Ethanol extract of Adiantum capillus-veneris L. suppresses the production of inflammatory mediators by inhibiting NF-κB activation.

Adiantum capillus-veneris L. is a wildly distributed plant species and has been extensively used in south of China as traditional folk medicine for the treatment of inflammatory diseases. To investigate the anti-inflammatory effect of ethanolic extracts of Adiantum capillus-veneris L. and the involvement of NF-κB signaling in the regulation of inflammation. The plant ethanolic extracts were initially tested against lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) production in RAW264.7 mouse macrophages, and interleukin 6 (IL-6) and tumor necrosis factor (TNF) production in human U937 monocytes. The effect of the plant extracts on the transcription factor nuclear factor kappa B (NF-κB) pathway was evaluated in TNF-α stimulated HepG2 cells by luciferase gene reporter assay and Western blotting at the transcriptional and translational levels. Subsequently, the inhibition of NF-κB downstream gene expression (IL-8 and ICAM-1) by the plant extracts was assessed via quantitative real time polymerase chain reaction (qPCR). Lastly, the anti-inflammatory activities of the plant extracts in vivo were evaluated by testing spleen index and NF-κB related protein expression in LPS-stimulated CD1 mice. The plant ethanolic extracts effectively suppressed PGE2, IL-6 and TNF release with an IC50 less than 50 μg/ml. Moreover, luciferase expression could be specifically blocked in HepG2 cells, not in HEK293 cells, showing that the plant extracts displayed a cell-specific pattern on NF-κB gene transcription. The assayed biological activity also depended on the order of adding TNF-α and the plant extracts because the plant extracts could only block the NF-κB activation if added earlier but were unable to stop the signal when added after TNF-α. However, the plant extracts did not exert any effect on ubiquitination which regulates several steps in the NF-κB pathway. Additionally, the plant extracts down-regulated phosphorylation of IKKα/β at S176/180, p38 at T180/Y182 and p65 at S536, but not p65 at S276. This was confirmed by their ability to selectively abrogate the induction of IL-8 transcription, whereas the ICAM-1 gene, which is not transcribed selectively by an NF-κB complex containing a form of p65 phosphorylated on Ser536, did not change. Finally, the plant extracts at 200 μg/mg could normalize the LPS-induced elevation of spleen index as well as NF-κB and p38 activations in CD1 mice. The present studies presents the potential utilization of this plant extracts, as a natural resources for the development of an anti-inflammatory medicine.

Yuan Q ，Zhang X ，Liu Z ，Song S ，Xue P ，Wang J ，Ruan J ... -  《-》

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 ... -  《-》

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 ... -  《-》

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 anti-nociceptive activities of ethanolic extract and its various fractions from Adiantum capillus veneris Linn.

To investigate the anti-inflammatory and anti-nociceptive activities of the crude ethanolic extract of Adiantum capillus veneris Linn. (Adiantaceae) and its various fractions. The ethanolic extract and its fractions were given at a dose of 200 mg/kg po and 300 mg/kg po for testing their anti-inflammatory activity by carrageenan induced hind paw edema. The analgesic activity of the ethanolic extract and its fractions has been carried out by tail-flick method and writhing test at a dosage of 300 mg/kg po. Gastric ulceration studies have been further carried out to study the antiulcer effect of the ethanolic extract and its various fractions at dose of 900 mg/kg body weight. Amongst the tested fractions, the ethyl acetate fraction exhibited better inhibition (67.27%) at 300 mg/kg po dosage when compared to the standard drug Indomethacin (63.63%) after 3h in the carrageenan induced hind paw edema. The anti-inflammatory activity of the ethanolic extract and its various fractions appear to be related to the inhibition of NO release, and the decreasing TNF-α level. The ethanolic extract and all its fractions especially the ethyl acetate (p<0.01) showed significant analgesic activity with insignificant ulceration as compared to the standard drug, i.e. ibuprofen. The histopathological study of ethanolic extract and its fractions reveals that none of them cause ulcer. The present study indicates that Adiantum capillus veneris Linn. has significant anti-inflammatory and analgesic effect.

Haider S ，Nazreen S ，Alam MM ，Gupta A ，Hamid H ，Alam MS ... -  《-》