Shikonin Isolated from Lithospermum erythrorhizon Downregulates Proinflammatory Mediators in Lipopolysaccharide-Stimulated BV2 Microglial Cells by Suppressing Crosstalk between Reactive Oxygen Species and NF-κB

According to the expansion of lifespan, neuronal disorder based on inflammation has been social problem. Therefore, we isolated shikonin from Lithospermum erythrorhizon and evaluated anti-inflammatory effects of shikonin in lipopolysaccharide (LSP)-stimulated BV2 microglial cells. Shikonin dose-dependently inhibits the expression of the proinflammatory mediators, nitric oxide (NO), prostaglandin E₂ (PGE₂), and tumor necrosis factor-α (TNF-α) as well as their main regulatory genes and products such as inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α in LPS-stimulated BV2 microglial cells. Additionally, shikonin suppressed the LPS-induced DNA-binding activity of nuclear factor-κB (NF-κB) to regulate the key regulatory genes of the proinflammatory mediators, such as iNOS, COX-2, and TNF-α, accompanied with downregulation of reactive oxygen species (ROS) generation. The results indicate that shikonin may downregulate the expression of proinflammatory genes involved in the synthesis of NO, PGE₂, and TNF-α in LPS-treated BV2 microglial cells by suppressing ROS and NF-κB. Taken together, our results revealed that shikonin exerts downregulation of proinflammatory mediators by interference the ROS and NF-κB signaling pathway.     

Anti-Inflammatory and Anti-Superbacterial Properties of Sulforaphane from Shepherd's Purse

Shepherd''s purse, Capsella bursa-pastoris (L.) Medik., has been considered a health food for centuries in Asia and is known to contain the isothiocyanate compound sulforaphane. In this study, we evaluated the anti-inflammatory and antibacterial properties of a sulforaphane-containing solution (SCS) isolated from shepherd''s purse. SCS had significant anti-inflammatory activity indicated by the decreased levels of nitric oxide (NO), cytokines (interleukin 1β [IL-1β], IL-6, and IL-10), and prostaglandin E₂ (PGE₂) in lipopolysaccharide-stimulated RAW 264.7 murine macrophages. In addition, SCS decreased the inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) levels, which confirmed the anti-inflammatory activity of SCS. Further, SCS inhibited vancomycin-resistant enterococci (VRE) and Bacillus anthracis. The minimal inhibitory concentration was 250 µg/ml for VRE and 1,000 µg/ml for B. anthracis. Taken together, these data indicate that SCS has potential anti-inflammatory and anti-superbacterial properties, and thus it can be used as a functional food or pharmaceutical.     

Ginsenoside-Rd exhibits anti-inflammatory activities through elevation of antioxidant enzyme activities and inhibition of JNK and ERK activation in vivo

Our previous study has reported that ginsenoside-Rd significantly inhibited the production of pro-inflammatory cytokines and mediators in carrageenan (Carr)-induced rat paw edema, which might be due to its blocking of the nuclear factor-κB (NF-κB) signaling pathway. The aim of the present study was to clarify the more detailed mechanisms of anti-inflammatory activity of ginsenoside-Rd in Carr-induced rat paw edema model. Rats were pretreated with dexamethasone or ginsenoside-Rd 1 h before the Carr injection. Six hours after Carr injection, the malondialdehyde (MDA) level and myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) activities in inflamed paw tissues were determined. The levels of nitric oxide (NO) and prostaglandin E₂ (PGE₂) in serum were measured. The expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and NF-κB were detected by western blot. In addition, the extent of phosphorylation of extracellular signal-regulated protein kinase (ERK), p38 and c-Jun NH₂-terminal kinase (JNK) was analyzed by western blot. The results showed that ginsenoside-Rd significantly attenuated MPO activity and MDA level, increased the activities of SOD, GPx and CAT, lowered the levels of NO and PGE₂, down-regulated the expressions of iNOS, COX-2 and NF-κB, and suppressed the phosphorylation of ERK and JNK. Taken together, the possible mechanisms of anti-inflammatory activity of ginsenoside-Rd were: it could reduce the inflammatory cell infiltration into inflammatory sites, inhibit the tissue lipid peroxidation, increase the antioxidant enzyme activities, and suppress the proinflammatory enzyme expressions through the downregulation of NF-κB activation via suppression of ERK and JNK phosphorylation.     

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide

1. In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E₂ (PGE₂) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated.
2. The effect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE₂ EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNγ) 100 u ml⁻¹, interleukin-1β (IL-1β) 1 u ml⁻¹ and lipopolysaccharide (LPS) 10 μg ml⁻¹ induced nitrite formation which could be inhibited by the competitive NOS inhibitor N^G-nitro-L-arginine-methyl-ester (L-NAME). IL-1β alone (1–50 u ml⁻¹) induced PGE₂ formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ to induce both the iNOS and COX-2 pathways, and IL-1β 3 u ml⁻¹ to induce COX-2 without iNOS activity.
3. Cells treated with IFNγ 100 u ml⁻¹, IL-1β 1 u ml⁻¹ and LPS 10 μg ml⁻¹ for 48 h either alone, or with the addition of L-NAME (0 to 10⁻² M), demonstrated inhibition by L-NAME of PGE₂ (3.61±0.55 to 0.51±0.04 pg/10⁴ cells; P<0.001) and nitrite (34.33±8.07 to 0 pmol/10⁴ cells; P<0.001) production. Restoration of the PGE₂ response (0.187±0.053 to 15.46±2.59 pg/10⁴ cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10⁻⁶ M, but with the addition of the NOS substrate L-arginine (0 to 10⁻⁵ M).
4. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h, either alone or with addition of the NO donor S-nitroso-acetyl-penicillamine (SNAP) (0 to 10⁻⁴ M), demonstrated increased PGE₂ formation (1.23±0.03 to 2.92±0.19 pg/10⁴ cells; P< 0.05). No increase in PGE₂ formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 μM). Cells treated with SNAP alone did not demonstrate an increased PGE₂ formation. Cells incubated with IL-1β 3 u ml⁻¹ for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10⁻³ M) also demonstrated an increased PGE₂ response (2.56±0.21 to 4.53±0.64 pg/10⁴ cells; P<0.05).
5. These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.

     

The Effect of Polyphenols Isolated from Cynanchi wilfordii Radix with Anti-inflammatory,Antioxidant, and Anti-bacterial Activity

Recently, Cynanchi wilfordii Radix has gained wide use in Asian countries as a functional food effective for relieving fatigue, osteoporosis, and constipation, particularly in menopausal disorders. However, its anti-inflammatory and anti-microbial activities have not been explored in detail to date. The anti-inflammatory, antioxidant, and anti-bacterial properties of the Cynanchi wilfordii Radix extracts obtained with water, methanol, ethanol, and acetone were compared. All 4 polyphenol-containing extracts exhibited anti-inflammatory and antioxidant effects. The ethanol extract was found to elicit the most potent reduction of nitric oxide (NO), prostaglandin E₂ (PGE₂), and cytokine (IL-1β, IL-6, IL-10, and TNF-α) levels, as well as inhibit the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in a concentration-dependent manner. The evaluation of antioxidant activity also revealed the ethanol extract to have the highest free radical scavenging activity, measured as 85.3±0.4%, which is equivalent to 99.9% of the activity of α -tocopherol. In the assessment of anti-bacterial activity, only ethanol extract was found to inhibit the growth of the Bacillus species Bacillus cereus and Bacillus anthracis. These results show that polyphenols of Cynanchi wilfordii Radix have anti-inflammatory, antioxidant, and anti-bacterial properties that can be exploited and further improved for use as a supplementary functional food, in cosmetics, and for pharmaceutical purposes.     

(-)-Nyasol (cis-hinokiresinol), a norneolignan from the rhizomes of <Emphasis Type="Italic">Anemarrhena asphodeloides</Emphasis>, is a broad spectrum inhibitor of eicosanoid and nitric oxide production

To assess the anti-inflammatory activity of constituents from the rhizomes of Anemarrhena asphodeloides, (-)-nyasol {cis-hinokiresinol, 4,4-[1Z,3R]-3-ethenyl-1-propene-1,3-diyl]bisphenol} was isolated and its anti-inflammatory activity was examined in lipopolysaccharide (LPS)-treated RAW 264.7 cells and A23187-treated RBL-1 cells. In vivo activity was measured using carrageenan-induced paw edema assay. At > 1 μM, (-)-nyasol significantly inhibited cyclooxygenase-2 (COX-2)-mediated PGE₂ production and inducible nitric oxide synthase (iNOS)-mediated NO production in LPS-treated RAW 264.7 cells, a mouse macrophage-like cell line, but did not affect the expression levels of COX-2 and iNOS. (-)-Nyasol also inhibited 5-lipoxygenase (5-LOX)-mediated leukotriene production in A23187-treated RBL-1 cells. Furthermore, (-)-nyasol potently inhibited carrageenan-induced paw edema in mice (28.6–77.1% inhibition at 24–120 mg/kg). Therefore, (-)-nyasol is a potential new lead compound and may contribute to the anti-inflammatory action of A. asphodeloides, possibly by inhibiting COX-2, iNOS and 5-LOX.     

Phosphorylation of Akt Mediates Anti-Inflammatory Activity of 1-p-Coumaroyl β-D-Glucoside Against Lipopolysaccharide-Induced Inflammation in RAW264.7 Cells

Hydroxycinnamic acids have been reported to possess numerous pharmacological activities such as antioxidant, anti-inflammatory, and anti-tumor properties. However, the biological activity of 1-p-coumaroyl β-D-glucoside (CG), a glucose ester derivative of p-coumaric acid, has not been clearly examined. The objective of this study is to elucidate the anti-inflammatory action of CG in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. In the present study, CG significantly suppressed LPS-induced excessive production of pro-inflammatory mediators such as nitric oxide (NO) and PGE₂ and the protein expression of iNOS and COX-2. CG also inhibited LPS-induced secretion of pro-inflammatory cytokines, IL-1β and TNF-α. In addition, CG significantly suppressed LPS-induced degradation of IκB. To elucidate the underlying mechanism by which CG exerts its anti-inflammatory action, involvement of various signaling pathways were examined. CG exhibited significantly increased Akt phosphorylation in a concentration-dependent manner, although MAPKs such as Erk, JNK, and p38 appeared not to be involved. Furthermore, inhibition of Akt/PI3K signaling pathway with wortmannin significantly, albeit not completely, abolished CG-induced Akt phosphorylation and anti-inflammatory actions. Taken together, the present study demonstrates that Akt signaling pathway might play a major role in CG-mediated anti-inflammatory activity in LPS-stimulated RAW264.7 macrophage cells.     

Formosanin C attenuates lipopolysaccharide-induced inflammation through nuclear factor-κB inhibition in macrophages

Extended inflammation and cytokine production pathogenically contribute to a number of inflammatory disorders. Formosanin C (FC) is the major diosgenin saponin found in herb Paris formosana Hayata (Liliaceae), which has been shown to exert anti-cancer and immunomodulatory functions. In this study, we aimed to investigate anti-inflammatory activity of FC and the underlying molecular mechanism. RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS) or pre-treated with FC prior to being stimulated with LPS. Thereafter, the macrophages were subjected to analysis of the expression levels of pro-inflammatory mediators, including nitric oxide (NO), prostaglandin E₂ (PGE), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6, as well as two relevant enzymes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). The analysis revealed that FC administration blunted LPS-induced production of NO and PGE in a dose-dependent manner, while the expression of iNOS and COX-2 at both mRNA and protein levels was inhibited in LPS-stimulated macrophages pre-treated with FC. Moreover, LPS stimulation upregulated mRNA expression and medium release of TNF-α, IL-1β, and IL-6, whereas this effect was blocked upon FC pre-administration. Mechanistic studies showed that inhibitory effects of FC on LPS-induced inflammation were associated with a downregulation of IκB kinase, IκB, and p65/NF-κB pathway. Taken together, these data suggest that FC possesses an inflammation-suppressing activity, thus being a potential agent for the treatment of inflammation-associated disorders.     

Flavocoxid,a dual inhibitor of cyclooxygenase and 5-lipoxygenase,blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages

Background and purpose:

The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated.

Experimental approach:

LPS-stimulated (1 µg·mL⁻¹) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32–128 µg·mL⁻¹) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein κB-α (IκB-α) levels were evaluated by Western blot analysis. Nuclear factor κB (NF-κB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-α (TNF-α) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated.

Key results:

LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 µg·mL⁻¹) significantly inhibited COX-2 (LPS = 18 ± 2.1; flavocoxid = 3.8 ± 0.9 integrated intensity), 5-LOX (LPS = 20 ± 3.8; flavocoxid = 3.1 ± 0.8 integrated intensity) and iNOS expression (LPS = 15 ± 1.1; flavocoxid = 4.1 ± 0.4 integrated intensity), but did not modify COX-1 expression. PGE₂ and LTB₄ levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IκB-α protein (LPS = 1.9 ± 0.2; flavocoxid = 7.2 ± 1.6 integrated intensity), blunted increased NF-κB binding activity (LPS = 9.2 ± 2; flavocoxid = 2.4 ± 0.7 integrated intensity) and the enhanced TNF-α mRNA levels (LPS = 8 ± 0.9; flavocoxid = 1.9 ± 0.8 n-fold/β-actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS-stimulated macrophages.

Conclusion and implications:

Flavocoxid might be useful as a potential anti-inflammatory agent, acting at the level of gene and protein expression.     

Effects of volatile oils of Angelica sinensis on an acute inflammation rat model

Context Despite several pharmacological studies of volatile oils of Angelica sinensis (Oliv.) Diels (Umbelliferae) (VOAS), its anti-inflammatory mechanism remains unknown.

Objective The study investigates the effects of VOAS on the lipopolysaccharide (LPS)-induced acute inflammation rat model and analyzes its possible anti-inflammatory mechanisms.

Materials and methods Fourty rats were randomly divided into the control, model, VOAS and dexamethasone (Dex) groups. The VOAS and Dex groups were given VOAS (0.176?mL/kg) and Dex (40?μg/kg), respectively. Rats in all groups except the control group were intraperitoneally injected with LPS (100?μg/kg), their exterior behaviour and liver pathological changes were observed, and the level of white blood cell (WBC), the number of neutrophils (NE)%, glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), alkaline phosphatase (ALP), tumour necrosis factor (TNF-α), interleukin (IL)-1β, IL-6, IL-10, histamine (HIS), 5-hydroxytryptamine (5-HT), nitric oxide (NO), prostaglandin E₂ (PGE₂), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) were detected.

Results Compared with the model group, VOAS and Dex significantly accelerated the recovery of the exterior behaviour, the liver pathological changes of rats, and increased the level of IL-10, but decreased the level of WBC, NE%, GOT, GPT, ALP, TNF-α, IL-1β, IL-6, HIS, 5-HT, NO, PGE₂, iNOS and COX-2 (p?<?0.05).

Conclusion VOAS exhibits anti-inflammatory and liver protection effects by inhibiting the secretion of the pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), the inflammatory mediators (HIS, 5-HT, PGE₂ and NO), the inflammation-related enzymes (iNOS and COX-2), as well as promoting the production of the anti-inflammatory cytokines IL-10.     

N-(p-Coumaryol)-Tryptamine Suppresses the Activation of JNK/c-Jun Signaling Pathway in LPS-Challenged RAW264.7 Cells

N-(p-Coumaryol) tryptamine (CT), a phenolic amide, has been reported to exhibit anti-oxidant and anti-inflammatory activities. However, the underlying mechanism by which CT exerts its pharmacological properties has not been clearly demonstrated. The objective of this study is to elucidate the anti-inflammatory mechanism of CT in lipopolysaccharide (LPS)-challenged RAW264.7 macrophage cells. CT significantly inhibited LPS-induced extracellular secretion of pro-inflammatory mediators such as nitric oxide (NO) and PGE₂, and protein expressions of iNOS and COX-2. In addition, CT significantly suppressed LPS-induced secretion of pro-inflammatory cytokines such as TNF-α and IL-1β. To elucidate the underlying anti-inflammatory mechanism of CT, involvement of MAPK and Akt signaling pathways was examined. CT significantly attenuated LPS-induced activation of JNK/c-Jun, but not ERK and p38, in a concentration-dependent manner. Interestingly, CT appeared to suppress LPS-induced Akt phosphorylation. However, JNK inhibition, but not Akt inhibition, resulted in the suppression of LPS-induced responses, suggesting that JNK/c-Jun signaling pathway significantly contributes to LPS-induced inflammatory responses and that LPS-induced Akt phosphorylation might be a compensatory response to a stress condition. Taken together, the present study clearly demonstrates CT exerts anti-inflammatory activity through the suppression of JNK/c-Jun signaling pathway in LPS-challenged RAW264.7 macrophage cells.     

Neocryptotanshinone inhibits lipopolysaccharide-induced inflammation in RAW264.7 macrophages by suppression of NF-κB and iNOS signaling pathways

Neocryptotanshinone (NCTS) is a natural product isolated from traditional Chinese herb Salvia miltiorrhiza Bunge. In this study, we investigated its anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) cells. MTT results showed that NCTS partly reversed LPS-induced cytotoxicity. Real-time PCR results showed that NCTS suppressed LPS-induced mRNA expression of inflammatory cytokines, including tumor necrosis factor α (TNFα), interleukin-6 (IL-6) and interleukin-1β (IL-1β). Moreover, NCTS could decrease LPS-induced nitric oxide (NO) production. Western blotting results showed that NCTS could down-regulate LPS-induced expression of inducible nitric oxide synthase (iNOS), p-IκBα, p-IKKβ and p-NF-κB p65 without affecting cyclooxygenase-2 (COX-2). In addition, NCTS inhibited LPS-induced p-NF-κB p65 nuclear translocation. In conclusion, these data demonstrated that NCTS showed anti-inflammatory effect by suppression of NF-κB and iNOS signaling pathways.KEY WORDS: Neocryptotanshinone, Inflammation, NF-κB, Inducible nitric oxide synthase     

Induction of cyclo-oxygenase-2 by cytokines in human cultured airway smooth muscle cells: novel inflammatory role of this cell type

1. Cyclo-oxygenase (COX) is the enzyme that converts arachidonic acid to prostaglandin H₂ (PGH₂) which can then be further metabolized to prostanoids which modulate various airway functions. COX exists in at least two isoforms. COX-1 is expressed constitutively, whereas COX-2 is expressed in response to pro-inflammatory stimuli. Prostanoids are produced under physiological and pathophysiological conditions by many cell types in the lung. However, the regulation of the different COX isoforms in human airway smooth muscle (HASM) cells has not yet been determined.
2. COX-1 and COX-2 protein were measured by Western blot analysis with specific antibodies for COX-1 and COX-2. COX-2 mRNA levels were assessed by Northern blot analysis by use of a COX-2 cDNA probe. COX activity was determined by measuring conversion of either endogenous or exogenous arachidonic acid to three metabolites, PGE₂, thromboxane B₂ or 6-ketoPGF_1α by radioimmunoassay.
3. Under control culture conditions HASM cells expressed COX-1, but not COX-2, protein. However, a mixture of cytokines (interleukin-1β (IL-1β), tumour necrosis factor α (TNFα) and interferon γ (IFNγ) each at 10 ng ml⁻¹) induced COX-2 mRNA expression, which was maximal at 12 h and inhibited by dexamethasone (1 μM; added 30 min before the cytokines). Furthermore, COX-2 protein was detected 24 h after the cytokine treatment and the expression of this protein was also inhibited by dexamethasone (1 μM) and cyclohexamide (10 μg ml⁻¹; added 30 min before the cytokines).
4. Untreated HASM cells released low or undetectable amounts of all COX metabolites measured over a 24 h period. Incubation of the cells with the cytokine mixture (IL-1β, TNFα, IFNγ each at 10 ng ml⁻¹ for 24 h) caused the accumulation of PGE₂ and 6-keto-PGF_1α.
5. In experiments where COX-2 metabolized endogenous stores of arachidonic acid, treatment of HASM cells with IL-1β in combination with TNFα caused a similar release of PGE₂ to that when the three cytokines were given in combination.
6. In other experiments designed to measure COX-2 activity directly, cells were treated with cytokines for 24 h before fresh culture medium was added containing exogenous arachidonic acid (30 μM for 15 min) after which PGE₂ was measured. IL-1β and TNFα increased COX-2 activity and an additional small increase was produced by the three cytokines in combination.
7. These findings suggest that the increased expression of COX-2 is intimately involved in the exaggerated release of prostanoids from HASM cells exposed to pro-inflammatory cytokines. These data indicate a role for airway smooth muscle cells, in addition to their contractile function, as inflammatory cells involved in the production of mediators which may contribute to the inflammatory response seen in diseases such as asthma.

     

Anti-inflammatory activity of Nerium indicum by inhibition of prostaglandin E2 in murine splenic lymphocytes

Objective:

Nerium indicum Mill (syn. N. oleander L. and N. odorum Aiton; family: Apocynaceae) is a medicinal plant, used in the treatment of diverse ailments including various chronic inflammatory diseases in traditional medicine. We have previously demonstrated the immunomodulatory activity of a bioactive fraction of Nerium indicum leaf (NILE) by studying up-regulation of interleukin-2 (IL-2), IL-10, interferon-gamma and down regulation of IL-4, tumor necrosis factor-alpha (TNF-α), nitric oxide, cyclooxygenase-1 (COX-1) and COX-2 activities. Therefore, this study aimed to confirm the anti-inflammatory activity of NILE by inhibition of prostaglandin E₂ (PGE₂) activity in murine splenic lymphocytes in vitro.

Materials and Methods:

Murine lymphocytes were isolated from spleen and stimulated with 5 ΅g/mL concanavalin A in RPMI-1640, supplemented with 50 U/mL penicillin, 50 U/mL streptomycin, 50 U/mL nystatin and 10% fetal bovine serum. Different concentrations (0–80 μg/mL) of NILE were added and the cells were cultured for 48 h. The culture supernatants were thereafter collected by centrifugation and assayed for expression of PGE₂ level. The data were analyzed statistically.

Results:

The results demonstrated a 2.26-fold inhibition of PGE₂ level at 80 μg/mL of NILE. Half maximum inhibitory concentration (IC₅₀) was calculated to be 44.95 ± 0.45 ΅g/mL. Linear correlation analysis of the dose-dependent PGE₂ inhibition with other pro- and anti-inflammatory mediators demonstrated high inter-correlation between the parameters.

Conclusions:

Thus, the present study remains in accordance with our previous report and confirms the anti-inflammatory claim of N. indicum, mentioned in the traditional medicine.KEY WORDS: Anti-inflammatory, cyclooxygenase, lymphocyte, nerium, prostaglandin     

Myrtucommulone,a natural acylphloroglucinol,inhibits microsomal prostaglandin E2 synthase-1

Background and purpose:

The selective inhibition of prostaglandin (PG)E₂ formation via interference with microsomal PGE₂ synthase (mPGES)-1 could have advantages in the treatment of PGE₂-associated diseases, such as inflammation, fever and pain, compared with a general suppression of all PG biosynthesis, provided by inhibition of cyclooxygenase (COX)-1 and 2. Here, we addressed whether the naturally occurring acylphloroglucinol myrtucommulone (MC) from Myrtus communis L. (myrtle) affected mPGES-1.

Experimental approach:

The effect of MC on PGE₂ formation was investigated in a cell-free assay by using microsomal preparations of interleukin-1β-stimulated A549 cells as the source of mPGES-1, in intact A549 cells, and in lipopolysaccharide-stimulated human whole blood. Inhibition of COX-1 and COX-2 activity in cellular and cell-free assays was assessed by measuring 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-oxo PGF_1α formation.

Key results:

MC concentration-dependently inhibited cell-free mPGES-1-mediated conversion of PGH₂ to PGE₂ (IC₅₀ = 1 µmol·L⁻¹). PGE₂ formation was also diminished in intact A549 cells as well as in human whole blood at low micromolar concentrations. Neither COX-2 activity in A549 cells nor isolated human recombinant COX-2 was significantly affected by MC up to 30 µmol·L⁻¹, and only moderate inhibition of cellular or cell-free COX-1 was evident (IC₅₀ > 15 µmol·L⁻¹).

Conclusions and implications:

MC is the first natural product to inhibit mPGES-1 that efficiently suppresses PGE₂ formation without significant inhibition of the COX enzymes. This provides an interesting pharmacological profile suitable for interventions in inflammatory disorders, without the typical side effects of coxibs and non-steroidal anti-inflammatory drugs.