Effects of compounds from bi-qi capsule on the expression of inflammatory mediators in lipopolysaccharide-stimulated RAW 264.7 macrophages.

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.

Wang QS ，Cui YL ，Wang YF ，Chi W ... -  《-》

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

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. 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. 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 E(2) (PGE(2)) 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. The secretions of NO, PGE(2) 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. 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.

Wang QS ，Cui YL ，Dong TJ ，Zhang XF ，Lin KM ... -  《-》

Amelioration of Danhong injection on the lipopolysaccharide-stimulated systemic acute inflammatory reaction via multi-target strategy.

Gao LN ，Cui YL ，Wang QS ，Wang SX ... -  《-》

Aqueous extract of Vitex trifolia L. (Labiatae) inhibits LPS-dependent regulation of inflammatory mediators in RAW 264.7 macrophages through inhibition of Nuclear Factor kappa B translocation and expression.

Vitex trifolia L. (Labiatae), a widespread tree found from the Asia-Pacific to the east Africa regions is used in the traditional medicine of the Pacific islands to treat inflammatory-associated conditions. We herein evaluated its in vitro regulatory effects on the expression profile of lipopolysaccharide (LPS)-induced inflammatory genes focusing on regulation of chemokines C-X-C motif 10 (CXCL-10) and C-C motif ligand 3 (CCL-3) and cyclo-oxygenase (COX)-2. Furthermore, the plant effect on the LPS-mediated activation of Nuclear Factor kappa B (NF-κB) was also studied. Aqueous extract of Vitex trifolia leaves was prepared and evaluated for its effect on LPS-induced stress and toxicity-related genes in RAW 264.7 macrophage cells using RT(2) Profiler Polymerase Chain Reaction (PCR) Array System. Effects of the extract on LPS-induced chemokines CCL-3 and CXCL-10, COX-2, and NF-κB p50 and p65 mRNA levels were also studied using Reverse Transcription quantitative PCR (RT-qPCR) technique. Translocation of the nuclear factor was further assessed by measuring its nuclear p65 subunit via an ELISA-based TransAM method. Vitex trifolia extract at 5000μg/ml exerted a significant inhibitory effect on the expression of various LPS-induced inflammatory genes in RAW 264.7 cells after 8h of incubation time. Using RT-qPCR, this anti-inflammatory effect was further confirmed by significant inhibition of CCL-3 and CXCL-10 mRNA production in LPS-stimulated RAW 264.7 cells upon treatment with 2500μg/ml of Vitex trifolia extract. Furthermore, the inhibitory activity of this plant on LPS-induced COX-2 mRNA was also observed at a concentration of 2500μg/ml in a time-dependent manner. TransAM assays showed that LPS-induced NF-κB translocation was also inhibited by Vitex trifolia extract even at a concentration of extract as low as 250μg/ml. RT-qPCR assays showed that aqueous extract of Vitex trifolia leaves had a significant inhibitory activity on LPS-induced p50 mRNA synthesis. Interestingly, however, no effect on p65 subunit mRNA expression was observed. Moreover, PCR array analysis showed that LPS-induced inflammatory and apoptosis genes under NF-κB control are also repressed by the extract. The anti-inflammatory properties of Vitex trifolia extract seem associated with inhibition of NF-κB translocation through a reduction in the expression level of NF-κB p50 but interestingly not p65 subunit mRNA. The regulatory effects of Vitex trifolia on NF-κB and consequently on inflammation mediators such as chemokines CCL-3 and CXCL-10, and COX-2 provide new evidence of its efficacy and emphasise its high potential therapeutic value. However, further in vivo experiments are still required to validate its utilization as a remedy against inflammatory diseases.

Matsui M ，Adib-Conquy M ，Coste A ，Kumar-Roiné S ，Pipy B ，Laurent D ，Pauillac S ... -  《-》

Zedoarondiol isolated from the rhizoma of Curcuma heyneana is involved in the inhibition of iNOS, COX-2 and pro-inflammatory cytokines via the downregulation of NF-kappaB pathway in LPS-stimulated murine macrophages.

Cho W ，Nam JW ，Kang HJ ，Windono T ，Seo EK ，Lee KT ... -  《-》