells could be seen in stroma.The results of electron microscope showed that the structure and organelles of thyroid follicular cells in the model group were destroyed,revealing the morphological characteristics of pyroptosis.After the intervention of XPXY recipe and tripterygium wilfordii polyglycoside,the thyroid injury and pyroptosis of thyroid follicular cells in rats were improved in varying degrees.② The mRNA expression of GSDMD in thyroid in model group was up-regulated compared with that in normal rats(P<0.05),while that in high-dose group was down-regulated compared with that in model group(P<0.01).The mRNA expression of GSDMD in thyroid in middle-dose group,low-dose group and tripterygium wilfordii polyglycoside group was down-regulated compared with that in model group,but there was no statistical difference among them.The protein expression of thyroid GSDMD in model group was up-regulated compared with that in normal rats(P<0.01),and the protein expression of thyroid GSDMD in each intervention group was down-regulated compared with that in model group(P<0.05).The mRNA expression of thyroid NLRP3 in model group was up-regulated compared with that in normal rats(P<0.01),and the mRNA expression of thyroid NLRP3 in high-dose group was down-regulated compared with that in model group(P<0.05).The mRNA expression of thyroid NLRP3 in middle-dose group,low-dose group and tripterygium wilfordii polyglycoside group was down-regulated compared with that in model group,but the difference among them was not statistically significant.The protein expression of thyroid NLRP3 in model group was up-regulated compared with that in normal rats(P<0.01),while the protein expression of thyroid NLRP3 in high dose group,middle dose group and tripterygium wilfordii polyglycoside group was down-regulated compared with that in model group(P<0.01),and the protein expression of thyroid NLRP3 in low dose group was down-regulated compared with that in model group,but the difference among them was not statistically significant.The mRNA expression of Caspase-1 in thyroid gland of rats in model group was up-regulated compared with that of normal rats(P<0.01),while the mRNA expression of Caspase-1 in thyroid gland of rats in high dose,middle dose and tripterygium wilfordii poly glycoside group was down-regulated compared with that in model group(P<0.01),and the mRNA expression of Caspase-1 in thyroid gland of rats in low dose group was down-regulated compared with that in model group,but there was no statistical difference among them.The protein expression of Caspase-1 in thyroid gland of rats in model group was up-regulated compared with that of normal rats(P<0.01),and the protein expression of Caspase-1 in thyroid gland of rats in each intervention group was down-regulated compared with that in model group(P<0.01).The mRNA expression of thyroid ASC in model group was up-regulated compared with that in normal rats,but the difference was not statistically significant.The mRNA expression of thyroid ASC in high-dose group was down-regulated compared with that in model group(P<0.05),and the mRNA expression of thyroid ASC in middle-dose group,low-dose group and tripterygium wilfordii polyglycoside group was down-regulated compared with that in model group,but the contrast among them was not statistically significant.Compared with normal rats,the protein expression of thyroid ASC in model group was up-regulated(P<0.01),and the protein expression of thyroid ASC in each intervention group was down-regulated(P<0.01).The mRNA expression of IL-18 in thyroid gland of rats in model group was up-regulated compared with that of normal rats(P<0.01),and the mRNA expression of IL-18 in thyroid gland of rats in each intervention group was down-regulated compared with that in model group(P<0.01).The protein expression of thyroid IL-18 in model group was up-regulated compared with that in normal rats(P<0.01),and the protein of thyroid IL-18 in all intervention groups was down-regulated compared with that in model group(P<0.01).There was no statistical difference in serum IL-18 level between the model group and the normal group,and there was no statistical difference in IL-18 level between the intervention groups compared with the model group.The mRNA expression of IL-1β in thyroid gland of rats in model group was up-regulated compared with that in normal rats,but the difference was not statistically significant.The mRNA expression of IL-1β in thyroid gland of rats in each intervention group was down-regulated compared with that in model group,but the difference was not statistically significant.The protein expression of IL-1β in thyroid gland of rats in model group was up-regulated compared with that of normal rats(P<0.05),and the protein expression of IL-1β in thyroid gland of rats in each intervention group was down-regulated compared with that in model group,but the difference was not statistically significant.The level of serum IL-1β in the model group was higher than that in the normal group(P<0.01),and the levels of serum IL-1β in the high-dose group,the middle-dose group and the tripterygium wilfordii polyglycoside group were all lower than those in the model group(P<0.05),while the levels of serum IL-1β in the low-dose group were all lower than those in the model group,but the difference was not statistically significant.③The expression level of miR-423-5p in thyroid tissue of rats in model group was lower than that in normal group(P<0.05),and the expression level of miR-423-5p in thyroid tissue of all intervention groups was up-regulated compared with that in model group,but the difference was not statistically significant.The mRNA expression of NOX4 in thyroid gland of rats in model group was up-regulated compared with that in normal rats(P<0.01),and the mRNA expression of NOX4 in thyroid gland of rats in each intervention group was down-regulated compared with that in model group(P<0.01).The protein expression of NOX4 in thyroid gland of rats in model group was up-regulated compared with that in normal rats(P<0.01),while the protein expression of NOX4 in thyroid gland of rats in high dose group,middle dose group and tripterygium wilfordii polyglycoside group was down-regulated compared with that in model group(P<0.01).The protein expression of NOX4 in thyroid gland of rats in low dose group was down-regulated compared with that in model group,but the difference was not statistically significant.The content of ROS in thyroid gland of model rats was significantly higher than that of normal rats(P<0.01).Compared with the model group,the content of thyroid ROS in each intervention group decreased significantly(P<0.01),and the content of thyroid ROS in the low-dose group decreased,but there was no statistical difference compared with the model group.The mRNA expression of NF-κB in thyroid gland of rats in model group was up-regulated compared with that of normal rats(P<0.01),and the mRNA expression of NF-κB in thyroid gland of rats in high dose group was down-regulated compared with that in model group(P<0.05).The mRNA expression of NF-κB in thyroid gland of rats in middle dose group,low dose group and tripterygium wilfordii polyglycoside group was down-regulated compared with that in model group,but the difference was not statistically significant.The protein expression of NF-κB in thyroid gland of model rats was up-regulated compared with that of normal rats(P<0.01),and the protein expression of NF-κB in thyroid gland of rats in each intervention group was down-regulated compared with that of model rats(P<0.01).Conclusions1.There are similarities and differences between the subjective and objective clinical characteristics of patients in different syndromes of HT.XGBoost algorithm can obtain the characteristic variables and objective rules of TCM syndrome classification,and establish an accurate and interpretable diagnostic model of HT syndrome types.The model of this study ranked the important characteristics of HT syndrome differentiation(top 5):TPOAb,lack of qi and laziness,TGAb,edema of lower limbs,and pale complexion.We can focus on the above characteristics in clinical practice and give corresponding diagnosis and treatment measures.2.XPXY recipe is a multi-component,target,and synergistic channel therapy for HT,and its interventional mechanism mainly involves active oxygen metabolism,inflammatory reaction,immune regulation,and cell death.3.XPXY recipe can effectively improve thyroid function and ameliorate thyroid autoantibody level in EAT rats,reduce thyroid pathological damage and protect thyroid follicular cells.XPXY recipe can up-regulate the expression of miR-423-5p in thyroid gland of EAT rats and negatively regulate NOX4/ROS/NF-κB signaling pathway,thus handling the Pyroptosis induced by NLRP3 Inflammasome and the subsequent inflammatory response,and reducing the expressions of NLRP3,Caspase-1,ASC,GSDMD and inflammatory factors IL-18 and IL-1β in EAT rats. |
PDF Full Text Request