Liang-Ge-San, a classic traditional Chinese medicine formula, attenuates acute inflammation in zebrafish and RAW 264.7 cells

Abstract

Ethnopharmacological relevance: Liang-Ge-San (LGS) is a traditional Chinese medicine formula that commonly used in acute inflammatory diseases. However, the anti-inflammatory effects and the underlying mechanisms of LGS are not fully studied.

Aim of the study: This study aims to investigate the anti-inflammatory activity and explore the underlying mechanisms of LGS in zebrafish and cell inflammation models.

Materials and methods: LPS-induced zebrafish inflammation model was established by LPS-yolk microinjection. The protective effect of LGS on zebrafish injected with LPS was observed using survival analysis. Infiltration of inflammatory cells was determined by H&E staining assay. Expression levels of key inflammatory cytokines TNF-α and IL-6 were measured by q-PCR assay. Recruitment of neutrophils and macrophages were observed by fluorescence microscopy, SB staining and NR staining. In vitro anti-inflammatory effects of LGS were evaluated on LPS-stimulated RAW 264.7 cells. The generation of IL-6 and TNF-α was detected by ELISA. The protein expression levels of JNK, p-JNK (Thr183/Tyr185), Nur77 and p-Nur77 (Ser351) were determined by Western blotting. Finally, two additional inflammatory models in zebrafish, which were induced by CuSO₄ or tail fin injury, were also established and the recruitment of neutrophils and macrophages were observed for the determination of the anti-inflammatory activity of LGS.

Results: LGS protected zebrafish against LPS-induced death and dose-dependently inhibited LPS-induced acute inflammatory response in zebrafish, as indicated by increased survival rate, reduced infiltration of inflammatory cells, decreased recruitment of macrophages and neutrophils, and downregulated expression levels of TNF-α and IL-6. Additionally, LGS inhibited the secretion of TNF-α and IL-6, increased the expression of Nur77, and reduced the expression of p-Nur77 (Ser351) and p-JNK (Thr183/Tyr185) in LPS-stimulated RAW 264.7 cells. The anti-inflammatory action of LGS was also observed in another two zebrafish inflammation models, which was supported by the inhibition on neutrophils and macrophages recruitment.

Conclusion: The present study demonstrates that LGS possesses anti-inflammatory activity in zebrafish inflammation models and LPS-stimulated RAW 264.7 cells, which is related to the inhibition on p-JNK and p-Nur77. This finding provides a pharmacological basis for LGS in the control of inflammatory disorder.

Keywords: Acute inflammation; LPS; Liang-Ge-San; RAW 264.7 cell; Zebrafish.