Isoquercetin Attenuates Graves’ Disease-Related Thyroid Epithelial Inflammatory Responses: Exploratory Evidence from Network Pharmacology, Mendelian Randomization, and Cellular Validation

Shihan Zhang, Qunwei Ma, Xiaoyu Ding, Xinzhong Zhang, Guang Wang, Jia LiuDepartment of Endocrinology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, 100020, People’s Republic of ChinaCorrespondence: Guang Wang; Jia Liu, Department of Endocrinology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, 100020, People’s Republic of China, Email [email protected]; [email protected]: Graves’ disease (GD) is initiated by thyroid-stimulating hormone receptor autoantibody signaling, whereas cytokine-mediated inflammation within the thyroid may amplify thyrocyte activation. The microbiota-related metabolites contributing to this inflammatory component remain incompletely defined.Methods: We integrated reported gut microbiota–metabolite associations with GD-associated genes, constructed protein–protein interaction and enrichment networks, prioritized candidate metabolites, and evaluated predicted docking of isoquercetin (ISO) with inflammatory targets. Two-sample Mendelian randomization (MR) used MiBioGen microbiota and FinnGen autoimmune hyperthyroidism data. A separate four-group experiment compared control, M22 alone, interferon-γ (IFN-γ) alone, and combined M22/IFN-γ stimulation in Nthy-ori 3– 1 thyroid follicular epithelial cells. ISO treatment was then evaluated under combined stimulation; inflammatory transcripts, NF-κB p65 phosphorylation, and cell viability were assessed.Results: Fifty-three shared targets were identified, and the highest-degree PPI nodes included IL6, TNF, AKT1, IL1B, JUN, NFKB1, TP53, and CASP3. The shared targets were enriched in TNF, NF-κB, MAPK, PI3K-Akt, and Th17-related pathways. After chemical-name standardization, ISO had a network degree of 8 and was connected with TNF and IL6; docking indicated predicted molecular compatibility rather than direct binding. Twelve taxa showed nominal IVW associations with GD, but none survived false discovery rate correction. In the four-group experiment, IFN-γ was the dominant stimulus for IL1B, IL6, and TNF expression; the combined M22/IFN-γ group had the highest mean expression, but no interaction term was significant. Under combined stimulation, ISO reduced inflammatory transcripts and the phospho-p65/total p65 ratio without markedly changing total p65.Conclusion: These exploratory, convergent findings identify ISO as a microbiota-related candidate modulator of thyroid epithelial inflammatory responses. They do not establish microbial production, direct target binding, or clinical efficacy, and require further mechanistic and in vivo validation.Keywords: Graves’ disease, gut microbiota, isoquercetin, network pharmacology, Mendelian randomization, NF-κB, thyroid inflammation