Experimental Study Of Ephedrans On Autoimmune Thyroiditis

ObjectiveHerba ephedrae and its preparations were proved to be effective to the disease ofautoimmune thyroiditis by the long-time clinic practice and experiment research ofprofessor Xu zhiyin. Yet their toxicity limits the dosage and thus influents their effect.The immunization of polysaccharides, ephedrans and their lower poisonous side effecthave already been known and extensively studied by clinic and experimentalresearches. In the study, Ephedra was drawn from herba ephedrae and its immuneeffects and curative dose on autoimmune thyroiditis were evaluatd. With the model ofexperimental autoimmune thyroiditis（EAT）of mice, thyroid hormone, autoantibodies,the analysis of sub-cluster of lymphoid cells of peripheral blood, the pathologic formsand the expression of Fas\Fasl、Bcl-2、Bax and VEGF-globulin of thyroiditis tissue ontheir functions and mechanism were detected. We also had compared the effects oflow dose group （33mg/kg）, middling dose group （66mg/kg） and large dose group（132mg/kg）, Prednisone Acetate Tablets（PAT） and Thyroid Tablet （TT） on miceEAT. We hope to provide experiment data for the clinical application of Herbaephedrae, make certain its immunization valid part, raise clinical curative effect andreduce its poisonous side effect for the development of a new immunosuppressant ofChinese herbal medicine.MethodWe took dry grass Herba ephedrae 4.0 kg on water to lift, condense, pure sink,eliminate the albumen, dry, smash to get 128g ephedrans.We took 40 ICR mice with half male and half female and divided random intonormal controlled group, ephedrae low dose group （33mg/kg）, ephedrae middlingdose group （66mg/kg） and ephedrae large dose group（132mg/kg）. The ICR mice ineach group were infused stomach with 0.2mls/10g of various medicine for 8 days.Then we injected the suspension of sheep red blood cell 0.2mls/mice to the abdomencavity twice after given the medicine. One hour after the last time given the medicine,we took blood from the eye sockets, using the method of serum colorimetry tomeasure the half cytolysis (HC₅₀).Mix pure mice thyroid globulin（TG） antigen and Complete Freund’Adjunt （CFA） by 2mg/ml equally to make the oil-wrap-water emulsion. Then take 60 female ICRmice and divided random for experiment. Inject the emulsion of TG and CFA intomulti-part of the model mice with 0.2ml （each time 100ug TG）, once a week andtotally 5 times, meanwhile, the mice in control group were injected with physiologicalsaline. The mice in control group were drunken distilled water, while the ones in othergroups were given high iodine water （formed by joining 0.64gram kalium iodide intollitre）. The volume infused into stomach in every group was 0.2mls/10g: TT group13.3mg/kg every day, PAT group 10 mg/kg every day, the low dose group 33mg/kg,the large dose group 66mg/kg every day. From the morning of the sixth week, themice were hungry for 24 hours then used for taking samples which were the viscerasand the blood taken from eyeballs.We assayed the sum levels of thyroxine,TT3、TT4、FT3、FT4、TSH、TGAb andTMAb, with Radioimmune assay（RIA）, Measured the subpopulations of sub-clusterof T-lymphoid cells with flow cytometer （FCM）. and deal the thyroid tissue withHaematoxylin-eosin staining. And we observed the expression of Fas\Fasl, Bcl-2,Bax and the VEGF-globulin of thyroid tissue with S-P immune histochemistry.Result1. We got brown crystal enphidrans from the dry grass Herba ephedrae after theprocess on water to lift, pure sink, eliminate the albumen and got 128g （getting3.2％） of it after dry, smash in the end.2. The experiment revealed that Ephedran low dose group, dose middling group anddose big group all could make the HC₅₀ of the mice’s sheep red cell descend（P＜0.01, compared with control group）.3. It indicated that we had successfully reproduced the EAT mice model from theascertainment of high drop level of thyroxine antibody TGAb and TMAb in themodel mice’s serum （P＜0.01, compared with control group） and the soakage ofmice’s lymphoid cells of thyroid tissue, which lay solid foundation for ourexperiment.4. The TGAb, TMAb of the model group were evidently higher than the controlgroup（P＜0.01）, and theephedran groups were lower than the model group（P＜0.01）yet their function were similar with PAT group and TT group.5. The mice weight had increased naturally in the control group while the modelgroup lost weight from the fifth week which had no statistic difference compare tomodel group. The weight of mice in enphedran groups were all lower from the fourth week after took medicine particularly the low dose group than that of modelgroup in the same period. The statistic difference were found betweenexperimental groups and model group （P＜0.05 or P＜0.01）6. CD4⁺ lymphoid cells of EAT model mice were higher than control groupcomparatively（P＜0.05）. Specific values of CD4⁺/CD8⁺ is obviously high（P＜0.01）.CD4⁺ lymphoid cells in ephedrans large dose group had obvious difference tomodel group（P＜0.05） and low dose group showed no special deference whichwas similar to TT group. CD8⁺ lymphoid cells and Specific values of CD4⁺/CD8⁺of enphedran large dose and low dose group all showed high difference（P＜0.01）.7. We observed the thyroid pathologic configuration of the mice in every group withlight microscope and found the ephedran groups had improved obviously in thethyroid harmful change than model group, control group and medicine controlgroup. It showed that soakage of lymphoid cell descended obviously and theproportion of A cells decreased.8. The thyroid follicle cells of EAT model mice contained Fas high expression（P＜0.01）; Ephedran low dose group adjusted the Fas expression of thyroid cellsdownwards（P＜0.05 compare with model group）; Dose big group showed distinctdifference （P＜0.01 compare with model group）. Fas-l globulin expression of EATmodel mice upwards （P＜0.01 compare with control group）; Fas-l Integral lightdensity of large dose group descended obviously （P＜0.05 compare with modelgroup）; other therapeutic groups showed no distinct difference.Bcl-2 expression of area of thyroid follicle cells: it showed difference in integrallight density between model group and control group（P＜0.05）; distinctdifference between Ephedran low dose group and model group（P＜0.01）;distinct difference betweenephedran large dose group and model group（P＜0.05）.It showed difference in surface density between model group and controlgroup（P＜0.05）. bax globulin expression: it showed distinct difference inintegral light density between model group and control group（P＜0.01）;difference between Ephedran low dose group and model group（P＜0.05）;distinct difference between Ephedran low dose group and model group（P＜0.01）.It showed distinct difference in surface density between model group and controlgroup（P＜0.01）; distinct difference between ephedran large dose group andmodel group（P＜0.05）.9. Expression of thyroid VEGF of EAT model mice showed obvious difference from control group （P＜0.01）. It showed overt difference betweenVEGF expression of PAT and control group（P＜0.05）. VEGF expression in othergroups showed no distinct difference. Integral light density of ephedran groupsshowed difference （P＜0.05）.