Research Of The Time-Effect And Does-effect Relationship And The Mechanism Of DanZhi Compound Recipe On Treating Graves Disease By "Regulating The Liver"

Objective:Graves'disease is an autoimmune disease characterized by overproduction of thyroid hormone.It is the most common cause of hyperthyroidism,accounting for 80%of the hyperthyroidism patients.Anti-thyroid drug is the most common therapy for Graves disease but facing with a major problem of high relapse rate at present,so exploring a method to improve the prognosis and reduce the recurrence rate of Graves disease is a hot spot in the field.Studies found that the high level of thyroid stimulating hormone receptor antibody and the size of thyroid is closely related to the relapse of Graves disease.Oxidative stress is the cause of the autoimmune disorder of Graves'disease and closely related to apoptosis and the size of thyroid.Therefore it deserves great significance to explore the preventive and therapeutic effect of Danzhi compound recipe.So this study took the oxidative stress as the entry point,researched the dose-effect and time-effect of Danzhi compound recipe on treating the Graves'disease,and explore the optimal dose and course of treatment for Danzhi compound recipe,so as to find an optimal and precision therapeutic scheme for Traditional Chinese medicine to treat Graves disease.At the same time we tried to explore the mechanism of Danzhi compound recipe on treating and improving the prognosis of Graves disease by modern molecular biology technology,and provide a reliable theoretical basis for treating Graves disease by“regulating the liver”.Methods:210 female,6 weeks old BALB/c mice were immuned with Ad-TSHR-289 three times to conduct Graves disease model.Every mice was injected with 1×10~8 PFU Ad-TSHR-289 a time,and every injection at three weeks interval.On the Fifth weeks,checking whether the model is successful conducted and starting drug therapy at the sixth weeks after giving the third injection of Ad-TSHR-289.The 150Graves'disease mice models were randomly divided into 5 groups:model group(M group,n=30),methimazole group(MM group,n=30),low dose of DanZhi compound recipe group(DL group,n=30),middle dose of DanZhi compound recipe group(DM group,n=30)and high dose of DanZhi compound recipe group(DH group,n=30).30normal BALB/c female mice of the same age were set as the black group(B group,n=30).DL group,DM group and DH group were respectively given Dan Zhi compound recipe according to the body weight of mice by the dose of13.65mg/kg,27.3mg/kg and 54.6mg/kg,MM group were given medicine by the dose of 3.9mg/kg.The blank group,model group were fed with sterile water by the same amount.During the period of experiment,the general condition of mice was observed every day,and the 24 hours of food intake,water intake,body temperature and body weight were measured regularly.The mice were sacrificed after treating 2 weeks,4weeks and 6weeks.Then tested the serum T3,T4,TSH and TRAb levels,observed the mice thyroid tissue by HE staining,tested the SOD,MDA and CAT level in mouse liver by Elisa assay,tested the expression of protein HO-1 and Nrf-2 in the liver and Bcl-2,Bax level in thyroid tissue by Western blot method,detect ed the expression of gene Nrf-2/HO-1 in mouse liver by real-time PCR.This research was conducted to find the dose-effect and time-effect relationship of Danzhi compound on treating Graves disease,and further explored the mechanism of it from the perspective of oxidative stress and cell apoptosis.Results:1.Construction of the mice model of Graves diseaseMolding the Graves disease mice model by Intramuscular injection of Ad-TSHR-289,the model mice gradually manifest as exciting and irritable mood,increased food intake and water intake,decreased body weight,increased body temperature and increased excretion.The serum T3,T4 and TRAb levels of model mice were increased.thyroid pathology showed that the thyroid was enlarge,the color was deepen,the thyroid follicular epithelium cells were hyperplasia and manifested as cuboidal or columnar follicular,irregular shape,the glial in thyroid follicular was less or even completely absorbed,vacuolar degeneration.2.The influence of Danzhi compound recipe on general condition of rats(1)Compared with the blank group,the M group mice were excited and Edgy,hair was lusterless.The food intake,water intake and body temperature were higher than B group,and the body weight was lower than B group(P<0.05).(2)The Dan Zhi compound recipe can improve the general situation of Graves disease mice,the food intake,water intake,body temperature and body weight of Dan Zhi compound recipe groups were more stable.Comparing with the M group,MM group,DL group,DM group and DH group manifest as good spirit,gentle temperament,Lustrous fur,less food intake and water intake and lower body temperature.The body weight of Dan Zhi compound recipe groups were higher than M group(P<0.05),but the body weight of the MM group were lower than the other groups(P<0.05).The Danzhi compound showed no significant dose and time dependence relation in improving the general situation in mice.3.The influence of Danzhi compound recipe on serum T3,T4,TSH and TRAb levels(1)The serum T3,T4 and TRAb level of M group were significantly higher than the B group,and the TSH level of M group was significantly lower than the B group(P<0.05).(2)The Danzhi compound recipe and methimazole can significantly improve the thyroid function by down-regulating the T3,T4 and TRAb level and up-regulating the TSH level in Graves disease mice(P<0.05).(3)The effect of methimazole is more rapidly and significantly,It can play a significant effect at the second weeks of treatment(P<0.05).Danzhi compound recipe worked slower than methimazole,the thyroid function indexes of Danzhi compound recipe were significantly improved after 6 weeks of treatment,the effect of 6 weeks is better than 2 weeks and 4 weeks,high does group and middle does group are better than the low does group(P<0.05).There is no significant difference between The DM group and MM group on the 6 th week.4.The influence of Danzhi compound on the HE coloration of the thyroid tissueThe size of the thyroid tissues of blank group was normal,the shape of thyroid follicles were regular round,oval or tabular.The thyroid follicles were filled with abundant pink colloid,there was no edema and hyperplasia in thyroid epithelial cells and interstitial substance.On the contrary,thyroids of the model group were enlarge,deepen colored,of which the follicular epithelial cells were hyperplasia and edematous,vacuolar degeneration,the thyroid tissue were invaded by vessels,thyroid follicularmorphologywasirregular,The infiltration ofof lymphocyte was not found.The above situation were significantly improved after treatment with Danzhi compound recipe or methimazole.5.The influence of Danzhi compound recipe on liver MDA,SOD,CAT level(1)Liver MDA,SOD and CAT of the blank group were maintained at a low level,and the levels of MDA,SOD and CAT in the model group were significantly higher than the blank group(P<0.05).(2)The Danzhi compound recipe and methimazole could significantly reduce SOD,MDA and CAT level in liver(P<0.05).(3)The effect of Danzhi compound recipe on down-regulating the MDA,SOD,CAT is better than methimazole group,the effect is time and does dependent,the DH group is better than the DL group,the effect was more significant when treating for more than 4 weeks.6.The influence of Danzhi compound recipe on the level of Bcl-2?Bax in throid(1)The DanZhi compound recipe and methimazole can significantly down-regulating the level of caspase-3 and Bax,up-regulating the level of Bcl-2 in thyroid of Graves'disease mice(P<0.05),which may play a protective effect for thyroid tissue.(2)The effect of regulating the level of Bcl-2?Bax in throid by DH group is more significant than DL group and DM group,but all of the Danzhi compound group were not better than MM group(P<0.05).7.The influence of Danzhi compound recipe on the expression of Nrf-2,HO-1mRNA and protein(1)In the liver of the normal mice,the expression of Nrf-2,HO-1 mRNA and protein were at a low level.The expression of Nrf-2,HO-1 mRNA and protein in liver of model group mice increased significantly(P<0.05).(2)Danzhi compound and methimazole can down-regulate the expression of Nrf-2,HO-1 mRNA and protein in Graves disease mice.When treated after 4weeks,the effect of MM group down-regulating the expression of Nrf-2 mRNA and HO-1mRNA is obvious than DL group,and the effect was also significant when compare to DM group in down-regulating the expression of HO-1mRNA.When treated after 6 weeks,the effect of MM group was significant compare with DL group in down-regulating the expression of HO-1mRNA.No significant difference was observed between MM group and DH group in regulating the expression of Nrf-2mRNA and HO-1 mRNA(P>0.05).(3)Danzhi compound recipe and methimazole can down-regulated the expression of Nrf-2?HO-1 protein in liver.Compare with Danzhi compound recipe groups,MM group can play a more significant role in down-regulating the expression of Nrf-2protein.There was no significant difference in down-regulating the expression of HO-1 protein between MM group and DH group(P>0.05).8.Correlational analyses between MDA?SOD?CAT and TRAb and Nrf-2/HO-1The level of TRAb is positive relate to the level of MDA?SOD and CAT significantly(P<0.01),The level of Nrf2 and HO-1 protein is positive relate to the level of MDA?SOD and CAT significantly(P<0.01),the level of Nrf2 and HO-1protein is positive relate to the level of SOD significantly(P<0.01),but with no significant relate to MDA and CAT(P>0.05).9.Correlational analyses between MDA,SOD,CAT and Bcl-2,BaxThe level of MDA,SOD,CAT is negative relate to the level of Bcl-2 and positive relate to Bax significantly(P<0.01).Conclusion:(1)Using Ad-TSHR-289 can successfully construct Graves disease mice model,the model manifested as typical symptoms of high metabolism and goiter.Which is with a stability quality and and Low mice mortality,became a good repetition molding method.(2)Danzhi Compound recipe can alleviate the general situation and high metabolism symptoms of Graves disease mice,which is better than MM group.(3)Danzhi Compound recipe can down-regulating the level of thyroid hormone and TRAb levels,up-regulating the level of TSH.(4)Danzhi Compound recipe can ameliorate the oxidative stress condition of Graves'disease mice,it can down-regulate the oxidative stress index such as MDA,SOD and CAT level in Graves'disease mice,restore the balance of oxidation/antioxidant system,which is better than MM group.(5)Danzhi compound can down-regulate The expression of Nrf-2,HO-1mRNA and protein in the important oxidative stress pathway.So we presume that regulating the Nrf-2/HO-1 pathway is one the mechanism of Danzhi compound treating Graves disease,but not the only one.(6)Danzhi compound can regulate the course of apoptosis and play anti-apoptosis effect and protect the thyroid cell.(7)The mechanism of Danzhi compound treating Graves disease and reduce recurrence by“regulating the liver”may refer to the regulation of oxidative stress and apoptosis.(8)Danzhi compound show dose and time dependent relation in treating Graves disease mice,it is more effective by increasing the dosage and prolong the course of treatment.