Effects of compounds from Bi-Qi Capsule on the expression of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 macrophages

Abstract

The Bi-Qi Capsule (Bi-Qi) has been used in clinic as prescribed drug for the treatment of rheumatic arthritis, rheumatoid arthritis and other osteoarticular diseases about 20 years in China. Pharmacological and clinical studies have confirmed the anti-inflammatory and analgesic action of Bi-Qi in vivo. However, its anti-inflammatory molecular mechanism is still unclear. The objective of the study is to reveal the anti-inflammatory molecular mechanism of Bi-Qi which would form an additional proof to the traditional experience of Bi-Qi in clinical administration. The aqueous extract of Bi-Qi was used to evaluate the anti-inflammatory action in murine macrophage cell line RAW 264.7 treated with lipopolysaccharide (LPS). Cell viability was evaluated by MTT assay. Production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) were measured by the Griess colorimetric method and enzyme-linked immunosorbent assay (ELISA), respectively. Protein expression levels of cyclooxygenase 2 (COX-2) were monitored by cell-based ELISA. Proteome profiler array was analyzed to evaluate 40 cytokines at protein level. In addition, interleukin 6 (IL-6) and tumor necrosis factor-alpha (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), 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). Bi-Qi significantly decreased the production of NO, PGE(2), and inhibited the protein expression of COX-2. The Proteome profiler array showed that eight protein cytokines were down-regulated and six protein cytokines were up-regulated by Bi-Qi. Furthermore, the results of TNF-α and IL-6 protein expression analyzed by ELISA were similar to those of Proteome profiler array. The results of real-time RT-PCR demonstrated that iNOS, COX-2, TNF-α, IL-6 and IL-1β gene expression were also significantly reduced by Bi-Qi. These results suggested that the anti-inflammatory molecular mechanism of Bi-Qi might be the results from modulating the LPS-mediated NO-iNOS pathway, COX-2 pathway via inhibition of iNOS, COX-2, TNF-α, IL-6 and IL-1β expression in activated macrophages. In addition, these results provided evidence to understand the therapeutic effects of Bi-Qi on various inflammatory diseases, e.g. rheumatoid arthritis, rheumatic arthritis and other osteoarticular diseases.