Abstract Body (Do not enter title and authors here): Background: Atrial fibrillation (AF) significantly increases the risk of stroke and heart failure, with high disability and mortality rates. Recent studies have shown that neutrophil extracellular traps (NETs) contribute to AF pathogenesis by aggravating myocardial injury via cytotoxic effects on cardiomyocytes and activation of inflammatory cascades. Fuzheng Kangfu Decoction (FZKF), a traditional Chinese herbal formula comprising Codonopsis pilosula, Poria cocos, and other herbs, has shown clinical efficacy in treating AF and AF with heart failure, but its mechanisms remain unclear. This study aimed to explore FZKF’s regulatory effects on NETs in vitro and in vivo to better understand its anti-AF mechanism.
Methods and Results: An AF mouse model was induced by intraperitoneal lipopolysaccharide and high-frequency (35 Hz) electrical stimulation. FZKF treatment significantly reduced AF inducibility, as shown by more regular RR intervals and fewer fibrillatory waves on electrocardiogram. It also mitigated atrial tissue injury and reduced pro-inflammatory cytokines, as assessed by H&E staining and ELISA. Immunostaining showed that FZKF suppressed NETs formation, with decreased expression of neutrophil markers (Ly6G, CD11b) and NETs components (ELA2, MPO) in left atrial tissue. Western blot confirmed reduced expression of citrullinated histone H3 (Cit-H3), a NET-specific marker. Atrial RNA-seq data revealed neutrophil involvement and implicated damage-associated molecular patterns (DAMPs), such as HMGB1 and HMGB2, suggesting possible underlying mechanisms.
At the cellular level, primary neutrophils were stimulated with conditioned medium (CM) from rapid-paced murine atrial HL-1 cells. FZKF treatment reduced both HMGB1 and HMGB2 mRNA in HL-1 cells, while a significant reduction was observed in HMGB2 protein levels, both intracellularly and in the CM. Importantly, CM from FZKF-treated HL-1 cells exhibited a markedly reduced ability to promote neutrophil chemotaxis (Transwell assay) and NETs formation (MPO-positive reticular structures and Cit-H3 expression).
Conclusion: This study demonstrates that FZKF’s anti-AF effect may involve suppression of cardiomyocyte-derived DAMPs and neutrophil NETs formation, thereby alleviating inflammation-mediated atrial injury and reducing AF susceptibility.