Abstract: Objective To investigate the therapeutic effect of the incompatible Haizao-Gancao herbal pair against goitre, and to explore whether this effect is associated with autophagy regulated by the JNK1/Bcl-2/Beclin1 pathway. Methods Rats were randomly assigned to six groups: control, model, Euthyrox, high-dose Haizao-Gancao(HG-H, 7.92 g·kg^-1), medium-dose Haizao-Gancao (HG-M, 3.96 g·kg^-1), and low-dose Haizao-Gancao (HG-L, 1.98 g·kg^-1) groups. Except the control group, all the groups were orally administrated with propylthiouracil (PTU) for 14 days to replicate a pathological model of goiter rats. After modeling, each treatment group received their respective medication of 14 days. To stabilize the model, the treatment groups received oral PTU every other day at the previous dosage. The efficacy of HG was assessed based on histopathology of thyroid tissues of rats. Key targets were validated via immunofluorescence, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), and Western blot (WB) analysis. Results Compared with the control group, the thyroid coefficient increased in the model group (P<0.05). Compared with the model group, the thyroid coefficient decreased in all the three HG groups (P<0.05). Under light microscopy, irregular follicular morphology, smaller areas of thyroid follicular cavities, increased height of follicular epithelial cells, and larger numbers of nuclei were observed in the model group compared with the control group. Compared with the model group, the thyroid pathological morphology was significantly improved in the Euthyrox and all HG groups. LC3 immunofluorescence staining revealed enhanced LC3 fluorescence intensity in both the model and HG-L groups compared with the control group (P<0.05). However, the intensity of LC3 fluorescence decreased in the HG-L group relative to the model group (P<0.05). RT-qPCR and WB analysis suggested that mRNA and protein expression levels of JNK1, Beclin1, PI3KC3, and LC3 were significantly elevated in thyroid tissues of the model group (P<0.05) compared with the control group, while Bcl-2 mRNA and protein expression levels were significantly reduced (P<0.05). Compared with the model group, mRNA and protein expression levels of JNK1, Beclin1, PI3KC3, and LC3 were significantly downregulated in thyroid tissues in the HG-L group (P<0.05), while mRNA and protein expression levels of Bcl-2 were significantly upregulated (P< 0.05). Conclusion Haizao-Gancao herbal pair can inhibit cellular autophagy by regulating the expression levels of key downstream factors along the JNK1 pathway—Bcl-2, Beclin1, PI3KC3, and LC3, thereby exerting its therapeutic effect on goiter. The JNK1/Bcl-2/Beclin1 signaling pathway may be a key molecular mechanism underlying the therapeutic efficacy of Haizao and Gancao against goiter.

Key words: Haizao, Gancao, Goiter, Autophagy, Mechanism of Action, JNK1, Beclin1, Rat