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.     

Shikonin derivatives inhibited LPS-induced NOS in RAW 264.7 cells via downregulation of MAPK/NF-kappaB signaling

AIM OF THE STUDY: Shikonin/alkannin (SA) derivatives, analogs of naphthoquinone pigments, are the major components of root extracts of the Chinese medicinal herb (Lithospermum erythrorhizon; LE) and widely distributed in several folk medicines. In the present study, the effect and the underline molecular mechanism of shikonin derivatives isolated from root extracts of Lithospermum euchroma on lipopolysaccharide (LPS)-induced inflammatory response were investigated. MATERIALS AND METHODS: Effects of five SA derivatives, including SA, acetylshikonin, beta,beta-dimethylacrylshikonin, 5,8-dihydroxy-1.4-naphthoquinone, and 1,4-naphthoquinone on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in mouse macrophage RAW264.7 cells were examined. RESULTS: Data suggested that SA derivatives inhibited LPS-induced NO and PGE(2) production, and iNOS protein expression. RT-PCR analysis showed that SA derivatives diminished LPS-induced iNOS mRNA expression. Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in LPS-stimulated RAW 264.7 cells was concentration-dependently suppressed by SA derivatives. SA inhibited NF-kappaB activation by prevention of the degradation of inhibitory factor-kappaB and p65 level in nuclear fractions induced by LPS. CONCLUSIONS: Taken together, these results suggest that the anti-inflammatory properties of SA derivatives might result from inhibition of iNOS protein expression through the downregulation of NF-kappaB activation via suppression of phosphorylation of ERK, in LPS-stimulated RAW 264.7 cells.     

Praeruptorin a inhibits lipopolysaccharide‐induced inflammatory response in murine macrophages through inhibition of NF‐κB pathway activation

Praeruptorin A (PA) is a pyranocoumarin compound isolated from the dried root of Peucedanum praeruptorum Dunn (Umbelliferae). However, the antiinflammatory effect of PA has not been reported. The present study investigated the antiinflammatory effect of PA in lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells. PA significantly inhibited the LPS‐induced production of nitric oxide (NO), interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α). The mRNA and protein expressions of inducible nitric oxide synthase (iNOS), IL‐1β and TNF‐α were also suppressed by this compound. Further study showed that PA decreased the cytoplasmic loss of inhibitor κB‐α (IκB‐α) protein and inhibited the translocation of NF‐κB from cytoplasm to nucleus. Taken together, the results suggest that PA may exert antiinflammatory effects in vitro in LPS‐stimulated RAW 264.7 macrophages through inhibition of NF‐κB signal pathway activation. Copyright © 2010 John Wiley & Sons, Ltd.     

Anti-inflammatory effect of Citrus Unshiu peel in LPS-stimulated RAW 264.7 macrophage cells

Citrus Unshiu peel (CUP) has been traditionally used in East Asia as a drug for the treatment of vomiting and dyspepsia. However, its effects on inflammation remain unknown. In this study, we investigated the effects of CUP on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The research focused on determining whether CUP could inhibit the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and the activation of nuclear factor (NF)-κB, mitogen-activated protein kinases (MAPKs), as well as the secretion of nitric oxide (NO), prostaglandin (PG) E(2), tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in LPS-stimulated RAW 264.7 cells. We found that CUP represses LPS-induced iNOS and COX-2 gene expression as well as NO, PGE(2), TNF-α and IL-6 production. Additionally, CUP inhibited the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal kinase (JNK) MAPK, and suppressed IκBα degradation and nuclear translocation of NF-κB. Collectively, our results indicate that CUP inhibits the production of various inflammatory mediators via blockade of MAPK phosphorylation pursuant to the inhibition of IκBα degradation and the nuclear translocation of NF-κB. These findings are the first to clarify the mechanism underlying the anti-inflammatory effect exerted by CUP in RAW 264.7 macrophage cells stimulated by inflammatory agents.     

Effect of Seabuckthorn (Hippophae rhamnoides) flavone on immune system: an in-vitro approach

There are several reports, which suggest that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavones, of Seabuckthorn (SBT) (Hippophae rhamnoides L.) fruit berry can modulate the production and level of several signaling molecules associated with immune function and inflammation in vitro, including several cytokines. We have evaluated the immunomodulatory activity of ethanolic solution of SBT flavone (FLV) in human peripheral blood mononuclear cells (PBMCs). The SBT flavone was found to stimulate production of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in PBMCs. However, increased expressions of p-IkappaB, NF-kappaB, and p-p38 were found in flavone-treated human PBMCs with significantly suppressed expression of CD25 (IL-2R). There was no alteration found in the nitric oxide (NO) production in mouse macrophage cell line RAW 264.7. These observations suggest that stimulation of IL-6 and TNF-alpha secretion may contribute to the putative beneficial effects of dietary flavone against microbial infection.     

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.     

SunghyangJungki-San Ga Pogongyoung inhibits IL-1 mediated tumour necrosis factor-alpha secretion in astrocytes

Astrocytes play an important role in initiating and modulating inflammatory responses within the central nervous system (CNS). Extensive studies in rodents have shown that substance P induces inflammatory cytokine production in astrocytes. In this study we have examined whether an aqueous extract of SunghyangJungki-San Ga Pogongyoung (SSGP) inhibits the secretion of TNF-alpha from primary cultures of rat astrocytes. SSGP (10-1,000 microg/mL) significantly inhibited the TNF-alpha secretion by astrocytes stimulated with lipopolysaccharide (LPS) and substance P (SP). Interleukin-1 (IL-1) has been shown to elevate TNF-alpha secretion from LPS-stimulated astrocytes while having no effect on astrocytes in the absence of LPS. We therefore examined whether IL-1 mediated inhibition of TNF-alpha secretion from primary astrocytes by SSGP. Treatment with SSGP (10-1,000 microg/mL) to astrocytes stimulated with both LPS and SP decreased IL-1 secretion significantly. Moreover, the secretion of TNF-alpha by LPS and SP in astrocytes was progressively inhibited with an increasing amount of IL-1 neutralizing antibody. Our results suggest that SSGP may inhibit TNF-alpha secretion by inhibiting IL-1 secretion and that SSGP has an antiinflammatory activity in the CNS.     

Canna indica L. attenuates high-glucose- and lipopolysaccharide-induced inflammatory mediators in monocyte/macrophage

Ethnopharmacological relevance

Canna indica L. (CI) has been widely used as a folklore medicine in tropical and subtropical areas with beneficial effects in numerous diseases, including infection, rheumatism, hepatitis, and it has also been identified as an antioxidant.

Materials and methods

The present study aimed to investigate the effect of Canna indica CI ethanolic extract (CIE) on productions of nitric oxide (NO), prostaglandin E₂ (PGE₂), and interleukin-1β (IL-1β) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. In addition, the effects of CIE in high glucose (HG)-induced U937 monocytes on mRNA expressions of IL-8 and monocyte chemoattractant protein-1 (MCP-1), and regulation of mitogen-activated protein kinase (MAPK) pathways were also identified.

Results

CIE was found to inhibit the production of inflammatory mediators including NO, IL-1β, and PGE₂ from LPS-induced RAW 264.7 macrophages. The increases in HG-induced mRNA expressions of IL-8 and MCP-1 were also significantly inhibited by CIE. Stimulation of HG in U937 monocytes resulted in activation of p38 MAPK, ERK1/2, and JNK. However, CIE treatment significantly decreased phosphorylation of p38 MAPK, ERK1/2, and JNK.

Conclusion

The present study demonstrated that CIE suppressed the LPS-induced inflammatory mediator production and also inhibited HG-induced inflammatory mediator expression by the regulation of MAPK pathway.     

Heme oxygenase-1 mediates the anti-inflammatory effect of mushroom Phellinus linteus in LPS-stimulated RAW264.7 macrophages

This work aimed to elucidate the anti-inflammatory mechanism of the n-BuOH subfraction (PL) prepared from fruiting bodies of Phellinus linteus. PL induced heme oxygenase-1 (HO-1) of the RAW264.7 macrophages in concentration- and time-dependent manner. It suppressed induction of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO) through down-regulation of iNOS promoter activity in lipopolysaccharide (LPS)-stimulated macrophages. Zn(II) protoporphyrin IX (ZnPP), a specific inhibitor of HO-1, partly blocked suppression by PL on iNOS promoter activity and NO production, which were elevated in LPS-stimulated macrophages. LPS was able to enhance NO production via reactive oxygen species (ROS) generation, c-Jun NH(2)-terminal kinase (JNK) and c-Jun induction. ZnPP prevented PL from down-regulating ROS generation and JNK activation in LPS-stimulated macrophages. Taken together, PL shows its anti-inflammatory activity via mediation of HO-1 in an in vitro inflammation model.