To investigate the effects of Sini Powder on Graves′ disease (GD) model mice and to explore its mechanism in regulating Th1/Th2/Th17 cells immune imbalance at three levels: immune center (spleen), humoral circulation (serum), and target organ (thyroid).

Twenty-four female BALB/c mice were randomly divided into normal group (n = 6) and modeling group (n = 18)using a randomized block design. GD model was established through intramuscular injection of AdTSHR289 adenovirus. After successful modeling, GD mice were randomly assigned to the model, methimazole (4.55×10^-3 g/kg), and Sini Powder (5.46 g/kg) groups using random number table, with corresponding interventions administered daily for 6 weeks. HE staining was used to assess pathological changes in thyroid follicular epithelial cells; serum levels of thyrotropin receptor antibody (TRAb) and thyroxine (T₄) were measured using radioimmunoassay. The proportions of Th1, Th2, and Th17 cells in spleen tissues were analyzed by flow cytometry; mRNA and protein expressions of Th1-derived interferon-γ (IFN-γ), Th2-derived interleukin-4 (IL-4), and Th17-derived interleukin-17A (IL-17A) in spleen were detected by quantitative reverse transcription PCR and Western blotting. Serum levels of IFN-γ, IL-4, IL-10, IL-17A, as well as regulated on activation, normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein-1β (MIP-1β), and monocyte chemoattractant protein-1 (MCP-1) were measured using Luminex protein biochip assay. Immunohistochemistry was performed to localize and assess the expression of IFN-γ, IL-4, and IL-17A in thyroid tissues.

Sini Powder reduced thyroid volume of model mice, as well as decreased serum TRAb and T₄ levels (P< 0.01), ameliorated pathological damage to thyroid follicular structures, and alleviated splenomegaly. Mechanistic studies revealed that compared with the normal group, the model group exhibited increased trends of the proportions of Th1 and Th17 cells, along with an elevated Th1/Th2 ratio, whereas Th2 cell proportion decreased. Expressions of IFN-γ and IL-17A mRNA, as well as IFN-γ protein expression in spleen increased(P<0.01), with an upward trend in IL-17A protein expression, while IL-4 mRNA expression was downregulated (P<0.01). The serum levels of IFN-γ, IL-10, and RANTES elevated (P<0.01), with upward trends in IL-17A, MIP-1β, and MCP-1, and a downward trend in IL-4 level. The expressions of IFN-γ, IL-4, and IL-17A in thyroid tissues increased. These findings suggest the presence of Th1/Th2/Th17 cells immune imbalance in GD model mice. The methimazole and Sini Powder modulated expressions of IFN-γ, IL-4, and IL-17A in spleen, thyroid, and serum, as well as regulated the expression of chemokines including RANTES, MIP-1β, and MCP-1 of the GD model mice.

Sini Powder may exert intervention effect on goiter induced by GD via regulating Th1/Th2/Th17 cells immune imbalance and modulating chemokines such as RANTES, MIP-1β, and MCP-1.

To explore the mechanism of Huatan Sanjie Fang (HTSJ) in regulating goiter in Graves' disease (GD) mice by detecting key factors of the Hippo signaling pathway.

A mouse model of GD was established by injecting Ad-TSHR289 adenovirus into the bilateral quadriceps femoris of female mice. Successful mouse models were then randomly divided into a model group, methimazole (MMI) group, and HTSJ group, and fed with deionized water, MMI (4.5 mg/kg per day), and HTSJ (35.10 g/kg per day), respectively, for 10 weeks. Histopathological changes of the thyroid gland were subsequently observed by hematoxylin-eosin staining. Radioimmunoassay was used to detect serum total thyroxine (T4) and thyrotrophin-receptor antibody (TRAb) levels. The relative expression of mRNA of Mst1, YAP, and TAZ were detected by quantitative real-time polymerase chain reaction, while the protein expression of Mst1, YAP, TAZ, pMst1, and pYAP were detected by western blot.

After 10 weeks of drug intervention, goiter and other pathological changes in the HTSJ group significantly improved compared with the model group, and the levels of serum T4 and TRAb significantly decreased (P =.002, P <.001, respectively). Decreased mRNA expression of Mst1, YAP, and TAZ, the key factors of the Hippo signaling transduction pathway, was also observed (P =.002, P =.022, P <.001, respectively). In contrast, protein expression of Mst1 (P =.046), pMst1 (P =.026), and pYAP (P =.004) increased, while protein expression of YAP and TAZ decreased (P =.041, P <.001, respectively).

HTSJ can effectively improve goiter in GD mice through the Hippo signaling pathway.