Study On The Immunomodulatory Mechanism Of The Method Of Dispersing Phlegm And Dispersing Nodules In Inhibiting Thyroid Angiogenesis In Graves' Diseas

Graves’ disease(GD)is a common clinical autoimmune disease characterized by diffuse goiter and hyperthyroidism.The tutor believed that the basic pathogenesis of GD was"phlegm and fire condensation" in front of the neck.He established the basic treatment method of "resolving phlegm and dispersing stasis,clearing heat and eliminating gall" with Prunella vulgaris,Fritillaria et al.as the core drugs,and successively led four National Natural Science Foundation Projects to study the mechanism of resolving phlegm and dispersing stasis in the treatment of GD.This study was funded by the National Natural Science Foundation of China "Effect of resolving phlegm and Dispersing stasis on immune tolerance switching mechanism and modification of mice with Graves’ disease "(No.81774293)and"To explore the mechanism of eliminating phlegm and dispersing stasis in the treatment of Graves’ disease from the perspective of primary cilia regulating the synthesis and decomposition of thyroglobulin"(No.82004337).Firstly,from the clinical study of thyroid vascularization and TCM syndrome characteristics of GD patients,the animal experiment takes the GD mouse model constructed by ad TSHR as the carrier to study the regulatory mechanism and effective components of Method of eliminating phlegm and dispersing stasis on GD thyroid angiogenesis and immune tolerance disorder.Objective1.To study the clinical significance of the elevated degree of vascularization of GD thyroid and the internal relationship between it and the pathological factors of TRADITIONAL Chinese medicine,and to explore the mechanism,immune regulation mechanism and main effective components of inhibiting GD thyroid angiogenesis by resolving phlegm and dispersing stasis method.2.To reveal the theoretical connotation of resolving phlegm and dispersing stasis to inhibit GD thyroid angiogenesis.Methods1.Clinical research(1)Subjects:GD patients treated in the endocrinology Department of xxxx from June 2021 to March 2022 were selected as the observation group,and were divided into normal thyroid function group(GDn)and abnormal thyroid function group(GDa)according to thyroid function,and normal thyroid ultrasound was selected as the control group.(2)Clinical data:General data were collected,TCM syndrome and symptoms questionnaire was filled in,and five thyroid functions(TSH,FT3,FT4,T3,T4)and TRAb were detected.The degree of goitre(TV,isthm thickness)and degree of vascularization(STA-PSV,TVI)were evaluated by thyroid ultrasound,and serum VEGF level was detected by Elisa.(3)Data analysis:the degree of goiter,vascularization,VEGF level and TCM syndromes(divided into heart and liver fire hyperactivity syndrome,yin deficiency and yang hyperactivity syndrome and qi stagnation and phlegm coagulation syndrome)were compared among the groups;The correlation between the degree of thyroid vascularization and thyroid function,TRAb and TV in the observation group was analyzed;The degree of thyroid vascularization in patients with TCM syndromes was compared.2.Experimental research(1)Preparation of GD mouse model:construct ad TSHR recombinant adenovirus and detect the infectivity;Sixty BALB/C female mice were randomly divided into 45 in the model group and 1 5 in the normal control(NC)group.The model group was intramuscularly injected with adenovirus 2.0 by one-time immunization × 109pfu/piece;Four weeks after immunization,the content of serum T3 after dilution(1:3)was detected by chemiluminescence,and the model was identified.(2)Group administration and materials:the model group was randomly divided into three groups(n=15):model(GD)group,methimazole(MMI)group and Huatan Sanjie(HTSJ)group.The intervention began 7 weeks after the model was established.The MMI group was given methimazole suspension of 5.2 mg/kg/day,the HTSJ group was given Huatan Sanjie traditional Chinese medicine(Xiakucao,Xuanshen and Fritillaria)Decoction of 7.02 g/kg/day,and the NC group and Gd group were given equal volume pure water;After 4 weeks of intervention,after anesthesia,the eyeballs were removed for blood,the thyroid and spleen tissues were stripped,and treated according to different experimental requirements for detection.(3)Thyroid function,TRAb detection and morphological observation:the contents of T4 and TRAb in serum were detected by radioimmunoassay;The shape and size of thyroid gland were observed directly,and the shape of thyroid gland was observed by HE staining.(4)Detection of thyroid angiogenesis,permeability and regulatory pathway:CD31 immunohistochemical staining was used to count microvessel density(MVD)and observe the morphology;CD34+FGG immunofluorescence double staining was used to detect the permeability;The localization and expression of VEGFA in thyroid were detected by immunohistochemical staining,and the protein expressions of VEGFA,VEGFR2,Akt and p-Akt were detected by Western blot.(5)Detection of immune tolerance disorder and regulatory molecules:observe the appearance of spleen and measure the spleen index;The ratio of CD4+T cells to Treg and the expression of PD-1 and ICOS on Treg were detected by flow cytometry;Spleen Foxp3,IL-10 and TGF-β1 were detected by qRT-PCR.MRNA expression of CTLA-4;The level of serum IL-17A was detected by liquid suspension chip.(6)Chemical component analysis and molecular docking of Huatan Sanjie traditional Chinese medicine:UPLC-q-exactive orbitrap-MS method was used to detect the main chemical components of Huatan Sanjie traditional Chinese medicine(Prunella vulgaris,Scrophularia Scrophularia and Fritillaria thunbergii);Molecular docking was conducted with the possible targets(VEGFA,VEGFR2,AKT11 AKT2,Foxp3,CTLA4,PDCD1,ICOS,IL10,TGFB1 and IL1 7A)of traditional Chinese medicine for resolving phlegm and dispersing nodules in the treatment of Gd in this study to evaluate the binding force.Results1.Clinical research(1)General data:121 cases in the observation group(71 cases in the GDa group and 50 cases in the GDn group)were collected,and the incidence ratio of male to female was 1:5.72;Average age:39.07±10.90 years;Control group:46 cases;The general data of each group were comparable(P>0.05).(2)Degree of goitre:Compared with the control group,TV and isthmus thickness,full shape and proportion of enlargement in GDa group and GDn group were significantly increased(P<0.05,P<0.01);Compared with THE GDn group,the proportion of full thyroid morphology in THE GDa group was higher(P<0.05),and there was no statistical difference.(3)Degree of thyroid vascularization:TVI of control group,GDn group and GDa group were 1.77(1.17-2.65),9.99(5.29-15.54)and 13.08(8.75-19.88),respectively.Sta-psv were 39.20(32.90-44.30)cm/s,72.30(51.20-89.50)cm/s and 95.95(78.10-122.50)cm/s,respectively.Compared with the control group,TVI and STA-PSV in GDn and GDa groups were significantly increased(P<0.05,P<0.01);Compared with GDn group,TVI and STA-PSV in GDa group were significantly increased(P<0.05,P<0.01).(4)Serum VEGF level:Compared with the control group,there was no statistical difference in serum VEGF level in the observation group(ALL P>0.05).(5)Distribution of TCM syndromes:GDn group:Yin deficiency Yang hyperactivity syndrome(44.68%)>heart and liver fire flourishing syndrome(31.91%)>Qi stagnation spittoon coagulation syndrome(23.40%);GDa group:heart and liver fire flourishing syndrome(57.50%)>Yin deficiency Yang hyperactivity syndrome(31.50%)>Qi stagnation spittoon coagulation syndrome(10.00%).(6)Correlation between degree of vascularization and THYROID function,TRAb and TV:TVI in GDa group was positively correlated with FT3,T3,TV and STA-PSV(P<0.05),the correlation coefficients were 0.239,0.268,0.353 and 0.453,respectively,especially with TV and STA-PSV(P<0.01);STA-PSV was positively correlated with FT3,FT4,T3,T4,TRAb,TV and isthmic thickness(P<0.05),the correlation coefficients were 0.373,0.323,0.352,0.276,0.242,0.494 and 0.298,respectively,especially with FT3,FT4,T3 and TV(P<0.01);TVI and STA-PSV were negatively correlated with TSH(P>0.05).TVI was positively correlated with TSH and STA-PSV in GDn group(P<0.05),the correlation coefficients were 0.295 and 0.609,especially with STA-PSV(P<0.01);STA-PSV was positively correlated with TSH and TV(P<0.05),the correlation coefficients were 0.341,0.621 and 0.537,respectively,and the correlation with TV was strong(P<0.01);STA-PSV was negatively correlated with FT4 in GDn group(P<0.05),and the correlation coefficient was-0.391.(7)The degree of thyroid vascularization of each TCM syndrome:the median of TVI and STA-PSV are heart and liver fire hyperactivity syndrome>Yin deficiency and yang hyperactivity syndrome>qi stagnation and phlegm coagulation syndrome.2.Experimental research(1)GD mouse model was successfully prepared:the modeling rate was 100%,and the serum T3 increased by 5～21 times 4 weeks after immunization.(2)Effects of resolving phlegm and dispersing stasis method on thyroid function,TRAb and morphology of GD mice:after the intervention,the content of T4 in MMI group and HTSJ group decreased significantly(P<0.01),the content of TRAb in MMI group decreased(P>0.05),and the content of TRAb in HTSJ group decreased significantly(P<0.01);Goiter,hyperemia and TW/BW decreased significantly in MMI group and HTSJ group(P<0.05,P<0.01);He staining showed that the proliferation and proliferation of follicular epithelial cells in MMI group and HTSJ group were reduced,the internal convex was significantly reduced,and the filling of follicular cavity was restored.(3)Effects of resolving phlegm and dispersing nodules on thyroid angiogenesis and regulatory pathway in GD mice:after the intervention,MVD decreased significantly in MMI group and HTSJ group(all P<0.01),microvascular bulge and small blood vessels in follicular stroma decreased significantly,lumen narrowed,and Fg exudation decreased significantly;The expression of VEGFA protein was significantly down regulated in MMI group and HTSJ group(all P<0.01),and the expression of VEGFR2,Akt and p-Akt were significantly down regulated(all P<0.05).(4)Effects of resolving phlegm and dispersing stasis method on immune tolerance disorder and regulatory molecules in GD mice:after the intervention,the spleen index of MMI group and HTSJ group decreased significantly(P<0.05,P<0.01);The proportion of CD4+T cells did not change significantly in MMI group(P>0.05),but decreased significantly in HTSJ group(P<0.01);The ratio of Treg/CD4+T cells in MMI group and HTSJ group did not increase(P>0.05);The expression of Foxp3 mRNA in MMI group had no increasing trend,while that in HTSJ group had an increasing trend(P>0.05);The mRNA expression of IL-10 increased in MMI group(P>0.05),and the mRNA expression of IL-10 increased significantly in HTSJ group(P<0.05);Spleen TGF in MMI group and HTSJ group-β The mRNA of 1 increased(P>0.05);The mRNA expression of CTLA-4 in MMI group had no increasing trend(P>0.05),while that in HTSJ group increased significantly(P<0.05);In MMI group,the expression of ICOS on Treg decreased(P>0.05),and the expression of PD-1 did not change.The expression of ICOS and PD-1 on Treg in HTSJ group increased significantly compared with NC group(P<0.01),and the expression of ICOS and PD-1 on Treg in HTSJ group increased significantly compared with Gd group(P>0.05);The level of IL-17 in serum decreased significantly in MMI group and HTSJ group(P<0.01).(5)The main chemical components and molecular docking of Huatan Sanjie Recipe:the main components are phenolic acids and triterpenoid saponins,including citric acid,syringic acid,harpagoside,p-hydroxycinnamic acid,caffeic acid,isoquercetin,hypericin,rosmarinic acid,trans cinnamic acid,harpagoside,tubeimoside B,tubeimoside A and tubeimoside C;Fritillaria thunbergii a,Fritillaria thunbergii B and Fritillaria thunbergii C have the lowest binding energy with each target,while habaside,habasioside,hypericin,isoquercetin and rosmarinic acid have lower binding energy with each target(especially VEGFA,VEGFR2,AKT1,IL10 and IL17A).Conclusions1.The significant increase of thyroid vascularization in GD patients may be involved in the formation of GD goiter,and may promote the development of GD by promoting the formation of pathological factors such as "fire" and "phlegm";TVI and STA-PSV can reflect the degree of thyroid function,goiter and autoimmune response;VEGF may play a role in promoting angiogenesis only in thyroid;"Fire" may be the initiating factor of GD thyroid angiogenesis,and thyroid angiogenesis may promote the development of GD goiter by promoting the formation of "phlegm".2.Method of resolving phlegm and dispersing stasis may directly inhibit thyroid angiogenesis in GD mice by down-regulating Akt/VEGFA/VEGFR2 signal pathway.3.Method of resolving phlegm and dispersing stasis may improve the immunosuppressive function of Treg,inhibit the proliferation and activation of CD4+T cells,inhibit Th17 proinflammatory cytokine IL-17A,correct the abnormal immune tolerance of GD mice,and may indirectly inhibit the thyroid angiogenesis of GD mice by up regulating the expression of Treg co signaling molecules(ICOS,PD-1,CTLA-4)and immunosuppressive cytokines(IL-10,etc.).4.Fritillaria thunbergii a,Fritillaria thunbergii B,Fritillaria thunbergii C,harpagoside,harpagoside,hypericin,isoquercetin and rosmarinic acid may be the main active components of Traditional Chinese medicine with the effect of resolving phlegm and dispersing stasis in regulating GD angiogenesis and immune tolerance disorders.