Baicalin attenuates myocardial ischemia-reperfusion injury by modulating TLR4/MD-2-associated inflammatory responses and reducing neutrophil‑related tissue injury

The sterile inflammatory response, particularly the self-amplifying axis involving complement, neutrophils, and neutrophil extracellular traps (NETs), is a critical driver of myocardial ischemia-reperfusion injury (MIRI). However, effective strategies to therapeutically target this amplification cascade are limited. Baicalin (BAI), a flavonoid with known anti-inflammatory properties, may modulate this intricate network, but its specific impact on the sterile immune amplification axis in MIRI remains undefined. An MIRI model was established in SD rats, which were randomly assigned to Sham, I/R, and I/R + BAI groups. Cardiac function and infarct size were assessed via echocardiography, TTC staining, and serum biomarkers. Transcriptome sequencing (RNA-seq) analyzed differentially expressed genes and enriched pathways. Key pathways were validated using immunohistochemistry, immunofluorescence, ELISA, and Western blot. In an HL-60 cell model, the interaction between BAI and TLR4/MD-2 was investigated via CCK-8, qPCR, Western blot, CETSA, and molecular docking. BAI significantly reduced myocardial infarct size (as assessed by IA/TA), improved cardiac function, and decreased serum cTnI and LDH levels induced by MIRI. Transcriptome analysis suggested that BAI reversed I/R-induced expression of genes related to complement‑related responses, neutrophil degranulation, and TLR signaling. Further experiments indicated that BAI attenuated complement C3a generation, neutrophil infiltration, and markers associated with NETs formation. Mechanistically, BAI interacted with the TLR4/MD-2 complex (likely via MD-2), suppressed downstream NF-κB/NLRP3 signaling activation, and reduced pro-inflammatory cytokine release. The TLR4 inhibitor TAK-242 mimicked the protective effects of BAI, whereas HMGB1 partially reversed the anti-inflammatory effects of BAI. This study shows that BAI attenuates the pathogenic sterile immune amplification axis, comprising complement‑related responses, neutrophil infiltration, and NETs‑associated markers, to confer cardioprotection against MIRI. Its mechanism of action is functionally linked to the interaction with the TLR4/MD-2 complex and subsequent inhibition of downstream inflammatory signaling. These findings highlight a promising immunomodulatory strategy for MIRI treatment by modulating a critical upstream node within this amplification network; however, direct binding between baicalin and the TLR4/MD-2 complex remains to be indicated in future studies.