Preliminary Study On The Efficacy And Pharmacology Of Jiawei Xiaoyao Pill Derived Formula In The Treatment Of Graves Disease

Graves' disease,as known as von Basedow disease,is an autoimmune disease primarily affects thyroid gland.The incidence peaks between 30 and 50 years of age,but people can be affected at any age.The lifetime risk is 3%for women and 0.5%for men.It's a syndrome characterized by an enlarged and overacvtive thyroid gland,Anterior tibial myxedema,hyperthyroidism and throid-associated ophthalmopathy.A mounting body of evidence demonstrated that Th17/Treg imbalance plays an important role in autoimmune diseases.Based on the change of TSHR structure,the Th17/Treg imbalance were carried out the pathogenesis.The pathological features are hyperplasia and hypertrophy of thyroid follicular epithelium accompanied and infiltration of lymphocytes.Antithyroid drugs,Radioactive iodine and surgery are used to treat GD.But the bad prognosis is the pivotal problem.Traditional Chinese Medical treatment has the advantages of syndrome differentiation,equal emphasis on prevention and overall conditioning.One of the most common syndrome is depressed liver and deficient spleen.Jiawei Xiaoyao Wan stems from Welfare Pharmacy.It has outstanding role in anti-inflammatory and immunoregulation with containing quercetin,beta-sitosterol and paeoniflorin.Diwu,the rhizome of Anemone flaccida Fr.Schmidt,has used to therapy autoimmune disease.Based on previous research,this study explored the pharmacodynamics and pharmacology of Diwu and Jiawei Xiaoyao wan in regulating immune system of GD by down-regulating JAK1-STAT3 pathway and then,regulating Th17/Treg balance.1.A pharmacodynamic model of Graves' disease induced by membrane antigen in ratsObjective:To establish a model of Graves' disease in rat by subcutaneous injection of membrane antigen for evaluating pharmacodynamics of drug candidates.Methods:The membrane protein extracted from thyroid gland and complete Freund's adjuvant(CFA)were emulsified by 1:1 in volume.The female rats were divided into control and 9 model groups with gradient intervals between the primary sensitization and the first challenge.The model group was subcutaneous injected with membrane antigen to induce autoimmune injuries of thyroid(three times for sensitization and challenge).The clinical symptoms were scored and at d110,and ECG was detected at d115.Serum level of T3,T4,TSH,thyrotropin receptor antibodies(TRAb)and IFN-y were measured.The thyroid and spleen pathological changes were observed by HE stains.Results:The regressive curve was set up between potency of indicators and logarithm intervals.The curve presented "S",with equation as Y=0.03+0.69/[1+10(7.71x-21.35)]and ET50 as 587.10h(24 days).Conclusion:The optical interval has been confirmed to set up the pharmacodynamic model of Graves' disease.The pathogenesis and clinical manifestations were similar to patients.This model could be used to evaluate therapeutic effects of drug candidates.2.To evaluate therapeutic effects in Graves' disease by Jiawei Xiaoyao wan derived prescriptionObjective:To evaluate therapeutic effects in Graves' disease by Diwu to Jiawei Xiaoyao wan.Methods:96 SD rats were randomly divided into 8 groups,the rats in 7 groups were administrated with membrane antigen(the primary sensitization at d0,d7,d14 and the first challenge d38,d45,d52).At d38,the rats were treated with MMI(0.0024g·kg-1),Diwu(0.24g·kg-1),the compound of Jiawei Xiaoyao wan(3.6g·kg-1),the low(1.21g·kg-1),middle(3.84g·kg-1)and high(12.14g·kg-1)level of the compound of Jiawei Xiaoyao wan and Diwu.At d72-76,the clinical symptoms were measured and at d82,ECG was detected.Serum level of T3,T4 and TRAb were measured.The thyroid gland pathological changes were observed by HE stains.The CD4 and CD8 in situ expression on spleen by immunohistochemical.Results:?Symptoms score:compared with Model group,the heart rate,food-intake,drink-intake,stool weight and urine volume were decreased(P<0.05)in each administration groups,among the heart rate of the QL group was abnormal;?Thyroid function:compared with Model group,the T3,T4,TRAb in serum level were declined(P<0.05)in each administration groups;?Pathological observation:compared with Model group,the thyroid follicles in each administration group were smaller and the epithelium is hyperplasia(P<0.05);?Immunohistochemistry:compared with Model group,the CD4 and CD8 in situ expression on spleen were positive and T lymphocyte infiltration was alleviated(P<0.05).Compared with MMI group,the QL group improved the high metabolic state,lowered the levels of serum hormones T3,T4,and TRAb,alleviated the proliferation of thyroid follicular epithelial cells,and reduced T cell infiltration.Conclusion:Jiawei Xiaoyao wan derived prescription treat Graves disease through improving hypermetabolic status,down-regulating T3,T4,TRAb level in serum,alleviating thyroid follicular epithelial cell proliferation and reducing T lymphocytes infiltration.3.To explore the pharmacological mechanism in Graves' disease by Jiawei Xiaoyao wan derived prescriptionObjective:To evaluate therapeutic effects in Graves'disease by Diwu to Jiawei Xiaoyao wan.Methods:96 SD rats were randomly divided into 8 groups,the rats in 7 groups were administrated with membrane antigen(the primary sensitization at d0,d7,d14 and the first challenge d38,d45,d52)At d38,the rats were treated with MMI(0.0024g·kg-1),Diwu(0.24g·kg-1),the compound of Jiawei Xiaoyao wan(3.6g·kg-1),the low(1.21g·kg-1),middle(3.84g·kg-1)and high(12.14g·kg-1)level of the compound of Jiawei Xiaoyao wan and Diwu.At d72-76,the clinical symptoms were measured and at d82,ECG was detected.Serum level of IFN-?,IL-10,IL-17 and IL-21 were measured.The JAK1,STAT3,p-STAT3,RORyt and FOXP3 in situ expression on spleen by immunohistochemical.The mRNA Expression level of JAK1,STAT3 were deter mined with RT-PCR.The protein expression level of JAK1,STAT3,RORyt and FOXP3 were tested by Western Blot.Results:?Cytokine levels in serum:Compared with the Model group,the levels of IFNy,IL-10,IL-17,and IL-21 in each administration group decreased significantly(P<0.05);compared with the MMI group,the level of IFN-y was no significant difference in DW,QL,and QM groups;there was no significant difference in the levels of the QM and QH groups of IL-10;there was no significant difference in the levels of the QL and QM groups of IL-17;there is no significant difference in the levels of the QM and QH groups of IL21.?Immunohistochemistry showed that compared with the Model group,the in situ expression levels of JAK1,STAT3,p-STAT3,ROR ?t,FOXP3 and ROR ?t/FOXP3 in each administration group decreased(P<0.05);compared with the MMI group,JAK1 was no significant difference in FF and QM groups;there was no significant difference in the in situ expression levels of STAT3 in DW,QL,and QM groups;there was no significant difference in p-STAT3 in the QM and QH groups;There was no significant difference in FOXP3 levels of QL and QM groups;there were significant differences in RORyt and the ratio of ROR ?t/FOXP3 in each group(P<0.05).?RT-PCR showed that JAK1 mRNA and STAT3 mRNA expression levels decreased compared with the Model group(P<0.05);compared with the MMI group,there was no significant difference in the JAK1 mRNA expression levels in QL and QM groups;the STAT3 mRNA expression levels in QM group were not significant differences.?Western Blot showed that compared with the Model group,the protein expression ratio levels of JAK1,STAT3,ROR ?t,FOXP3 and ROR ?t/FOXP3 in each administration group decreased(P<0.05).Compared with the MMI group,there was no significant difference in the expression levels of JAK1 in FF and QM groups;there was no significant difference in STAT3 expression levels in QL and QM groups;there was no significant difference in the expression levels in the QM group of RORyt;the expression levels in the QM and QH groups of FOXP3 was no significant difference;there was no significant difference in the expression ratio of RORyt/FOXP3 in the QM group.Conclusion:Jiawei Xiaoyao wan derived prescription inhibits in Graves' disease by downregulating JAK1-STAT3 pathway in CD4+T lymphocytes and then,regulating Th17/Treg cell balance.The full text conclusionTo evaluate therapeutic effects and the pharmacological mechanism in Graves' disease by Jiawei Xiaoyao wan derived prescription.To establish a novel Graves' disease model,the membrane protein extracted from thyroid gland and complete Freund's adjuvant were emulsified.MMI used as positive drug,compared with FF,DW,QL,QM and QH.IFN-?,IL-10,IL-17 and IL-21 in serum level were tested by Elisa.The protein expression level of JAK1,STAT3,pSTAT3,ROR ?t and FOXP3 were tested by immumohistochemical,Western Blot and Real-time PCR.All used to evaluate the pharmacological mechanism.The membrane antigen used to establish a novel Graves' disease model.To analyst the therapeutic effects and the pharmacological mechanism,QM group performed best in pharmacodynamics.To explored the pharmacological mechanism,similar with MMI group,saw that Jiawei Xiaoyao wan derived prescription treat Graves disease through improving hypermetabolic status,down-regulating T3,T4,TRAb level in serum,alleviating thyroid follicular epithelial cell proliferation and reducing T lymphocytes infiltration.