甲亢宁胶囊对Graves病小鼠甲状腺功能及Akt/mTOR信号通路的影响

Effect of Jiakangning Capsule on Thyroid Function and Akt/mTOR Signal Pathway of Graves' Disease Mice:an Experimental Study

目的观察甲亢宁胶囊干预下Graves病小鼠甲状腺功能的改善情况及Akt、磷酸化Akt(p-Akt)、雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)及磷酸化mTOR(p-mTOR)表达变化,探讨甲亢宁胶囊治疗Graves病可能的分子机制。方法用表达促甲状腺激素受体(thyroid stimulating hormone receptor)A亚单位的重组腺病毒(Ad-TSHRα-289)免疫BALB/c雌鼠建立Graves病动物模型。将70只造模成功小鼠按随机数字表法分为模型组(20只)、甲亢宁胶囊干预组(中药组,25只)、甲巯咪唑片干预组(西药组,25只),另设正常对照组(正常组,15只)和空载病毒对照组(空载组,20只,注射Ad-null)。中药组予1.5 g/(kg·d)甲亢宁胶囊混悬液灌胃,西药组予2.5 mg/(kg·d)甲巯咪唑混悬液灌胃,其余各组每日予同体积生理盐水灌胃,药物干预5周。各组最终选取6个样本,观察小鼠甲状腺组织的病理变化;放射免疫法测定小鼠血清甲状腺素(thyroxine,T4),三碘甲状腺素原氨酸(triiodothyronine,T3),促甲状腺激素(thyroid stimulating hormone,TSH)及促甲状腺激素受体抗体(thyrotropin receptor antibody,TRAb)水平;Western blot法测定小鼠甲状腺组织Akt、p-Akt、mTOR及p-mTOR的表达量。结果(1)模型组甲状腺呈增生性改变,滤泡增大,大小不一,间质血管浸润。中药和西药组甲状腺组织结构明显恢复,滤泡增生有所缓解。(2)与正常组、空载组比较,模型组TRAb、T4及T3水平升高(P<0.01),p-Akt/β-actin、p-Akt/Akt、p-mTOR/β-actin及p-mTOR/mTOR比值均升高(P<O.01);与模型组比较,中药和西药组TRAb、T4及T3水平均降低(P<0.01),中药组p-mTOR/β-actin、p-mTOR/mTOR比值均降低(P<0.01),西药组p-Akt/β-actin、p-Akt/Akt、p-mTOR/β-actin及p-mTOR/mTOR比值均降低(P<0.05,P<0.01)。结论甲亢宁胶囊能降低Graves病小鼠甲状腺激素水平,降低mTOR的表达,其改善Graves病小鼠甲状腺功能的机制可能与这一影响有关。

Objective To observe the improvement of thyroid function and changes of Akt,pAkt,mammalian target of rapamycin（mTOR）,and para-mTOR（p-mTOR） expression in Graves＇ disease（GD） mice after intervened by Jiakangning Capsule（JC）,and to explore possible mechanism for JC in treating GD.Methods GD model was established by immunizing female BALB/c mice with thyroid stimulating hormone receptor A subunit（Ad-TSHRα-289）.Totally 70 successfully modeled mice were divided into the model group（n =20）,the JC intervened group（n =25）,the Methimazole Tablet intervened group（n =25） according to random digit table.A normal control group（n =15） and a vehicle control group（n =20,injected with Ad-null） were also set up.Mice in the JC intervened group were administered with JC suspension at the daily dose of 1.5 g/kg by gastrogavag.Mice in the Methimazole intervened group were administered with Methimazole suspension at the daily dose of 2.5 g/kg by gastrogavage.Equal volume of normal saline was administered to mice in the rest 3 groups by gastrogavage.All intervention lasted for 5 weeks.Six mice were selected from each group to observe pathological changes of thyroid tissues.Serum levels of thyroxine（T4）,triiodothyronine（T3）,thyroid stimulating hormone（TSH）,and thyrotropin receptor antibody（TRAb） were analyzed by radioimmunoassay.Expression levels of Akt,p-Akt,mTOR,and p-mTOR in thyroid tissues were etermined by Western blot.Results（1） The thyroid gland in the GD model group showed proliferative changes,with enlarged follicles of various sizes.Interstitial stroma was filled with blood vessels.Structures of thyroid tissues in the JC intervened group and the Methimazole intervened group were significantly restored,and follicular hyperplasia was relieved.（2） Compared with the normal control group and the vehicle control group,levels of TRAb,T4,and T3increased;ratios of P-Akt/p-actin,p-Akt/Akt,p-mTOR/β-actin,and p-mTOR/mTOR also increased in the model group（all P 0.01）.Compared with the model group,levels of TRAb,T4,and T3 decreased in the JC intervened group and the Methimazole intervened group（P 0.01）;ratios of p-mTOR/p-actin and pmTOR/mTOR decreased in the JC intervened group（P 0.01）;ratios of P-Akt/p-actin,p-Akt/Akt,pmTOR/p-actin,and p-mTOR/mTOR decreased in the Methimazole intervened group（P0.05,P 0.01）.Conclusion JC could reduce thyroid hormonc levels of GD mice and lower expression levels of mTOR,and its mechanism for improving thyroid function of GD mice might be associated with this influence.