OBJECTIVE To evaluate the anti-inflammatory effects of Guanxinjing(a proprietary Chinese medicine for promoting blood circulation and removing blood stasis) and to investigate its bioactive components and mechanisms of action by using zebrafish and RAW264.7 macrophage inflammation models.

METHODS Lipopolysaccharide(LPS)-induced inflammation models were established in macrophage-labeled transgenic zebrafish line Tg(mpeg:eGFP) and RAW264.7 cells to assess the regulatory effects of Guanxinjing on macrophage aggregation in vivo and macrophage activation in vitro. HPLC was employed to identify the main chemical components of Guanxinjing. Representative active components were screened using the zebrafish inflammation model and the nitric oxide(NO) release level of RAW264.7 cells. For the key identified component, ligustilide’s anti-inflammatory effects were validated in both in vivo and in vitro models. Western blotting was performed to determine the protein levels of c-Jun N-terminal kinase(JNK) and phosphorylated JNK(p-JNK) to explore the molecular mechanism.

RESULTS Compared with the model group, Guanxinjing significantly inhibited LPS-induced macrophage aggregation in the zebrafish yolk sac and reduced NO production, inducible nitric oxide synthase(iNOS) gene expression, and tumor necrosis factor-alpha(TNF-α) secretion in RAW264.7 cells, these results indicated that Guanxinjing could effectively inhibit macrophage activation. HPLC identified 7 major components including danshensu, albiflorin, paeoniflorin, salvianolic acid B, salvianolic acid A, senkyunolide A, and ligustilide. Anti-inflammatory screening revealed that ligustilide significantly reduced macrophage aggregation and reactive oxygen species(ROS) levels in zebrafish. Furthermore, ligustilide exhibited concentration-dependent inhibition of LPS-induced NO release, iNOS expression, and both transcriptional and protein levels of the inflammatory cytokine interleukin-6(IL-6) in macrophages, while suppressing the activation of the inflammation-related JNK signaling pathway via downregulation of p-JNK protein expression.

CONCLUSION Guanxinjing effectively alleviates inflammatory responses, among which ligustilide is the key anti-inflammatory component. Ligustilide significantly regulates the release of inflammatory mediators and inhibits macrophage activation, and its mechanism of action may be related to the JNK signaling pathway.