Yigongsan decoction ameliorates anemia of chronic disease by attenuating mothers against decapentaplegic homolog-signal transducer and activator of transcription 3 interaction to regulate iron homeostasis.

OBJECTIVE: To investigate how Yigongsan decoction (YGS) regulates iron metabolism in anemia of chronic disease (ACD) by modulating the hepcidin-ferroportin axis, with particular focus on crosstalk between the mothers against decapentaplegic homolog (SMAD) and signal transducer and activator of transcription 3 (STAT3) signaling pathways. METHODS: In vivo: a lipopolysaccharide (LPS)-induced ACD mouse model was treated with YGS (15.413 g·kg-1·d-1) for 7 d. Iron metabolism was evaluated by measuring serum and splenic iron levels, as well as hepcidin antimicrobial peptide (HAMP) and ferroportin (Fpn) mRNA expression (quantitative real-time polymerase chain reaction, qRT-PCR), and protein-protein interactions (co-immunoprecipitation, Co-IP). In vitro: HepG2 cells were stimulated with interleukin-6 (IL-6) or activin B and treated with 10% YGS-medicated serum. Iron metabolism was evaluated by intracellular and extracellular iron. STAT3/SMAD pathway activity was assessed by siRNA knockdown, Western blotting of phosphorylated SMAD1/5/8 (p-SMAD1/5/8) and phosphorylated STAT3 (p-STAT3), and Co-IP. RESULTS: LPS-treated mice, YGS significantly decreased splenic iron content (P <0.01) and increased serum iron levels (P <0.05). Hepatic HAMP mRNA was downregulated, while Fpn mRNA was upregulated. In HepG2 cells, YGS counteracted IL-6/activin B-induced dysregulation, reducing hepcidin and intracellular iron while increasing ferroportin and extracellular iron. siRNA experiments confirmed that YGS restored the hepcidin-ferroportin balance independently of direct SMAD or STAT3 inhibition. Co-IP and immunofluorescence revealed that YGS attenuated the competitive interaction between phosphorylated SMAD1/5/8 (p-SMAD1/5/8) and phosphorylated STAT3 (p-STAT3), reducing their nuclear co-localization and subsequent HAMP transcription. CONCLUSION: Our results suggest that YGS may alleviate ACD by attenuating the interaction between the SMAD and STAT3 pathways, which appears to lead to reduced STAT3 phosphorylation and hepcidin expression, thereby re-establishing iron homeostasis. This identifies YGS as a potential therapeutic agent targeting inflammation-induced iron dysregulation.