DXXK exerts anti-inflammatory effects by inhibiting the lipopolysaccharide-induced NF-κB/COX-2 signalling pathway and the expression of inflammatory mediators.

Diao Xin Xue Kang (DXXK) is the active pharmaceutical ingredient of the traditional Chinese medicinal product DXXK capsules, which have been approved for the treatment of cardiovascular disease and have been widely used clinically in China for many years with distinct curative effects. In March 2012, DXXK capsules were approved in the Netherlands, making them the first traditional herbal medicinal product (THMP) made outside of Europe. To assess the anti-inflammatory effects of DXXK and the underlying mechanisms at the cellular and molecular levels. In this study, lipopolysaccharide (LPS) was used to induce inflammation in RAW 264.7 cells. The sulphorhodamine B (SRB) assay was used to study the effect of DXXK on the proliferation of RAW 264.7 cells. Gene expression levels of cyclooxygenase 1 (COX-1), COX-2, tumour necrosis factor-alpha (TNF-α), IL-1β and IL-6 were assessed by reverse transcription polymerase chain reaction (RT-PCR), while COX-2 protein levels were evaluated using western blotting. The levels of PGE2 in the culture media were detected by enzyme-linked immunosorbent assay (ELISA), while TNF-α, IL-1β and IL-6 levels were detected using a Milliplex Map Mouse Cytokine Panel system. The activation and nuclear translocation of nuclear transcription factor κB (NF-κB) were studied using western blotting. In vivo studies in mice were carried out using the carrageenan-air pouch models of inflammation. In exudates, leucocytes were counted, total protein was determined using the Bradford assay, nitric oxide(NO) levels were assessed using the Griess reagent, and PGE2 and TNF-α levels were quantified by ELISA. The SRB assay showed that at the doses used in this study (10, 20 and 40 μg/mL), DXXK did not affect the proliferation of RAW 264.7 cells. DXXK (10, 20 and 40 μg/mL) inhibited LPS-induced PGE2 production by down-regulating the expression of COX-2, without influencing COX-1 expression. We also demonstrated that DXXK reduced the expression of pro-inflammatory cytokines, including TNF-α, IL-1β and IL-6, at both the gene and protein levels. Furthermore, DXXK inhibited LPS-induced NF-κB activation and nuclear translocation by suppressing the phosphorylation of IκB. Consistent with the in vitro results, the in vivo studies demonstrated that DXXK reduced leucocyte counts as well as total protein, NO, PGE2 and TNF-α levels in the exudates of mice with carrageenan-air pouch inflammation. The current study revealed that DXXK has a significant anti-inflammatory effect that may be attributed to its inhibitory effect on the NF-κB/COX-2 pathway and associated inflammatory mediators, including PGE2, NO, TNF-α, IL-1β and IL-6. The current study provides additional evidence of the effects of DXXK in inflammation. Based on the combination of our results and previously reported data, we propose that DXXK has multiple pharmacological effects that could be harnessed to treat systemic diseases.

Yu Y ，Li X ，Qu L ，Chen Y ，Dai Y ，Wang M ，Zou W ... -  《-》

Anti-inflammatory and antitumoural effects of Uncaria guianensis bark.

Uncaria guianensis (Aublet) Gmell (Rubiaceae) is a medicinal plant from the jungles of South and Central America, used to treat cancer, arthritis, diabetes, and inflammation. Evaluate the anti-inflammatory and anti-tumor effects of Uncaria guianensis preparations. Bio-guided fractionation of a hydroethanolic extract of Uncaria guianensis was performed, evaluating the fractions and subfractions for their effect on inflammatory mediators, tumour necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) by ELISA and nitric oxide (NO) by the Griess reaction in cultured supernatant from RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). The expression of cyclooxygenase 2 (COX-2), inducible nitric oxide synthase (iNOS) and inhibitor of κB (IκB) were investigated in RAW 264.7 macrophages by flow cytometry. The activity of NF-κB in HeLa cells transfected with a luciferase reporter system was determined. The effect of Uncaria guianensis on the inflammatory response in vivo was assessed in BALB/c mice stimulated with LPS, on rat paw oedema induced by carrageenan, and on tumour growth and lung metastasis in BALB/c mice inoculated with 4T1 mammary tumour cells. Immune cell infiltrates and inflammatory mediators were evaluated in the tumour by immunohistochemistry. Sub-fraction Ug AIV inhibited, to varying degrees, NO, TNF-α, IL-6 and PGE2 production by macrophages in vitro (30 μg/ml) and in the serum of LPS-challenged mice (5 mg/kg). Macrophage expression of Cox-2 was inhibited (35%), IκB degradation was completely inhibited and NF-κB activation was inhibited (70%) by Ug AIV at 30 μg/ml. Ug AIV decreased paw oedema by 86% (5 mg/kg) and serum NO and TNF-α by 45% and 65% respectively. Ug AIV reduced 4T1 mammary tumour growth by 91% on day 33 post-inoculation as well as the levels of serum NO, IL-6 and TNF-α in the same animals. Ug AIV decreased the number of tumour-infiltrating T lymphocytes, macrophages and neutrophils as well as the number of cells positive for COX-2, iNOS, IL-6, TNF-α and p65. As Ug AIV was not cytotoxic for tumour cells or macrophages, its anti-tumour effect may be due to a reduction in pro-tumoural inflammatory processes in the tumour microenvironment, possibly mediated through NF-κB.

Urdanibia I ，Michelangeli F ，Ruiz MC ，Milano B ，Taylor P ... -  《-》

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 ... -  《-》

Nauclea officinalis inhibits inflammation in LPS-mediated RAW 264.7 macrophages by suppressing the NF-κB signaling pathway.

Nauclea officinalis has been traditionally used in China for the treatment of fever, pneumonia and enteritidis etc. This study aims to investigate effects of N. officinalis on the inflammatory response as well as the possible molecular mechanism in LPS-stimulated RAW 264.7 murine macrophage cells. Anti-inflammatory activity of N. officinalis (10, 20, 50, and 100µg/mL) was investigated by using LPS-induced RAW 264.7 macrophages. The NO production was determined by assaying nitrite in culture supernatants with the Griess reagent. The levels of TNF-α, IL-6 and IL-1β in culture media were measured with ELISA kits. Real time fluorescence quantitative PCR was detected for mRNA expression of iNOS, TNF-α, IL-6 and IL-1β. Western blot assay was performed to illustrate the inhibitory effects of N. officinalis on phosphorylation of IκB-α and NF-κB p65. Treatment with N. officinalis (10-100µg/mL) dose-dependently inhibited the production as well as mRNA expression of NO, TNF-α, IL-6 and IL-1β in RAW 264.7 macrophages. Western blot assay suggested that the mechanism of the anti-inflammatory effect was associated with the inhibition of phosphorylation of IκB-α and NF-κB p65. The results indicated that N. officinalis potentially inhibited the activation of upstream mediator NF-κB signaling pathway via suppressing phosphorylation of IκB-α and NF-κB p65 to inhibit LPS-stimulated inflammation.

Zhai XT ，Zhang ZY ，Jiang CH ，Chen JQ ，Ye JQ ，Jia XB ，Yang Y ，Ni Q ，Wang SX ，Song J ，Zhu FX ... -  《-》

Nodakenin suppresses lipopolysaccharide-induced inflammatory responses in macrophage cells by inhibiting tumor necrosis factor receptor-associated factor 6 and nuclear factor-κB pathways and protects mice from lethal endotoxin shock.

Rim HK ，Cho W ，Sung SH ，Lee KT ... -  《-》