Effects of taraxasterol on inflammatory responses in lipopolysaccharide-induced RAW 264.7 macrophages

Aim of the study

Taraxasterol, a pentacyclic-triterpene, was isolated from the Chinese medicinal herb Taraxacum officinale. In the present study, we investigated the in vitro anti-inflammatory activity of taraxasterol in lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages.

Materials and methods

RAW 264.7 cells were pretreated with 2.5, 5, or 12.5 μg/ml of taraxasterol 1 h prior to treatment with 1 μg/ml of LPS. Nitric oxide (NO) level in supernatants from cells was examined by Griess reaction, the concentrations of prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were measured by ELISA. Nuclear factor kappa B (NF-κB) activation was evaluated by immunocytochemical analysis.

Results

We found that taraxasterol inhibited NO, PGE₂, TNF-α, IL-1β and IL-6 production in LPS-induced RAW 264.7 macrophages in a dose-dependent manner. Further studies revealed that taraxasterol prevented the LPS-induced NF-κB translocation from cytoplasm into nuclear.

Conclusions

These results indicate that taraxasterol has anti-inflammatory effect by blocking NF-κB pathway.     

Chrysanthemum indicum Linné extract inhibits the inflammatory response by suppressing NF-κB and MAPKs activation in lipopolysaccharide-induced RAW 264.7 macrophages

Aims of study

Although the flowers of Chrysanthemum indicum Linné (Asteraceae) have long been used in traditional Korean and Chinese medicine to treat inflammatory diseases, the underlying mechanism(s) by which these effects are induced remains to be defined. We investigated the effects of a 70% ethanolic extract of C. indicum (CIE) on the activities of cellular signaling molecules that mediate inflammatory responses.

Materials and methods

Production of NO, PGE₂, TNF-α, and IL-1β by ELISA, mRNA and protein expression of iNOS and COX-2, phosphorylation of MAPKs, and activation of NF-κB by RT-PCR and Western blotting were examined in LPS-induced RAW 264.7 macrophages.

Results

The CIE strongly inhibited NO, PGE₂, TNF-α, and IL-1β production, and also significantly inhibited mRNA and protein expression of iNOS and COX-2 in LPS-induced RAW 264.7 macrophages, in a dose-dependent manner. Furthermore, the CIE clearly suppressed nuclear translocation of NF-κB p65 subunits, which correlated with an inhibitory effect on IκBα phosphorylation. The CIE also attenuated the activation of ERK1/2 and JNK in a dose-dependent manner.

Conclusion

Our results suggest that the anti-inflammatory properties of CIE might result from the inhibition of inflammatory mediators, such as NO, PGE₂, TNF-α, and IL-1β, via suppression of MAPKs and NF-κB-dependent pathways.     

Anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium on LPS-stimulated Raw264.7 cells

Aim of the study

Lilium lancifolium is commonly used to treat bronchitis, pneumonia, etc. In this study, we investigated the anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium (LL extracts) in LPS-stimulated Raw264.7 cells.

Material and methods

Levels of NO, PGE₂ and pro-inflammatory cytokines (IL-6 and TNF-α) in the supernatant fraction were determined using sandwich ELISA. Expression of COX-2 and iNOS, phosphorylation of MAPK subgroups (ERK and JNK), and NF-κB activation in extracts were detected via Western blot and immunocytochemistry assays.

Results

The LL extract significantly inhibited NO, PGE₂, IL-6 and TNF-α production in LPS-stimulated cells, and suppressed iNOS and COX-2 expression. A mechanism-based study showed that phosphorylation of ERK1/2 and JNK and translocation of the NF-κB p65 subunit into nuclei were inhibited by the LL extract. Furthermore, interleukin-4 and interleukin-13 production in Con A-induced splenocytes was suppressed.

Conclusion

These results indicate that anti-inflammatory effects of methanol extracts from Lilium lancifolium are due to downregulation of iNOS and COX-2 via suppression of NF-κB activation and nuclear translocation as well as blocking of ERK and JNK signaling in LPS-stimulated Raw264.7 cells.     

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.     

A herbal formula containing roots of Salvia miltiorrhiza (Danshen) and Pueraria lobata (Gegen) inhibits inflammatory mediators in LPS-stimulated RAW 264.7 macrophages through inhibition of nuclear factor κB (NFκB) pathway

Ethnopharmacological relevance

The herbal formula DG, containing roots of Salvia miltiorrhiza (Danshen) and Pueraria lobata (Gegen), has long history in treating cardiovascular diseases. It has been shown to be able to reduce intima-media thickening in coronary patients in our previous clinical study. Since intima-media thickening is the hallmark of atherosclerotic disease, the etiology of which is inflammation of the arterial wall, the mechanism underlying the effect of DG may be related to its anti-inflammatory activities.

Aim of study

The present study aims to determine the anti-inflammatory activity of DG and elucidate its underlying mechanisms with regards to its molecular basis of action.

Materials and method

The anti-inflammatory effect of DG was studied by using lipopolysaccharide (LPS)-stimulated activation of nuclear factor κB (NFκB) pathway and subsequent production of inflammatory mediators, including nitric oxide (NO), prostaglandin E₂ (PGE₂), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and macrophage chemotactic protein-1 (MCP-1), in mouse RAW 264.7 macrophages.

Results

The present study demonstrated that DG could suppress the production of NO and PGE₂ through the inhibition of iNOS and COX-2 genes. DG could also inhibit the production of IL-1β, IL-6 and MCP-1, but not TNF-α, through the inhibition of respective mRNA expressions. Further investigations showed the inhibitory effect of DG on activation of IKKα/β and degradation of IκBα, thus preventing nuclear translocation of NFκB. All these results suggested the inhibitory effects of DG on the production of inflammatory mediators through the inhibition of the NFκB pathway.

Conclusions

The inhibitory effects of DG on the production of inflammatory mediators by LPS-stimulated RAW 264.7 macrophages, are accomplished by inhibiting the nuclear translocation of NFκB through inactivating IKKα/β and preventing degradation of IκBα.     

Anti-inflammatory effect of Phellinus linteus grown on germinated brown rice on dextran sodium sulfate-induced acute colitis in mice and LPS-activated macrophages

Ethnopharmacological relevance and aim of the study

Phellinus linteus is a herb used in traditional Asian medicine to treat stomachache, inflammation, and tumors. Recent studies show that the extract of Phellinus linteus has anti-inflammatory and antitumor activities. However, Phellinus linteus extract has limitation of high cost and limited availability because of supply shortage. Here, we grew Phellinus linteus on germinated brown rice to address the issue of supply shortage and investigated anti-inflammatory effect in vivo as well as in vitro.

Materials and methods

Phellinus linteus grown on germinated brown rice (PBR) were extracted using filtration steps, which included γ-aminobutyric acid (GABA). The PBR (200, 500 mg/kg/day) was applied into the mouse model of dextran sodium sulfate (DSS)-induced colitis and lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells. We used sulfasalazine as a reference drug. In addition, mechanism related to anti-inflammatory was investigated by Western blotting.

Results

In the mouse model of DSS-induced colitis, PBR ameliorated the pathological characteristics of colitis such as shortening of colon length and improved the disease activity index score. In addition, we showed that PBR reduced the expression of nuclear factor-kappa B (NF-κB) in colitis. Western blotting showed that PBR decreased the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (Cox-2) proteins. Further, PBR treatment reduced the expression of mitogen-activated protein kinases (MAPKs) (e.g., extracellular signal-regulated protein kinase (ERK) and p38) in the mouse model of DSS-induced colitis.

Conclusions

Treatment of RAW 264.7 macrophages with a combination of PBR and LPS showed a significant concentration-dependent inhibition of nitric oxide (NO) and prostaglandin E2 (PGE₂) production. In addition, we determined the ability of PBR to reduce the iNOS and tumor necrosis factor (TNF)-α expression. PBR inhibited the expression of iNOS, NF-κB, and Cox-2 proteins in LPS-stimulated RAW264.7 macrophages. This study presents the potential use of PBR as a drug candidate against colitis.     

Biomolecular evidence of anti-inflammatory effects by Clematis mandshurica Ruprecht root extract in rodent cells

Ethnopharmacological relevance

Clematis mandshurica Ruprecht root is widely used in Asia as an analgesic and anti-inflammatory agent. This research investigated the anti-inflammatory effects of Clematis mandshurica Ruprecht root extract (CRE) using RAW 264.7 macrophage cells and carrageenan- (CA-) induced rat paw edema.

Materials and methods

Production of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, nitric oxide (NO) and prostaglandin E₂ (PGE₂) in the culture supernatant, mRNA expression of TNF-α, IL-1β, IL-6, iNOS and COX-2, protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in the extract were assayed. In addition, the effect of CRE on acute inflammation in vivo was observed using CA-induced rat hind paw edema assay. The changes on the histopathology and histomorphometry of hind paw skins—dorsum and ventrum pedis were observed using CA-treated rats.

Results

Treatment with CRE (0.25, 0.5, and 1 mg/mL) resulted in inhibited levels of protein expression of lipopolysaccharide- (LPS-) induced iNOS, COX-2, NF-κB, and MAPKs (ERK, JNK, and p38) as well as production of TNF-α, IL-1β, IL-6, NO, and PGE₂ induced by LPS. Consistent with these results, CRE reduced the LPS-induced expressions of these cytokines, iNOS and COX-2 at the mRNA levels in a dose-dependent manner. In particular, results of the CA-induced rat hind paw edema assay showed an anti-edema effect of CRE. In addition, treatment with CRE resulted in dose-dependent inhibition of CA-induced increases of skin thickness, mast cell degranulation, and infiltrated inflammatory, TNF-α, IL-1β, iNOS, and COX-2-positive cells in both dorsum and ventrum pedis skin, respectively.

Conclusions

These results demonstrate that CRE exhibits anti-inflammatory activities via decreasing production of pro-inflammatory mediators through suppression of the pathways of NF-κB and MAPKs in LPS-induced macrophage cells. In addition, results of the CA-induced rat hind paw edema assay show an anti-edema effect of CRE. Our findings also support the traditional use of CRE in the inflammatory symptoms of rheumatic arthritis and acute icteric hepatitis. Thus, CRE may have therapeutic potential for a variety of inflammation-mediated diseases and may be developed into potent anti-inflammatory drugs.     

Anti-inflammatory effects of YeongyoSeungma-tang

Ethnopharmacological relevance

YeongyoSeungma-tang which includes Fructus Forsythia, has been used in oriental traditional medicine for treatment of early smallpox and atopic dermatitis.

Aim of the study

YeongyoSeungma-tang was carried out to investigate for anti-inflammatory effects.

Materials and methods

YeongyoSeungma-tang was tested for anti-inflammatory effects against lipopolysaccharide (LPS)-induced nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor alpha (TNF-α) releases as well as nuclear factor kappa B (NF-κB) expression using RAW264.7 macrophage cells.

Results

YeongyoSeungma-tang significantly inhibited generation of NO (42% and 59% inhibition at doses of 5 μg/mL and 10 μg/mL, respectively), PGE₂ (46% and 80% inhibition at doses of 5 μg/mL and 10 μg/mL, respectively) and TNF-α (6% and 23% inhibition at doses of 5 μg/mL and 10 μg/mL, respectively) on LPS-stimulated RAW 264.7 cells in a concentration-dependent manner. Consistently in these observations, the expression of inducible NO synthase (iNOS) enzyme was also inhibited by YeongyoSeungma-tang. However, YeongyoSeungma-tang did not show any influence on the expression of cyclooxygenase-2. The cream containing 0.075% YeongyoSeungma-tang showed good skin moisturizing effect without any irritation.

Conclusion

The present study may support the fact that YeongyoSeungma-tang can have the good possibility as an anti-inflammatory agent for troubled skins.     

Achyranthes japonica exhibits anti-inflammatory effect via NF-κB suppression and HO-1 induction in macrophages

Ethnopharmacological relevance

The roots of Achyranthes japonica Nakai have been used in traditional herbal medicine for the treatment of edema and arthritis in Korea.

Aim of the study

In this study, we investigated the molecular mechanism responsible for anti-inflammatory effects of the aqueous extract of A. japonica roots (AJ) in LPS-stimulated macrophages.

Materials and methods

Nitric oxide (NO) production and as inducible nitric oxide synthase (iNOS) expression were examined in TG-elicited peritoneal macrophages and RAW 264.7 cells. Cell viability was monitored by MTT assay. Protein and mRNA expressions were determined by Western blotting and RT-PCR, respectively. The activity of NF-κB and Nrf2 were examined by EMSA, immunocytochemistry or reporter assay.

Results

AJ inhibited LPS-induced NO secretion as well as iNOS expression, without affecting cell viability. Furthermore, AJ suppressed LPS-induced NF-κB activation, degradation of IκB-α, phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. Further study demonstrated that AJ induced heme oxygenase-1 (HO-1) gene expression via nuclear translocation and transactivation of Nrf2. In addition, the inhibitory effects of AJ on iNOS expression were abrogated by small interfering RNA-mediated knock-down of HO-1.

Conclusions

These results suggest that AJ suppresses LPS-induced NO production and iNOS expression in macrophages through the inhibition of IκB/NF-κB and MAPK as well as the Nrf2-mediated HO-1 induction. These findings provide the scientific rationale for anti-inflammatory therapeutic use of A. japonica roots.