Flower extract of Panax notoginseng attenuates lipopolysaccharide-induced inflammatory response via blocking of NF-κB signaling pathway in murine macrophages

Aim of the study

The root of Panax notoginseng (PN) is commonly used to treat chronic liver disease with its therapeutic abilities to stop haemorrhage in the circulation, while the PN flower (PN-F) is largely unknown in the biological activities on inflammation and mechanisms of its actions. In this study, the pharmacologic effects of PN-F methanol extract on inflammation were investigated to address potential therapeutic or toxic effects in LPS-stimulated mouse macrophage cells, RAW264.7 cells.

Materials and methods

Production of NO, PGE₂ and pro-inflammatory cytokines (TNF-α and IL-1β) in supernatant, the expression of iNOS, COX-2 and cytokines, the phosphorylation of MAPK moleduces (ERK1/2, JNK and p38 MAPK), and the activation of NF-κB in PN-F extract were assayed in LPS-stimulated RAW264.7 cells.

Results

PN-F extract significantly inhibited the productions of NO, PGE₂, TNF-α and IL-1β on the LPS-stimulated RAW264.7 cells. In addition, PN-F extract suppressed the mRNA and protein expressions of iNOS, COX-2, TNF-α and IL-1β in LPS-stimulated RAW264.7 cells. The molecular mechanism of PN-F extract-mediated attenuation in RAW264.7 cells has close a relationship to suppressing the phosphorylation of MAPK molecules such as ERK1/2, JNK and p38 MAPK, and the translocation of NF-κB p65 subunit into nuclear.

Conclusion

These results indicate that PN-F extract inhibits LPS-induced inflammatory response via the blocking of NF-κB signaling pathway in macrophages, and demonstrated that PN-F extract possesses anti-inflammatory properties in vitro.     

Anti-inflammatory and anti-arthritic activity of total flavonoids of the roots of Sophora flavescens

Ethnopharmacological relevance

The roots of Sophora flavescens have long been used in Chinese medicine for the treatment of fever, inflammatory disorders, ulcers and skin burns. Sophora flavescens contains flavonoids and alkaloids.

Aim of the study

This study was conducted to develop a plant-based anti-inflammatory agent focused on chronic inflammatory disorders. To accomplish this, the alkaloid-free prenylated flavonoid-enriched fraction (PFS) of rhizomes of Sophora flavescens was prepared and its in vitro and in vivo anti-inflammatory activities were then evaluated for the first time.

Materials and methods

The inhibitory activity of PFS on PGE₂, NO, IL-6 and TNF-α production of lipopolysaccharide (LPS)-treated RAW 264.7 cells was measured. Additionally, adjuvant-induced arthritis in rats was used as an animal model of chronic inflammation to establish the in vivo anti-inflammatory effects of PFS.

Result

PFS inhibited cyclooxygenase-2 (COX-2)-catalyzed PGE₂ and inducible nitric oxide synthase (iNOS)-catalyzed NO production by lipopolysaccharide (LPS)-treated RAW 264.7 cells at 10–50 μg/ml, and these effects primarily occurred via COX-2 inhibition and iNOS down-regulation, respectively. PFS also inhibited IL-6 and TNF-α production. When tested against adjuvant-induced arthritis in rats (chronic inflammation), PFS strongly inhibited arthritic inflammation when administered orally at doses of 10–100 mg/kg/day. In addition, PFS administered orally potently inhibited acetic acid-induced writhing in mice.

Conclusions

Our results suggest that PFS inhibits chronic inflammatory response and the inhibition of proinflammatory molecules such as COX-2, iNOS and IL-6 may contribute, at least in part, to the anti-inflammatory activity in vivo. Overall, these results indicate that PFS from Sophora flavescens may have the potential for treatment of chronic inflammatory disorders such as rheumatoid arthritis.     

Anti-inflammatory effects of Z23 on LPS-induced inflammatory responses in RAW264.7 macrophages

Aim of the study

Fissistigma oldhamii (Hemsl.) Merr, a traditional Chinese herb medicine, is used for treating rheumatoid arthritis in China. In our previous study, an effective compound, 7′-(3′,4′-dihydroxyphenyl)-N-[(4-methoxyphenyl) ethyl] propenamide (Z23), from this herb has showed potent immunosuppressive effects both in vitro and in vivo. However, its anti-inflammatory effect and mechanism is still need to explore.

Materials and methods

We examined the in vitro effects of Z23 on the production of nitric oxide (NO), prostaglandin E2 (PGE2) and cytokines by lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.

Results

Z23 significantly decreased the production of PGE2, NO, tumour necrosis factor alpha (TNFalpha) and IL6 production. Inducible nitric oxide synthase (iNOS) and cyclooxygenase2 (COX2) gene expression were also significantly reduced.

Conclusions

These results demonstrated that Z23 exerted an anti-inflammatory effect through modulating the synthesis of several mediators and cytokines involved in the inflammatory process. This study provided evidence to understand the therapeutic effects of Fissistigma oldhamii (Hemsl.) Merr and indicated that Z23 has the potential for treatment of various inflammatory diseases where the overproduction of NO, PGE2 and inflammatory cytokines has been shown to play a role, e.g. rheumatoid arthritis.     

Wogonoside displays anti-inflammatory effects through modulating inflammatory mediator expression using RAW264.7 cells

Ethnopharmacological relevance

The root of Scutellaria baicalensis Georgi, also called Huangqin in China, is an herbal-based nutraceutical which is usually used in Chinese medicated diet (CMD). As an abundant ingredient in Huangqin, wogonoside is a flavonoid glycoside. The present work investigated the anti-inflammatory activities of wogonoside in lipopolysaccharides (LPS)-induced RAW264.7 cells.

Materials and methods

RAW264.7 cells were used. The inhibition of wogonoside against nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in LPS-induced RAW264.7 cells were measured. Additionally, the effects of wogonoside on mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), TNF-α and IL-6 were also investigated.

Results and discussion

Wogonoside not only dose-dependently decreased the production of inflammatory mediators including NO and PGE₂ but also inhibited the release of pro-inflammatory cytokines including TNF-α and IL-6 in LPS-induced RAW264.7 cells. Furthermore, wogonoside possessed significantly in vitro inhibitory effects on the gene expression of iNOS, COX2, TNF-α and IL-6.

Conclusion

These results suggest that wogonoside may be used as a functional food component for prevention and treatment of inflammation.     

Aloe-emodin from rhubarb (Rheum rhabarbarum) inhibits lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages

Ethnopharmacological relevance

Rheum rhabarbarum (rhubarb) has long been used for the treatment of inflammation in China and other Asian countries. However, the mechanism underlying the anti-inflammatory activity of this medicinal plant is not fully understood. The present study was designed to investigate the anti-inflammatory effects of anthraquinones, the major constituents in rhubarb, and the molecular mechanism involved in their anti-inflammatory effects.

Materials and methods

RAW264.7 cells were stimulated by lipopolysaccharide (LPS) in the presence or absence of the compounds examined. The proliferation of RAW264.7 cells was assayed by the Alamar-Blue method. The quantity of nitric oxide (NO) was determined by Griess assay. The expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR. Inducible nitric oxide synthase (iNOS), inhibitor of nuclear factor κBα (IκBα), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), and Akt/phosphoinositide 3-kinase (PI3K) protein expression levels were determined by Western blotting.

Results

Aloe-emodin markedly suppressed the production of NO, interleukin-6 (IL-6), and interleukin-1β (IL-1β) in LPS-stimulated RAW264.7 cells with no apparent cytotoxicity. The mRNA expression levels of iNOS, IL-6, and IL-1β genes were also significantly inhibited by aloe-emodin. Western blot analysis showed that aloe-emodin suppressed LPS-induced iNOS protein expression, IκBα degradation, and the phosphorylation of ERK, p38, JNK, and Akt.

Conclusions

These results demonstrate that aloe-emodin is the bioactive component of rhubarb that confers an anti-inflammatory effect through a likely mechanism involving a decrease in pro-inflammatory cytokine production in LPS-induced RAW264.7 macrophages via inhibition of NF-κB, MAPK, and PI3K pathways.     

Echinacea increases arginase activity and has anti-inflammatory properties in RAW 264.7 macrophage cells,indicative of alternative macrophage activation

Ethnopharmacological relevance

The genus Echinacea is a popular herbal immunomodulator. Recent reports indicate that Echinacea products inhibit nitric oxide (NO) production in activated macrophages.

Aim of the study

In the present study we determined the inhibitory effects of alcohol extracts and individual fractions of alcohol extracts of Echinacea on NO production, and explored the mechanism underlying the pharmacological anti-inflammatory activity.

Materials and methods

Alcohol extracts of three medicinal Echinacea species, Echinacea angustifolia, Echinacea pallida and Echinacea purpurea, were prepared using Soxhlet apparatus and fractionated using HPLC. NO production by LPS activated RAW 264.7 macrophage cells was measured using a Griess reagent and iNOS detected using immunoblotting. In addition, effects on arginase activity were measured in RAW 264.7 cells stimulated with 8-bromo-cAMP +/− LPS.

Results

Alcohol extracts of all three Echinacea species significantly inhibited NO production by lipopolysaccharide (LPS)-activated the RAW 264.7 macrophage cell line; among them Echinacea pallida was the most active. The Echinacea-mediated decrease in NO production was unlikely due to a direct scavenging of NO because the extracts did not directly inhibit NO released from an NO donor, sodium nitroprusside. An immunoblotting assay demonstrated that the extract of Echinacea pallida inhibited inducible nitric oxide synthase (iNOS) protein expression in LPS-treated macrophages. The enzymes iNOS and arginase metabolize a common substrate, l-arginine, but produce distinct biological effects. While iNOS is involved in inflammatory response and host defense, arginase participates actively in anti-inflammatory activation. Arginase activity of RAW 264.7 cells stimulated with 8-bromo-cAMP was significantly increased by alcohol extracts of all three Echinacea species. The polar fraction containing caffeic acid derivatives enhanced arginase activity, while the lipophilic fraction containing alkamides exhibited a potential of inhibiting NO production and iNOS expression.

Conclusions

These results suggest that the anti-inflammatory activity of Echinacea might be due to multiple active metabolites, which work together to switch macrophage activation from classical activation towards alternative activation.     

Anti-inflammatory and immune-modulating effect of Ulmus davidiana var. japonica Nakai extract on a macrophage cell line and immune cells in the mouse small intestine

Ethnopharmacological relevance

Traditional Oriental medicine has utilized the barks of the stem and root of Ulmus davidiana var. japonica Nakai (UD) to treat inflammatory disorders.

Aim of the study

The purpose of the present study was to evaluate UD's anti-inflammatory and immune-modulating effects on a lipopolysaccharide (LPS)-stimulated macrophage cell line and small-intestinal lamina propria (LP) cells, respectively.

Materials and methods

RAW 264.7 cells were stimulated with LPS in the presence of various concentrations of a UD water-soluble extract. Cell viability, nitric oxide (NO) production, and the level of inflammatory cytokines synthesis were measured. Among the mice receiving the UD water-soluble extract, changes in the LP cell populations and immunoglobulin (Ig)A production were evaluated.

Results

The UD water-soluble extract inhibited LPS-induced NO synthesis and inflammatory cytokine production in a RAW264.7 macrophage-like cell line. Small-intestinal LP cells isolated from mice that received the UD extract displayed a decrease in the side scatter of medium-to-high cells. Those LP cells isolated from the UD-treated mice also showed a marked decrease of intracellular IgA. However, UD administration had no apparent effect on the synthesis of systemic inflammatory cytokines.

Conclusions

These results suggest that UD water-soluble extracts have anti-inflammatory properties and, as such, can be used to promote intestinal immune-homeostatic conditions.     

Mechanism of anti-inflammatory activity of umbelliferone 6-carboxylic acid isolated from Angelica decursiva

Aims of the study

We recently reported the potential antioxidant and anti-inflammatory activities of umbelliferone 6-carboxylic acid (UMC) isolated from the whole plants of Angelica decursiva. In this study, we elucidated the anti-inflammatory mechanisms of UMC in vitro and in vivo.

Methods

The inhibitory effects of UMC on the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor-α (TNF-α), the expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the activation of nuclear factor kappa B (NF-κB) were evaluated using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The reactive oxygen species (ROS) generation inhibitory activity of UMC was evaluated using t-butyl hydroperoxide (t-BHP)-induced RAW 264.7 cells. Furthermore, the in vivo anti-inflammatory activity of UMC was evaluated using carrageenan induced mouse paw edema model.

Results

UMC dose-dependently inhibited NO and PGE₂ production by down-regulating iNOS and COX-2 protein expression in LPS-stimulated RAW 264.7 macrophages. UMC also suppressed the production of the proinflammatory cytokine TNF-α in LPS stimulated RAW 264.7 cells in a concentration dependent manner. In addition, UMC dose-dependently prevented LPS-induced nuclear translocation of NF-κB in RAW 264.7 macrophages. Furthermore, UMC exhibited the inhibitory activity against t-BHP-induced ROS generation in RAW 264.7 cells with an IC₅₀ value of 705.1 μg/ml. Moreover, UMC inhibited λ-carrageenan induced mouse paw edema by 70.40 and 60.20% at doses of 50 and 25 mg/kg body weight, respectively.

Conclusion

The combined results of this study indicate that UMC is an important anti-inflammatory constituent of A. decursiva and its anti-inflammatory effect was due to its ability to inhibit the production of inflammatory mediators via inhibition of NF-κB activation pathway.     

In vitro anti-inflammatory effects of arctigenin,a lignan from Arctium lappa L., through inhibition on iNOS pathway

Ethnopharmacological relevance

Arctigenin, a bioactive constituent from dried seeds of Arctium lappa L. (Compositae) which has been widely used as a Traditional Chinese Medicine for dispelling wind and heat included in Chinese Pharmacophere, was found to exhibit anti-inflammatory activities but its molecular mechanism remains unknown yet.

Aim of the study

To investigate the anti-inflammatory mechanism of arctigenin.

Materials and methods

Cultured macrophage RAW 264.7 cells and THP-1 cells were used for the experiments. Griess assay was used to evaluate the inhibitory effect of arctigenin on the overproduction of nitric oxide (NO). ELISA was used to determine the level of pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). The inhibitory effect on the enzymatic activity of cyclooxygenase-2 (COX-2) was tested by colorimetric method. Western blot was used to detect the expression of inducible nitric oxide synthase (iNOS) and COX-2.

Results

Arctigenin suppressed lipopolysaccharide (LPS)-stimulated NO production and pro-inflammatory cytokines secretion, including TNF-α and IL-6 in a dose-dependent manner. Arctigenin also strongly inhibited the expression of iNOS and iNOS enzymatic activity, whereas the expression of COX-2 and COX-2 enzymatic activity were not affected by arctigenin.

Conclusions

These results indicated that potent inhibition on NO, TNF-α and IL-6, but not COX-2 expression and COX-2 activity, might constitute the anti-inflammatory mechanism of arctigenin. Arctigenin suppressed the overproduction of NO through down-regulation of iNOS expression and iNOS enzymatic activity in LPS-stimulated macrophage.     

Ethanol extract of Alismatis Rhizoma reduces acute lung inflammation by suppressing NF-κB and activating Nrf2

Ethnopharmacological relevance

The tuber of Alisma orientale Juzepzuk, a medicinal herb that has been used for the treatment of various disorders in Korea, has an anti-inflammatory effect. Here, we investigated a possible underlying mechanism and a protective effect on acute lung injury (ALI).

Materials and methods

Alisma orientale tuber was extracted in 80% ethanol and dried. The powder of the ethanol extract of Alisma orientale tuber (EEAO) was dissolved in PBS. The effect of EEAO on NF-κB and Nrf2 activities was analyzed with RAW 264.7 cells. The effect of EEAO on lung inflammation was determined by histologic and molecular biological analyses of the lung tissue of C57BL/6 mice that were gavaged once a day with 0.3 or 1.2 g/kg of EEAO for 14 days, prior to an intranasal administration of LPS (0.01 g/kg) for inducing ALI.

Results

EEAO pre-treatment of RAW 264.7 cells suppressed NF-κB activity and the expression of its dependent genes including COX-2, IL-1β and iNOS. Similar treatment enhanced Nrf2 activity and the expression of Nrf2-regulated genes including NQO-1, HO-1 and GCLC. LPS instillation induced acute neutrophilic lung inflammation, which was significantly suppressed by pre-treatment with EEAO. Analysis of the lungs revealed that EEAO pre-treatment induced the expression of Nrf2-regulated genes, with concomitant down-regulation of inflammatory gene expression.

Conclusions

EEAO attenuated lung inflammation in LPS-induced ALI mice, which was associated with differential regulation of NF-κB and Nrf2 activities. We suggest that EEAO can be developed as a potential therapeutics for the treatment of ALI.     

Anti-inflammatory effects of ethyl acetate fraction from Melilotus suaveolens Ledeb on LPS-stimulated RAW 264.7 cells

Aim of the study

This paper aimed to elucidate the anti-inflammatory effects of EtOAc fraction prepared from Melilotus suaveolens Ledeb ethanol extract with a cellular model of LPS-stimulated RAW 264.7 cell.

Materials and methods

Some key pro-inflammatory cytokines and mediators including IL-1β, IL-6, NO, iNOS, COX-2 and TNF-α, two important anti-inflammatory cytokines and mediators IL-10 and HO-1, I-κB and NF-κB were studied by sandwich ELISA, real-time PCR, western blot analysis and immunocytochemistry. At last a HPLC fingerprint was taken to evaluate the fraction.

Results

The EtOAc fraction could significantly inhibit the production of IL-1β, IL-6, NO, TNF-α, COX-2 in LPS-stimulated cell than that of single LPS-stimulated cell (p < 0.01 or p < 0.05), and the extract could increase the production of IL-10 and HO-1 than that of single LPS intervention cell (p < 0.01 or p < 0.05). Meanwhile, the extract also could inhibit the production of NF-κB compared to single LPS-stimulated cell. All the results showed that the extract had a good anti-inflammatory effect on LPS-stimulated RAW264.7 cell.

Conclusions

Taken together, the anti-inflammatory actions of M. suaveolens Ledeb EtOAc fraction might be due to the down-regulation of IL-1β, IL-6, NO, TNF-α and COX-2 via the suppression of NF-κB activation, and another pathway was up regulating the production of IL-10 and HO-1. Meanwhile, the EtOAc fraction might be further studied to isolate the active anti-inflammatory ingredients besides coumarin.