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.     

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.     

Tanshinones isolated from the rhizome of Salvia miltiorrhiza inhibit passive cutaneous anaphylaxis reaction in mice

Ethnopharmacological relevance

The rhizome of Salvia miltiorrhiza Bunge (SM, family Labiatae), which contains tanshinones as main constituents, has been used as a cardiovascular and anti-inflammatory agent in Chinese medicine.

Aim of the study

This study aimed to elucidate anti-allergic effects of the root of Salvia miltiorrhiza Bunge (SM, family Labiatae) and its main constituents, tanshinones, against passive cutaneous anaphylaxis (PCA) reaction.

Materials and methods

PCA reaction was induced by IgE-antigen complex (IAC) in ICR mice. Protein expression of IL-4 and TNF-α in rat basophilic leukemia (RBL)-2H3 cells was performed by enzyme-linked immunosorbent assay and NF-κB and c-jun (AP-1) activation assayed by immunoblot.

Results

Tanshinones inhibited the PCA reaction and reduced IL-4 and TNF-α production in mice as well as in IAC-stimulated RBL-2H3 cells. Tanshinones also inhibited NF-κB and AP-1 activation in RBL-2H3 cells stimulated with IAC. Among tested tanshinones, tanshinone I exhibited the most potent inhibition, followed by 15,16-dihydrotanshinone I, tanshinone IIA and cryptotanshinone.

Conclusions

SM and tanshinones may ameliorate the PCA reaction by inhibiting the allergic cytokines IL-4 and TNF-α via NF-κB and AP-1 pathways.     

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.     

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.     

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.     

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.     

Evaluation of the immunomodulatory properties in mice and in vitro anti-inflammatory activity of cycloartane type saponins from Astragalus species

Ethnopharmacological relevance

Astragalus roots are used to treat leukemia and for their wound healing properties in Southeast Anatolia-Turkey.

Materials and methods

In vivo studies to investigate the effects of two Astragalus saponins were carried out on the immune response cytokines by using six to eight weeks old male Swiss albino mice. The production of IL-1β, TGF-1β, TNF-α, IL-2, IL-4 and IFN-γ cytokines was determined by ELISA. The spleen and lymph nodes, isolated from the mice subjects, were analyzed to realize induction of the surface antigen productions for IL-2Rα (CD25) and CD69. In addition, their effects on the targets of inflammation such as NF κB, iNOS and NAG-1 were investigated in cell-based assays.

Results

The results suggested that AST VII and Mac B had positive effect on Th1 cytokine release (IL-2 and IFN-γ), and suppression on Th2 cytokine production (IL-4). The immunohistochemical results exhibited induction of both IL-Rα (CD25) and CD69 surface receptors justifying the Th1 cytokine release. The compounds did not affect NF-κB or NAG-1 activity but iNOS activity was inhibited by Mac B with an IC₅₀ of 156 μg/ml.

Conclusions

The results show that Ast VII and Mac B create powerful immunoregulatory effects without the stimulation of inflammatory cytokines in mice, and have no significant effect on the inflammatory cellular targets in vitro.     

Euonymus alatus extract attenuates LPS-induced NF-κB activation via IKKβ inhibition in RAW 264.7 cells

Aim of the study

The present study was performed to investigate the underlying mechanisms of anti-inflammatory effects with the extract of Euonymus alatus (EEA), and specially focused on nuclear factor κB (NF-κB) signaling pathway by targeting the IκB kinase β (IKKβ).

Materials and methods

The effect of EEA for IKKβ activity was analyzed using an immobilized metal affinity for phosphochemicals (IMAP)-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay. The effect of EEA on lipopolysaccharide (LPS)-induced NF-κB activation in murine macrophage RAW 264.7 cells with western blotting and immunofluorescent staining was evaluated.

Results

IKKβ studies based on IMAP-TR-FRET showed that EEA possesses a potent IKKβ inhibitory activity with IC₅₀ value of 11.83 μg/ml. EEA (10, 30 μg/ml) also attenuated the LPS-induced IκBα phosphorylation/degradation, NF-κB translocation and subsequent NO synthesis in RAW 264.7 cells.

Conclusions

These results suggest that EEA abrogates LPS-induced NF-κB signaling pathway by targeting the IKKβ in RAW 264.7 cells and these properties may provide a molecular basis for understanding the inhibitory effects of EEA on LPS-mediated inflammation.     

Modulation of LPS-stimulated neuroinflammation in BV-2 microglia by Gastrodia elata: 4-hydroxybenzyl alcohol is the bioactive candidate

Ethnopharmacological relevance

Gastrodia elata Blume (Orchidaceae, GE) a traditional plant in Oriental countries is known for its enormous benefits to treat headaches, dizziness, vertigo and convulsive illnesses. In the present study, the ethnopharmacological role of GE in neuroinflammation mediated by activated microglia and the mechanisms underlying were reported.

Materials and methods

BV-2 microglia activated by lipopolysaccharide (LPS) was employed and the effects of GE on corresponding neuroinflammatory parameters were assessed.

Results

GE extract inhibited LPS-stimulated production of inflammatory cytokines and down regulated the c-Jun NH₂-Terminal Kinase (JNK) and nuclear factor-kappa B (NF-κB) signaling pathways, which are known to be involved in neuroinflammation. Further, inhibition of NO and iNOS by 4-hydroxybenzyl alcohol (4-HBA), one of the active constituent of GE in LPS-stimulated BV-2 cells suggest that 4-HBA might be the bioactive candidate.

Conclusion

GE extract and its active constituent 4-HBA could be further exploited to mitigate microglial activation and may be developed as a new therapeutic remedy in treating various neuroinflammatory diseases.     

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.     

TLR4-mediated activation of macrophages by the polysaccharide fraction from Polyporus umbellatus(pers.) Fries

Aim of the Study

Zhu Ling (Polyporus umbellatus) is well-known to reduce the risk of a variety of diseases. In this study, we explored the molecular mechanism of its immunostimulatory potency in immune responses of macrophages, using polysaccharides prepared from Polyporus umbellatus (PPS).

Materials and methods

Splenocyte proliferation was analyzed with ³H-TdR incorporation method. Nitric oxide (NO) was measured by Griess method and cytokines of culture supernatants was detected by enzyme linked immunosorbent assay (ELISA). The fluoresceinamine-labeled PPS (Flu-PPS) and dextran (Flu-dextran) were prepared by the cyanogen bromide activation method. The cell-binding activity of Flu-PPS was analyzed with FACS and confocal microscopy. NF-κB activity was measured by ELISA assay.

Results

We found that PPS is able to strongly upregulate the functions of macrophages such as Nitric oxide (NO) production and cytokine expression. Compared with C3H/HeJ group, PPS significantly stimulated the proliferation of splenocytes and the production of TNF-α, IL-1β and NO of peritoneal macrophages from C3H/HeN mice. The function blocking antibodies to TLR-4, but not TLR-2 and CR3, markedly suppressed PPS-mediated TNF-α and IL-1β production. Flow cytometric and confocal laser-scanning microscopy analysis shown that fluorescence-labeled PPS (f-PPS) can bind specifically to the target cells, and the binding can blocked by unlabeled PPS and anti-TLR4, but not anti-TLR2 and CR3 monoclonal antibodies. Nuclear translocation and DNA binding activity of NF-κB was significantly induced by PPS.

Conclusions

Therefore, our data suggest that PPS may exert its immunostimulating potency via TLR-4 activation of signaling pathway.     

Anti-inflammatory action of methanol extract of Carthamus tinctorius involves in heme oxygenase-1 induction

Ethnopharmacological relevance

The methanol extracts of Carthamus tinctorius (MEC) have long been used in traditional medicine as anti-inflammatory agent, however, the molecular mechanism by which MEC shows anti-inflammatory action is not investigated.

Aim of the study

Induction of heme oxygenase-1 (HO-1) by many medicinal herbs has been reported excellent anti-inflammatory action. Thus, the aim of the study is to explore whether anti-inflammatory action of MEC is related with HO-1 induction in RAW 264.7 cells.

Materials and methods

The present study was designed to investigate as to MEC induces HO-1 expression so that it reduces inflammation by suppression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in cells activated with lipopolysaccharide (LPS).

Results

Expression of HO-1 protein by MEC in macrophages was increased in a concentration- and time-dependent manner. Treatment with MEC significantly inhibited upregulation of both iNOS and COX-2 in LPS-activated macrophages and consequently reduced production of NO and PGE₂, respectively. The reduced expression of iNOS and COX-2 by MEC was reversed by siHO-1 RNA transfection. In addition, NF-E2-related factor (Nrf2) was translocated from cytosol to nucleus by MEC. The binding of NF-κB as well as NF-κB luciferase activity was also significantly diminished by MEC. Finally, tumor necrosis factor (TNF)-α-mediated VCAM-1 expression in endothelial cell was significantly inhibited by MEC.

Conclusions

The present results show that MEC induces HO-1 expression via Nrf2 translocation and inhibits NF-κB activity, which may be responsible for anti-inflammatory action. Therefore, we propose that anti-inflammatory action of MEC involves at least HO-1 induction.