Effects of taraxasterol on iNOS and COX-2 expression in LPS-induced RAW 264.7 macrophages

Ethnopharmacological relevance

Taraxasterol was isolated from the Chinese medicinal herb Taraxacum officinale which has been frequently used as a remedy for inflammatory diseases. Our previous study has shown that taraxasterol inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) production in RAW 264.7 macrophages. To elucidate the underlying mechanism responsible for these effects, in the present study, we investigated the effects of taraxasterol on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, and mitogen-activated protein kinases (MAPKs) signaling pathway in LPS-induced RAW 264.7 macrophages.

Materials and methods

RAW 264.7 cells were pretreated with 2.5, 5 and 12.5 μg/ml of taraxasterol 1 h prior to treatment with 1 μg/ml of LPS. The mRNA expression levels of iNOS and COX-2 were examined by RT-PCR. The protein expression levels of iNOS and COX-2, and the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK) MAPKs were measured by Western blot.

Results

The mRNA and protein expression levels of iNOS and COX-2 were inhibited by taraxasterol in a concentration-dependent manner. Further studies revealed that taraxasterol suppressed the phosphorylation of ERK1/2 and p38 in LPS-induced RAW 264.7 macrophages.

Conclusions

These results indicate that taraxasterol inhibits iNOS and COX-2 expression by blocking ERK1/2 and p38 MAPKs signaling pathway.     

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.     

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.     

Mechanisms of anti-inflammatory property of Anacardium occidentale stem bark: Inhibition of NF-κB and MAPK signalling in the microglia

Ethnopharmacological relevance

Anacardium occidentale is used in traditional African medicine for the treatment of arthritis, fever, aches, pains, and inflammation of the extremities.

Aim of the study

In this study, we investigated the molecular mechanisms responsible for anti-inflammatory effects of a stem bark extract of A. occidentale (ANE) in LPS-stimulated microglia.

Materials and methods

Nitric oxide (NO), prostaglandin E₂ and cytokine (TNFα and IL-6) production were evaluated in supernatants from LPS-stimulated BV-2 cells. Cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and microsomal prostaglandin E2 synthase (mPGES-1) protein expressions in rat primary microglia were measured using western blot. The effects of ANE on NF-κB activation and nuclear translocation were evaluated in the luciferase reporter gene assay and ELISA, while ability of ANE to influence IκB phosphorylation was determined using ELISA specific for phospho-IκB. The involvement of MAPK phosphorylation in the anti-inflammatory actions of ANE was evaluated using specific ELISA for phospho-p38, phospho-p42/44 and phospho-JNK. The MTT assay was used to determine the effect of ANE on BV-2 microglia viability.

Results

ANE (25–100 μg/ml) produced significant (p<0.05) reduction in the production of NO, PGE₂, TNFα and IL-6 in BV-2 microglia stimulated with LPS for 24 h. Pre-treatment with ANE caused a significant (p<0.05) inhibition of COX-2, iNOS and mPGES-1 protein expressions in the rat primary microglia. Further experiments showed that ANE inhibited COX-2 and iNOS protein expression via IκB-mediated nuclear translocation and transactivation of NF-κB. Our studies also revealed that ANE produced significant (p<0.05) and dose-dependent inhibition of p38, p42/44 and JNK MAPK phosphorylation in LPS-activated BV-2 microglia.

Conclusions

We conclude that ANE has an anti-inflammatory property related to inhibition of inflammation-associated cytokine production as well as iNOS and COX-2 gene expression by blocking NF-κB and MAPK pathways in the microglia. It is also suggested that mPGES-1 inhibition contributes to the effect of ANE on PGE₂ production in the microglia.     

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.     

Effect of Rhizoma Polygonati on 12-O-tetradecanoylphorbol-acetate-induced ear edema in mice

Ethnopharmacological relevance

Rhizoma Polygonati is originated from the dried rhizomes of Polygonatum sibircum Red. It has long been used in traditional Chinese medicine for the treatment of inflammatory disorders.

Aim of the study

The present study aims to investigate the anti-inflammatory effect of aqueous extract of Rhizoma Polygonati (ERP) in a mouse model of inflammation induced by 12-O-tetradecanoylphorbol-acetate (TPA).

Materials and methods

The anti-inflammatory effect was evaluated by measuring the ear thickness and activity of myeloperoxidase (MPO). The anti-inflammatory mechanism was explored by determining the protein and mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6.

Results

The results showed that ERP significantly decreased the ear thickness and MPO activity in mouse model of inflammation induced by TPA. In addition, ERP also remarkably inhibited the protein and mRNA levels of iNOS, COX-2, TNF-α, IL-1β, and IL-6.

Conclusions

These results indicate that ERP has potential anti-inflammatory effect on TPA-induced inflammatory in mice, and the anti-inflammatory effect may be mediated, at least in part, by inhibiting the mRNA expression of a panel of inflammatory mediators including iNOS, COX-2, TNF-α, IL-1β, and IL-6.     

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.     

Drosera rotundifolia and Drosera tokaiensis suppress the activation of HMC-1 human mast cells

Ethnopharmacological relevance

Several Northern Hemisphere Drosera species have been used in the therapy of respiratory tract infections as the traditional medicine Droserae Herba.

Aim of the study

To determine the anti-inflammatory effects of Drosera species and to investigate a substitute material for Droserae Herba, we examined the effect of extracts of Drosera rotundifolia, Drosera tokaiensis and Drosera spatulata on activated T cell membrane (aTc-m)-induced inflammatory gene expression in HMC-1 human mast cells.

Materials and methods

Drosera rotundifolia, Drosera spatulata and Drosera tokaiensis were collected in Japan. Herbs were extracted with 80% EtOH, and subsequently applied to OASIS HLB column. HMC-1 cells were treated with each Drosera column-adsorbed fraction for 15 min, and subsequently added to aTc-m and incubated for 16 h. Inflammatory gene and protein expressions were determined by DNA microarray, RT-PCR and Western blotting.

Results

Drosera rotundifolia and Drosera tokaiensis fractions, but not the Drosera spatulata fraction, suppressed inflammatory gene expression induced by aTc-m in HMC-1 cells.

Conclusions

Drosera rotundifolia and Drosera tokaiensis suppressed activation of HMC-1 cells induced by aTc-m. Since the Drosera tokaiensis fraction was more effective than the traditionally used Drosera rotundifolia, Drosera tokaiensis is a likely substitute as a source of Droserae Herba.     

Inhibitory effect of Physalis alkekengi L. var. franchetii extract and its chloroform fraction on LPS or LPS/IFN-γ-stimulated inflammatory response in peritoneal macrophages

Ethnopharmacological relevance

The aerial parts of the plant Physalis alkekengi L. var. franchetii (PA) are traditionally used in folk medicine to treat cough, middle ear infection, sore throat, abscesses, and urinary problems. However, the cellular mechanisms underlying PA's possible anti-inflammatory effects are unknown.

Materials and methods

We examined the effect of a methanol extract of PA on the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-6. We also examined the extract's effect on the activity of IκBα, mitogen-activated protein kinases (MAPKs) and STAT1 in macrophages stimulated by lipopolysaccharide (LPS) or LPS/interferon-γ (IFN-γ). We further fractioned the extract into chloroform and water to investigate which fraction possessed anti-inflammatory activity.

Results

We found that the PA methanol extract significantly reduced the production of NO, iNOS, COX-2, TNF-α, and IL-6. The extract also inhibited LPS-induced IκBα degradation and MAPK activation as well as LPS/IFN-γ-induced STAT1 activation, effects observed at a higher concentration than that required to suppress iNOS. The chloroform fraction possessed the anti-inflammatory activity of PA by inhibiting the expression of iNOS, TNF-α and IL-6 through downregulation of IκBα degradation and MAPK activation.

Conclusion

Our findings indicate that the PA methanol extract contains different anti-inflammatory compounds, some of which suppressed iNOS expression and some of which inhibited IκBα degradation and MAPK activity. Further research is warranted to identify these anti-inflammatory components of PA and validate its use in animal studies.     

Tetramethylpyrazine inhibits production of nitric oxide and inducible nitric oxide synthase in lipopolysaccharide-induced N9 microglial cells through blockade of MAPK and PI3K/Akt signaling pathways,and suppression of intracellular reactive oxygen species

Aim of the study

To determine the inhibitory effect of tetramethylpyrazine (TMP) on lipopolysaccharide (LPS)-induced over-production of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in N9 microglial cells.

Materials and methods

N9 cells were pretreated with vehicle or TMP and then exposed to LPS for the time indicated. Cell viability was determined by methylthiazoyltetrazolium (MTT) assay. Nitrite assay was performed by Griess reaction. Expression of iNOS mRNA was examined by RT-PCR. Protein levels of iNOS, p38 mitogen-activated protein kinase (MAPK), ERK1/2, JNK, phosphatidylinositol 3-kinase (PI3K) and Akt were determined by western blot analysis. Formation of reactive oxygen species (ROS) was evaluated by fluorescence image system.

Results

TMP inhibited LPS-induced over-production of NO and iNOS in N9 cells. TMP also inhibited the NF-κB translocation from cytoplasm into nucleus of N9 cells. In addition, TMP showed blocking effect on the phosphorylation of p38 MAPK, ERK1/2, JNK and Akt, but not PI3K. Further, TMP suppressed the formation of intracellular ROS in LPS-induced N9 cells.

Conclusions

TMP inhibited production of NO and iNOS in LPS-induced N9 cells through blocking MAPK and PI3K/Akt activation and suppressing ROS production.     

Abarema cochliacarpos reduces LPS-induced inflammatory response in murine peritoneal macrophages regulating ROS-MAPK signal pathway

Ethnopharmacological relevance

Abarema cochliacarpos (Gomes) Barneby and Grimes (Fabaceae), known by the vulgar name of Babatenã, has been traditionally used in Northeast Brazil, as an anti-inflammatory remedy. Previous studies have demonstrated its anti-inflammatory and antiulcer effects in skin lesion, alcohol gastric ulcer and acute and chronic colitis.

Aims

The present study was designed to evaluate the antioxidant and anti-inflammatory effects of the butanolic fraction from A. cochliacarpos (BFAC) and its major flavonoid, (+)-catechin, in LPS-stimulated murine peritoneal macrophages. Moreover, we studied the role of mitogen-activated protein kinase (MAPK)s and NF-kB signaling pathways possibly involved in the beneficial effects.

Materials and methods

The quantification of the extract was carried out by ultra-performance liquid chromatography analysis. Cell viability was determined using SRB assay. Nitric oxide (NO) production was analyzed by Griess method and intracellular reactive oxygen species (ROS) by fluorescence analysis. In addition, cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) expression, MAPK activation and IkappaBalpha (IKBα) degradation, were determined by Western blot.

Results

After BFAC characterization, (+)-catechin was revealed as its major constituent. Both BFAC and (+)-catechin, exerted significant anti-oxidant and anti-inflammatory effects inhibiting LPS-induced intracellular ROS and NO production in peritoneal macrophages. Additionally, the extract but also its major component reduced pro-inflammatory proteins expression probably through c-Jun N-terminal kinase and p38 MAPK signaling pathways.

Conclusion

These data suggest that the beneficial effects of BFAC might be mediated, at least in part, by the presence of (+)-catechin. Conclusively our findings confirm the potential of A. cochliacarpos as a new therapeutic strategy for the management of inflammatory and oxidative stress-related diseases.