Involvement of heme oxygenase-1 in the anti-inflammatory activity of Chrysanthemum boreale Makino extracts on the expression of inducible nitric oxide synthase in RAW264.7 macrophages

Aim of the study

This study is to elucidate the involvement of anti-inflammatory heme oxygenase-1 (HO-1) in the inhibitory activity of a Chrysanthemum boreale Makino (CB) extract on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.

Materials and methods

Cell viability and NO assay were performed. In addition, iNOS expression was detected by Western blotting and real-time PCR. HO-1 expression was also evaluated by Western blotting, and blocking HO-1 activity on NO production was performed.

Results

The CB extract at the highest concentration (100 μg/ml) significantly inhibited NO production by approximately 90% and suppressed iNOS protein expression by approximately 84.8% compared to LPS-stimulated cells. Furthermore, the CB extract (100 μg/ml) inhibited iNOS mRNA expression in a concentration-dependant manner and suppressed iNOS mRNA expression by 94.8%. The CB extract induced the expression of HO-1 in a dose-dependent manner, and blocking HO-1 activity abolished the inhibitory effects of the CB extract. Moreover, the addition of carbon monoxide such as tricarbonyl dichlororuthenium (II) dimmer (RuCO), a byproduct derived from heme degradation, mimicked the inhibitory action of low concentrations of CB extract.

Conclusion

These results suggest that a CB extract has potent anti-inflammatory activity in RAW264.7 macrophages involving the induction of HO-1.     

Immunomodulatory properties of Xylaria nigripes in peritoneal macrophage cells of Balb/c mice

Ethnopharmacological relevance

Wu Ling Shen, a folklore name for Xylaria nigripes (XN), is a high value medicinal fungus used in traditional Chinese medicine.

Aim of study

The present study aimed to examine the immunomodulatory properties of aqueous (XN-H) and ethanol (XN-E) XN extracts in lipopolysaccharide (LPS)-induced peritoneal macrophage cells of Balb/c mice.

Materials and methods

After treating the macrophage cells with LPS (1 μg/ml) and different XN extracts, the immunomodulatory properties were determined by the responses of inflammatory mediators, namely nitrite oxide (NO), prostaglandin E2 (PGE₂) and cytokine (IL-1β, IL-6, TNF-α and IFN-γ) production, iNOS, COX-2 and IκB-α expression, and NF-κB activation.

Results

Results showed that treatment of macrophages with 5-30 μg/ml of XN-H or XN-E plus 1 μg/ml LPS exhibited no cytotoxic effect on cell viability. At these concentrations, although both XN-H and XN-E showed a dose-dependent inhibitory effect on NO, PGE₂, IL-1β, IL-6, TNF-α and IFN-γ production in LPS-stimulated macrophages, a greater potency was noted in the XN-H treated group. RT-PCR assay also showed that XN-H possessed a greater inhibition than XN-E on iNOS and COX-2 RNA expression. Furthermore, XN-H also showed a significant stronger suppression than XN-E on the LPS-induced IκB-α phosphorylation and NF-κB activation. XN-E showed a higher total flavonoid and phenol contents but a lower β-glucan content than XN-H.

Conclusion

Taken together, these results conclude that XN-H possesses a stronger anti-inflammatory activity than XN-E, and its mechanism of action could be mediated by inhibiting iNOS and COX-2 expression via the NF-κB signaling pathway, and these activities could be contributed by the β-glucan content.     

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.     

In vitro anti-inflammatory and anti-cancer activities of Cuscuta reflexa Roxb

Aim of the study

To determine anti-inflammatory and anti-cancer activities of Cuscuta reflexa in cell lines (in vitro).

Materials and methods

Anti-inflammatory activity of the water extract was analysed in vitro using lipopolysaccharide (LPS) induced inflammatory reactions in murine macrophage cell line RAW264.7. The expression of COX-2 and TNF-α genes involved in inflammation was analysed by SQ RT-PCR. EMSA was conducted to analyse the influence of the extract on NF-κB signalling. Anti-cancer activity was analysed on Hep3B cells by MTT assay, DAPI staining, annexin V staining and SQ-RT PCR analysis of BAX, Bcl-2, p53 and survivin.

Results

The extract down regulated LPS induced over expression of TNF-α and COX-2 in RAW264.7 cells; blocked NF-κB binding to its motifs and induced apoptosis in Hep3B cells as evidenced from MTT, DAPI staining and annexin V staining assays. The extract up regulated pro-apoptotic factors BAX and p53, and down regulated anti-apoptotic factors Bcl-2 and survivin.

Conclusions

The study showed that Cuscuta reflexa inhibits LPS induced inflammatory responses in RAW264.7 cells through interplay of TNF-α, COX-2 and NF-κB signalling. It induced apoptosis in Hep3B cells through the up regulation of p53, BAX and down regulation of Bcl-2 and survivin.     

Anti-inflammatory and antinociceptive properties of the leaves of Eriobotrya japonica

Aim of the study

The leaves of Eriobotrya japonica Lindl. have been widely used as a traditional medicine for the treatment of many diseases including coughs and asthma. The present study was designed to validate the anti-inflammatory and antinociceptive properties of the n-BuOH fraction of E. japonica (LEJ) leaves.

Materials and methods

The anti-inflammatory properties of LEJ were studied using IFN-γ/LPS activated murine peritoneal macrophage model. The antinociceptive effects of LEJ were assessed using experimental models of pain, including thermal nociception methods, such as the tail immersion test and the hotplate test, and chemical nociception induced by intraperitoneal acetic acid and subplantar formalin in mice. To examine the possible connection of the opioid receptor to the antinociceptive activity of LEJ, we performed a combination test with naloxone, a nonselective opioid receptor antagonist.

Results

In the IFN-γ and LPS-activated murine peritoneal macrophage model, LEJ suppressed NO production and iNOS expression via down-regulation of NF-κB activation. It also attenuated the expression of COX-2 and the secretion of pro-inflammatory cytokines like TNF-α and IL-6. Moreover, LEJ also demonstrated strong and dose-dependent antinociceptive activity compared to tramadol and indomethacin in various experimental pain models. In a combination test using naloxone, diminished analgesic activities of LEJ were observed, indicating that the antinociceptive activity of LEJ is connected with the opioid receptor.

Conclusions

The results indicate that LEJ had potent inhibitory effects on the inflammatory mediators including nitric oxide, iNOS, COX-2, TNF-α and IL-6 via the attenuation of NF-κB translocation to the nucleus. LEJ also showed excellent antinociceptive activity in both central and peripheral mechanism as a weak opioid agonist. Based on these results, LEJ may possibly be used as an anti-inflammatory and an analgesic agent for the treatment of pains and inflammatory diseases.     

Methylene chloride fraction of the leaves of Thuja orientalis inhibits in vitro inflammatory biomarkers by blocking NF-κB and p38 MAPK signaling and protects mice from lethal endotoxemia

Aim of the study

Thuja orientalis (TO) has been a recognized herbal medicine across Northeast Asian countries for thousands of years and used for the treatment of various inflammatory diseases through as yet undefined mechanisms. In this study, we set out to determine whether the anti-inflammatory effects of this plant are mediated to suppress mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) activation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.

Materials and methods

RAW 264.7 cells were pretreated with the methylene chloride fraction of TO (MTO) and stimulated with LPS. Nitric oxide (NO) release was determined by the accumulation of nitrite in the culture supernatants and tumor necrosis factor-α (TNF-α) and IL-6 secretion were determined by immunoenzymatic assay. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression were evaluated via RT-PCR and Western blotting. NF-κB activation was also evaluated by reporter gene assay and electrophoretic mobility shift assay (EMSA). In addition, the protective effect of MTO was evaluated by use of the LPS-induced endotoxin shock model in mice.

Results

We found that MTO significantly suppressed LPS-stimulated NO and IL-6 production without affecting cell viability. MTO inhibited the expression of LPS-induced iNOS and COX-2 protein and their mRNA expression. Also, TNF-α and IL-6 secretion were decreased by MTO in both PMA and ionomycin-stimulated splenocytes. As a result, MTO inhibited pro-inflammatory cytokines such as TNF-α and IL-6, which is hypothesized as being due to the suppression of LPS-induced p38 MAPK and NF-κB activation. Moreover, MTO improved the survival rate during lethal endotoxemia by inhibiting the production of TNF-α in an animal model and our LC-MS analysis showed that a major component of MTO was pinusolide.

Conclusions

We demonstrate here the evidence that the methylene chloride fraction of Thuja orientalis (MTO) potentially inhibits the biomarkers related to inflammation in vitro and in vivo, and might be provided as a potential candidate for the treatment of inflammatory diseases.     

Anti-inflammatory effects of Polygala tenuifolia root through inhibition of NF-κB activation in lipopolysaccharide-induced BV2 microglial cells

Ethnopharmacological relevance

The root of Polygala tenuifolia Willd is a well-known traditional Oriental medicine and has been prescribed for treatment of dysfunction in memorial systems and various brain inflammatory diseases. The present study was designed to validate the anti-inflammatory effects of the water extract of Polygala tenuifolia root (WEPT).

Materials and methods

The anti-inflammatory properties of WEPT were studied using lipopolysaccharide (LPS)-stimulated murine BV2 microglia model. As inflammatory parameters, the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E₂ (PGE₂), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β were evaluated. We also examined the extract's effect on the activity of nuclear factor-kappaB (NF-κB), and toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (Myd-88) expression.

Results

WEPT suppressed LPS-induced production of NO, PGE₂, and expression of iNOS and COX-2 in a dose-dependent manner, without causing cytotoxicity. It also significantly reduced generation of proinflammatory cytokines, including IL-1β and TNF-α. In addition, WEPT suppressed NF-κB translocation by blockade of IkappaB-α (IκB-α) degradation and inhibited TLR4 and Myd-88 expression in LPS-stimulated BV2 cells.

Conclusions

These results indicate that the inhibitory effects of WEPT on LPS-stimulated inflammatory mediator production in BV2 microglia are associated with suppression of the NF-κB and toll-like receptor signaling pathways. Therefore, Polygala tenuifolia extracts may be useful in treatment of neurodegenerative diseases by inhibition of inflammatory mediator production in activated microglia.     

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.     

2-methoxycinnamaldehyde from Cinnamomum cassia reduces rat myocardial ischemia and reperfusion injury in vivo due to HO-1 induction

Ethnopharmacological relevance

Cinnamomum cassia Blume has been used as a traditional Chinese herbal medicine for alleviation of fever, inflammation, chronic bronchitis, and to improve blood circulation.

Aim of the study

We addressed whether 2-methoxycinnamaldehyde (2-MCA), one of active ingredients of Cinnamomum cassia, reduces vascular cell adhesion molecule-1 (VCAM-1) expression in tumor necrosis factor-alpha (TNF-α)-activated endothelial cells and protects ischemia/reperfusion (I/R)-injury due to heme oxygenase (HO)-1 induction.

Materials and methods

Adult male rats were subjected to 30 min of ischemia by occlusion of the left anterior descending coronary artery followed by 24 h of reperfusion. Rats were randomized to receive vehicle or 2-MCA (i.v.) 10 min before reperfusion.

Results

Administration of 2-MCA significantly improved I/R-induced myocardial dysfunction by increasing the values of the first derivative (±dp/dt) of left ventricular pressure and decreased infarct size. In addition, 2-MCA reduced the expression of high mobility group box 1 (HMGB1), an activator of the inflammatory cascade when released into the extracellular space, and VCAM-1 in I/R myocardium along with increase of HO-1 induction. The reduced injury was accompanied by significantly reduction of neutrophils infiltration and increased SOD activity in ischemic tissues and reduced serum level of cardiac troponin I (cTnI). Furthermore, 2-MCA significantly increased HO-1 induction by translocation of Nrf-2 from cytosol to nucleus in endothelial cells. Inhibition of VCAM-1 expression by 2-MCA was reversed both by SnPPIX, a HO-1 inhibitor and siHO-1 RNA trasfection in TNF-α-activated cells. In addition, 2-MCA significantly inhibited NF-κB luciferase activity in TNF-α-activated endothelial cells. As expected, 2-MCA significantly inhibited monocyte (U937) adhesion to endothelial cells.

Conclusion

We concluded that 2-MCA protects of myocardial I/R-injury due to antioxidant and anti-inflammatory action possibly by HO-1 induction which can be explained why Cinnamomum cassia has been used in inflammatory disorders.     

(+)-Nootkatone and (+)-valencene from rhizomes of Cyperus rotundus increase survival rates in septic mice due to heme oxygenase-1 induction

Ethnopharmacological relevance

The rhizomes of Cyperus rotundus have been used as traditional folk medicine for the treatment of inflammatory diseases. However, the mechanism by which extract of rhizomes of Cyperus rotundus (ECR) elicits anti-inflammation has not been extensively investigated so far. The aim of the present study was to test whether heme oxygenase (HO)-1 induction is involved in the anti-inflammatory action of ECR.

Materials and methods

Induction of HO-1 and inhibition of inducible nitric oxide synthase (iNOS)/NO production by ECR and its 12 constituents (3 monoterpenes, 5 sesquiterpenes, and 4 aromatic compounds) were investigated using RAW264.7 cells in vitro. In addition, anti-inflammatory action of ECR and its two active ingredients (nookkatone, valencene) were confirmed in sepsis animal model in vivo.

Results

ECR increased HO-1 expression in a concentration-dependent manner, which was correlated with significant inhibition of iNOS/NO production in LPS-activated RAW264.7 cells. Among 12 compounds isolated from ECR, mostly sesquiterpenes induced stronger HO-1 expression than monoterpenes in macrophage cells. Nootkatone and valencene (sesquiterpenes) significantly inhibited iNOS expression and NO production in LPS-simulated RAW264.7 cells. Inhibition of iNOS expression by nootkatone, valencene, and ECR were significantly reduced in siHO-1 RNA transfected cells. Furthermore, all three showed marked inhibition of high mobility group box-1 (HMGB1) in LPS-activated macrophages and increased survival rates in cecal ligation and puncture (CLP)-induced sepsis in mice.

Conclusions

Taken together, we concluded that possible anti-inflammatory mechanism of ECR is, at least, due to HO-1 induction, in which sesquiterpenes such as nootkatone and valencene play a crucial role.     

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.     

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.     

Anti-inflammatory effect of ethyl acetate extract from Cissus quadrangularis Linn may be involved with induction of heme oxygenase-1 and suppression of NF-κB activation

Aim of the study

Cissus quadrangularis (family: Vitaceae) has been widely used in traditional herbal medicine for the treatment of hemorrhoids, gastric ulcers and bone healing. In the present study, we determined the anti-inflammatory activity and the molecular mechanism of the ethyl acetate extract of Cissus quadrangularis stem (CQE) in LPS-stimulated RAW 264.7 macrophage cells.

Materials and methods

The inhibitory effect of CQE on LPS-induced nitric oxide (NO) production was evaluated in conditioned media. Cell viability was monitored by MTT assay. Protein and mRNA expressions were determined by RT-PCR and Western blotting analysis, respectively.

Results

CQE potently inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 macrophage cells in a dose-dependent manner. The mRNA and protein expressions of inducible nitric oxide synthase (iNOS) were suppressed also by CQE as was p65 NF-κB nuclear translocation. Further study demonstrated that CQE by itself induced heme oxygenase-1 (HO-1) gene expression at the protein and mRNA levels in dose- and time-dependent manner. In addition, the inhibitory effects of CQE on NO production were abrogated by a HO-1 inhibitor, zinc protoporphyrin IX (ZnPP).

Conclusions

Collectively, these results suggest that CQE exerts an anti-inflammatory effect in macrophages, at least in part, through the induction of HO-1 expression. These findings provide the scientific rationale for anti-inflammatory therapeutic use of Cissus quadrangularis stem.