Ethanol extract from a Chinese herbal formula, Zuojin Pill, inhibit the expression of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 mouse macrophages

Ethnopharmacological relevance

Zuojin Pill (ZJP), a traditional Chinese medicinal decoction that has been used in treating gastritis, gastric ulcer since 15th century, contains two herbs: Rhizoma Coptidis and Fructus Evodiae in the ratio of 6:1 (w/w). Alkaloids are the main active principles contributing to ZJP's efficacy, but anti-inflammatory mechanism has not been fully clarified.

Aim of the study

The objective of the study is to reveal anti-inflammatory molecular mechanism of ethanol extract from ZJP, which would form an additional proof to the traditional experience of ZJP in clinical administration.

Materials and methods

Seven alkaloids were determined from the ethanol extract of ZJP using High Performance Liquid Chromatography (HPLC) with the gradient mobile phase. The ethanol extract from ZJP were used to evaluate the anti-inflammatory action in murine macrophage cell line RAW 264.7 treated with lipopolysaccharide (LPS). Production of nitric oxide (NO) and prostaglandin E₂ (PGE₂) were measured by the Griess colorimetric method and enzyme-linked immunosorbent assay (ELISA), respectively. Proteome profiler array was analyzed to evaluate 40 cytokines at protein level. In addition, interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) synthesis were analyzed using ELISA to confirm the result of the Proteome profiler array. The gene expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-6, and interleukin 1β (IL-1β) were detected by quantitative real-time reverse-transcription polymerase chain reaction (real-time RT-PCR). Furthermore, the nuclear translocation of the NF-κB p50 and p65 subunits was detected with ELISA.

Results

The secretions of NO, PGE₂ and the mRNA expression of iNOS, COX-2 were significantly inhibited, moreover, the protein and mRNA expressions of IL-6, IL-1β and TNF-α were inhibited by preventing the nuclear translocation of the NF-κB p50 and p65 subunits. The proteome profiler array showed that 15 cytokines and chemokines involved in the inflammatory process were down-regulated by ZJP.

Conclusion

These results suggest that the anti-inflammatory properties of ethanol extract from ZJP might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1β, and TNF-α expression through preventing the nuclear translocation of the NF-κB p50 and p65 subunits in RAW 264.7 cells. In addition, these results provided evidence to understand the therapeutic effects of ZJP on gastritis, gastric ulcer, and other inflammatory diseases in clinic.