Daidzein attenuates hepatic ischemia/reperfusion injury via HMGB1-TLR4 signaling pathway.

Daidzein, a soy isoflavone, has been demonstrated to mitigate cerebral and myocardial ischemia- reperfusion injury. However, its potential protective effects against hepatic ischemia-reperfusion injury (HIRI) remain unclear. This study aimed to investigate the hepatoprotective role of daidzein in HIRI and elucidate its underlying mechanisms. In our study, a HIRI model was established in male C57BL/6 mice, and intraperitoneal administration of daidzein was initiated 3 days prior to modeling. The extent of liver injury was assessed by measuring serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Histopathological changes in liver tissues were evaluated via HE staining, while hepatocyte apoptosis was detected using the TUNEL assay. Immunohistochemistry was employed to analyze high-mobility group box 1 (HMGB1) translocation. Western blotting was performed to quantify the protein expression levels of BCL-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), TLR4 (Toll-like receptor 4), myeloid differentiation factor 88 (MyD88), and phosphorylated p65 (p-p65) in liver tissues. Real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to measure proinflammatory cytokine levels in liver tissues and serum, respectively. To validate whether daidzein exerts its protective effects by inhibiting HMGB1 release, exogenous HMGB1 was administered post- reperfusion. For in vitro experiments, the AML-12 cell line was subjected to cobalt chloride (CoCl2)-induced cell death, and HMGB1 nucleocytoplasmic translocation was analyzed via immunofluorescence and Western blotting. The results showed that daidzein treatment significantly reduced serum AST and ALT levels, ameliorated histopathological liver damage, suppressed HMGB1 release, and attenuated proinflammatory cytokine production. Mechanistically, daidzein downregulated the expression of the proapoptotic protein Bax while upregulating the antiapoptotic protein Bcl-2. It also inhibited HMGB1 nucleocytoplasmic translocation and reduced the protein expression levels of TLR4, MyD88, and p-NF-κB. Exogenous HMGB1 partially reversed the protective effects of daidzein against HIRI. In vitro experiments confirmed that daidzein alleviated CoCl2-induced AML-12 cell death and suppressed HMGB1 nucleocytoplasmic translocation. In conclusion, daidzein attenuates HIRI by modulating the HMGB1-TLR4/MyD88/NF-κB signaling pathway. These findings provide a theoretical foundation for its clinical application in the prevention and treatment of HIRI.