海藻玉壶汤加减海藻甘草反药组合对甲状腺肿大模型大鼠甲状腺功能及mTOR蛋白表达的影响

Effects of a modified sargassum and glycyrrhiza anti-drug combination of the Haizao Yuhu decoction on thyroid function and mTOR protein expression in rats with goiter

目的通过研究海藻甘草反药组合在海藻玉壶汤中的加减应用对甲状腺肿大模型大鼠甲状腺功能及哺乳动物雷帕霉素靶蛋白(mTOR)表达的影响,为海藻甘草反药组合配伍应用提供实验依据。方法选取雄性Wistar大鼠84只,随机分为空白组、模型组、优甲乐组(20.00μg/kg)、海藻玉壶汤组(10.08g/kg)、海藻玉壶汤减海藻组(9.00g/kg)、海藻玉壶汤减甘草组(9.18g/kg)和海藻玉壶汤减海藻甘草组(8.10g/kg),除空白组其余各组给予丙硫氧嘧啶复制甲状腺肿大病理模型,以优甲乐作为阳性对照药,其余各组给予相应药液。采用放射免疫法检测各组大鼠血清三碘甲状腺原氨酸(T3)、甲状腺素(T4)、促甲状腺激素(TSH)、游离三碘甲状腺原氨酸(FT3)、游离甲状腺素(FT4)水平,免疫组化法检测甲状腺组织Ki67蛋白表达情况,Western blotting检测甲状腺组织磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、mTOR蛋白表达情况。结果与模型组比较,优甲乐组大鼠血清T3、T4、FT3、FT4水平升高,TSH水平降低(P<0.01);海藻玉壶汤组及各拆方组大鼠血清T3、T4、FT3、FT4水平升高(P<0.05,P<0.01),TSH水平降低(P<0.05,P<0.01)。与模型组比较,海藻玉壶汤组、海藻玉壶汤减海藻组大鼠甲状腺组织Ki67蛋白阳性表达率降低(P<0.01),海藻玉壶汤减甘草组、海藻玉壶汤减海藻甘草组大鼠甲状腺组织Ki67蛋白阳性表达率降低(P<0.05)。与模型组比较,优甲乐组、海藻玉壶汤减海藻组、海藻玉壶汤减甘草组p-mTOR蛋白表达水平降低(P<0.05),海藻玉壶汤组p-mTOR蛋白表达水平降低(P<0.01)。mTOR蛋白表达水平各组之间比较差异无统计学意义。海藻玉壶汤减海藻组和海藻玉壶汤减甘草组p-mTOR/mTOR降低(P<0.05)。结论海藻玉壶汤对甲状腺肿大模型大鼠甲状腺激素水平具有明显的回调作用,且可能通过抑制mTOR信号通路的激活进而抑制细胞增殖,起到纠正甲状腺肿大的作用。

Objective To study the effect of a seaweed and licorice anti-drug combination on thyroid function and the expression of the mammalian target of rapamycin(mTOR)in rats with goiter.This is done to provide an experimental basis for the application of an anti-drug combination of sargassum and glycyrrhiza.Methods A total of 84 male Wistar rats were selected and randomly divided into a blank group,a model group,a euthyrox group(20.00μg/kg),a Haizao Yuhu decoction group(10.08 g/kg),a Haizao Yuhu decoction without sargassum group(9.00 g/kg),a Haizao Yuhu decoction without glycyrrhiza group(9.18 g/kg),and a Haizao Yuhu decoction without sargassum and glycyrrhiza group(8.10 g/kg).Except for the blank group,the others were given propylthiouracil to replicate the goiter pathological model and the corresponding drug solution.The serum levels of triiodothyronine(T3),tetraiodothyronine(T4),thyroid stimulating hormone(TSH),free triiodothyronine(FT3),and free tetraiodothyronine(FT4)in rats were determined using a radioimmunoassay,and the expression of the Ki67 protein was detected using immunohistochemistry.The p-mTOR and mTOR proteins were detected using Western blotting assay.Results Compared with the model group,the serum levels of T3,T4,FT3,and FT4 in the euthyrox group were higher,and the TSH level was lower(P<0.01).Serum levels of T3,T4,FT3,and FT4 were increased in the Haizao Yuhu decoction group,the Haizao Yuhu decoction without the sargassum group,the Haizao Yuhu decoction without glycyrrhiza group,and the Haizao Yuhu decoction without sargassum and glycyrrhiza group(P<0.05,P<0.01),the TSH level was decreased(P<0.05,P<0.01).Compared with the model group,the positive expression rate of the Ki67 protein in the thyroid tissue of rats in the Haizao Yuhu decoction group and the Haizao Yuhu decoction without sargassum group was lower(P<0.01).The positive expression rate of the Ki67 protein in the thyroid tissues of the rats in the Haizao Yuhu decoction without glycyrrhiza group and the Haizao Yuhu decoction without sargassum and glycyrrhiza group was decreased(P<0.05).Compared with the model group,the p-mTOR protein expression level in the euthyrox group,the Haizao Yuhu decoction without sargassum group,and the Haizao Yuhu decoction without glycyrrhiza group was decreased(P<0.05).In addition,that in the Haizao Yuhu decoction group was decreased(P<0.01).Furthermore,there was no significant difference in the expression level of the mTOR protein,but p-mTOR/mTOR decreased in the Haizao Yuhu decoction without sargassum group and the Haizao Yuhu decoction without glycyrrhiza group(P<0.05).Conclusion The Haizao Yuhu decoction had a significant thyroid hormone effect on the level of thyroid enlargement in rats and may inhibit cell proliferation by inhibiting the activation of the mTOR signaling pathway.Hence,it had the effect of correcting goiter.