海藻-甘草药配对通过调节Beclin1介导的自噬改善丙硫氧嘧啶引起的甲状腺肿大

海藻（Sargassum）广泛用于治疗甲状腺肿大。甘草（Glycyrrhizae radix et rhizome）在传统中医中以减少毒性或增强疗效而闻名。作为经典药材配对，海藻-甘草（HG）是常用且有效的联合治疗甲状腺肿大的方法。关于HG对甲状腺肿大的作用机制尚不清楚。为探究HG的作用机制，采用分子对接结合实验验证，阐明其分子机制。通过灌胃丙硫氧嘧啶（PTU）建立大鼠甲状腺肿模型，持续14天。大鼠被分为六组：对照组、模型组、优甲乐（Euthyrox）组、HG高剂量（HG-H）组、中剂量（HG-M）组和低剂量（HG-L）组。根据一般观察（如大鼠的生活状态、体重和直肠温度）、甲状腺相对重、甲状腺功能、苏木精-伊红（HE）染色及透射电子显微镜（TEM）观察甲状腺的病理变化，评估HG的作用。采用UPLC-MS/MS分析HG的化学成分，并通过多个数据库预测潜在靶点。随后利用分子对接探索其药理机制，并用Western Blot（WB）和免疫共沉淀（Co-IP）验证关键靶点。结果显示，HG显著提高PTU诱导大鼠的三碘甲腺原氨酸（T3）、游离三碘甲腺原氨酸（FT3）、游离甲状腺素（FT4）水平，增加体重，减少甲状腺肿大。模型组显示滤泡上皮细胞大小不均、形态不规则、排列紊乱等病理变化，而HG组甲状腺滤泡和上皮细胞基本正常。HG组表现出多种自噬泡的特征性变化。结论中，HG-L组表现出最佳的治疗效果。通过UPLC-MS/MS鉴定出HG的主要化学成分，发现其含有类黄酮、碱性成分、有机酸、酚酸和倍半萜等。分子对接显示，Formononetin与Beclin1的结合能为-5.38 kcal/mol，Naringenin与Beclin1的结合能为-5.25 kcal/mol，提示这些成分可能通过调控Beclin1介导的自噬发挥抗甲状腺肿作用。WB和Co-IP实验表明，HG可以破坏Beclin1与类磷脂酰肌醇3-激酶（PI3KC3）的结合，促进Beclin1与B细胞淋巴瘤-2（Bcl-2）复合物的形成。综上所述，HG通过抑制Beclin1/PI3KC3复合物的形成并增强Beclin1/Bcl-2结合，调节自噬反应，从而缓解丙硫氧嘧啶引起的甲状腺肿大。

Haizao, Sargassum, is widely used to treat goiter. Gancao, Glycyrrhizae radix et rhizome, is renowned for reducing toxicity or increasing effects in traditional Chinese medicine. As a classic herb pair, Haizao-Gancao (HG) is a commonly used and effective combined therapy for goiter. The underlying biological mechanisms of HG on goiter is still unclear.To explore the effect of HG on goiter, employing molecular docking combined with experimental validation to elucidate the molecular mechanism.The rat goiter model was created by gavageing propylthiouracil (PTU) intragastrically for a duration of 14 days. The rats had been separated into six groups: control, model, euthyrox, HG high-dose (HG-H), medium-dose (HG-M) and low-dose (HG-L) group. Based on general observations (such as the rats' living status, body weight, and rectal temperature), the relative weight of the thyroid, thyroid function, hematoxylin-eosin (HE) staining, and transmission electron microscopy (TEM) to view the pathological variations of the thyroid glands, the effect of HG was evaluated. Discovered the chemical composition of HG by UPLC-MS/MS and the possible targets were predicted adopting several databases. Next, we explored their pharmacological mechanisms using molecular docking and validated key targets using western blotting (WB) and co-immunoprecipitation (Co-IP).HG significantly increased the levels of triiodothyronine(T3), free triiodothyronine (FT3), free thyroxine (FT4), gained body weight and reduced tumescent thyroid glands in PTU-induced rats. The model group pathological changes such as uneven size, irregular shape and disordered arrangement of follicular epithelial cells occurred. However, HG groups thyroid follicles and epithelial cells appeared apparently normal. A variety of characteristic changes of autophagic vesicles appeared in the HG groups as opposed to the model group. In conclusion, the HG-L showed the best therapeutic effect. By UPLC-MS/MS, the major chemical components of HG were identified. The result revealed that HG contained flavonoids, alkaloids, organic acids, phenolic acidsand and terpenoids, etc. The molecular docking results of formononetin and naringenin and Beclin1 protein showed a high interaction of -5.38 kcal/mol and -5.25 kcal/mol. This implies that formononetin and naringenin may have a therapeutic effect in goiter rats by controlling the Beclin1-mediated autophagy. Western Blot (WB) and co-immunoprecipitation (Co-IP) results showed that HG can disrupt Beclin1/class III phosphatidylinositol 3-kinase(PI3KC3) binding and promote Beclin1/B-cell leukemia/lymphoma-2(Bcl-2) complex formation. Taken together, results demonstrate that autophagy inhibition via reducing Beclin1/PI3KC3 formation and increasing Beclin1/Bcl-2 binding activity.HG ameliorates propylthiouracil-induced goiter by regulating the Beclin1-mediated autophagy, thus promoting the autophagy of thyroid cells.