LPS-stimulated Production Of Inflammatory Mediators Research Articles

Synthesis of pyranochalcone derivatives and their inhibitory effect on NF-κB activation

In an effort to identify novel inhibitors of nuclear factor kappa B (NF-κB), twenty five pyranochalcone derivatives were synthesized and evaluated for their in vitro activities against TNF-α induced NF-κB inhibition in HEK293T cells. Among all of these derivatives, several displaying the same acrylate moiety on the B ring exhibited potent inhibition, with IC50 values ranging from 0.29 to 10.46 μM. A functional study of the most potent of these compounds, designated 6b, revealed that it significantly suppressed the transcriptional expression of inflammatory factor IL-1β in lipopolysaccharide-induced RAW 264.7 macrophages, and also mildly inhibited CCL2, IL6 and TNF-α. In addition, compound 6b was found to inhibit IL-1β released in LPS-induced BMDM cells. This study demonstrates that the inhibitory effect of 6b on LPS-stimulated inflammatory mediator production in the mouse macrophage cell line RAW 264.7 correlates with the suppression of the NF-κB and MAPK signaling pathways.

A Novel 1,8-Naphthyridine-2-Carboxamide Derivative Attenuates Inflammatory Responses and Cell Migration in LPS-Treated BV2 Cells via the Suppression of ROS Generation and TLR4/Myd88/NF-κB Signaling Pathway.

Novel 1,8-naphthyridine-2-carboxamide derivatives with various substituents (HSR2101-HSR2113) were synthesized and evaluated for their effects on the production of pro-inflammatory mediators and cell migration in lipopolysaccharide (LPS)-treated BV2 microglial cells. Among the tested compounds, HSR2104 exhibited the most potent inhibitory effects on the LPS-stimulated production of inflammatory mediators, including nitric oxide (NO), tumor necrosis factor-α, and interleukin-6. Therefore, this compound was chosen for further investigation. We found that HSR2104 attenuated levels of inducible NO synthase and cyclooxygenase 2 in LPS-treated BV2 cells. In addition, it markedly suppressed LPS-induced cell migration as well as the generation of intracellular reactive oxygen species (ROS). Moreover, HSR2104 abated the LPS-triggered nuclear translocation of nuclear factor-κB (NF-κB) through inhibition of inhibitor kappa Bα phosphorylation. Furthermore, it reduced the expressions of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in LPS-treated BV2 cells. Similar results were observed with TAK242, a specific inhibitor of TLR4, suggesting that TLR4 is an upstream regulator of NF-κB signaling in BV2 cells. Collectively, our findings demonstrate that HSR2104 exhibits anti-inflammatory and anti-migratory activities in LPS-treated BV2 cells via the suppression of ROS and TLR4/MyD88/NF-κB signaling pathway. Based on our observations, HSR2104 may have a beneficial impact on inflammatory responses and microglial cell migration involved in the pathogenesis of various neurodegenerative disorders.

Suppression of Inflammatory Mediators by Ethanol Extracts of <i>Pennisetum purpureum </i>S. (Napiergrass Taishigrass no. 2) in Activated RAW 264.7 Macrophages

Napiergrass Taishigrass no. 2 (NT2) is a new hybrid of napiergrass (Pennisetum purpureum S.), and is commonly added as an ingredient in bioorganic energy soup, a plant-based drink which is thought to have health-promoting effects. Thus, NT2 is hypothesized to possess biological functions. In this study, we explored the effects of NT2 ethanol extracts on in vitro antioxidant activities and on modulation of immune mediators in RAW 264.7 macrophages. Results showed that ethanol extracts contained significant amount of polyphenols and chlorophylls, and possessed in vitro ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidative activities. The ethanol extracts did not show immunomodulatory activities in macrophages without lipopolysaccharide (LPS, 100 ng/ml) treatment, but significantly suppressed the LPS-stimulated production of inflammatory mediators, including nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α, and such effects were associated with decreased expression of inducible NO synthase (iNOS) protein and cluster of differentiation 14 (CD14) mRNA, suggesting that NT2 has anti-inflammatory activities. On the other hand, the extracts increased matrix metalloproteinase (MMP)-9 activity, but showed no effects on Toll-like receptor (TLR) expression. In conclusion, ethanol extracts of NT2 possessed in vitro antioxidative activities and exhibited anti-inflammatory activities by decreasing NO, IL-6, and TNF-α production in association with decreased CD14 mRNA expression in LPS-induced RAW 264.7 macrophages, indicating its potential roles on health promotion and prentive effects against chronic diseases and such effects were and it’s in vitro antioxidant activities, suggesting its potential roles on health promotion.

Standardized extract of Centella asiatica ECa 233 inhibits lipopolysaccharide-induced cytokine release in skin keratinocytes by suppressing ERK1/2 pathways

Objective: To evaluate the effect of standardized extract of Centella asiatica ECa 233 on inflammatory mediator production through cyclooxygenase-2 (COX-2), extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-κB (NF-κB) pathway in keratinocyte HaCaT cells. Methods: HaCaT cells were treated with 0.1, 1, 10 and 100 μg/mL ECa 233 in the presence of lipopolysaccharide (LPS). Proinflammatory cytokines and prostaglandin E2 were assessed with ELISA. Western blotting was used to determine the inhibition of COX-2, ERK1/2 and NF-κB protein expression. Results: ECa 233 suppressed LPS-induced release of interleukin- 1β, tumor necrosis factor-α, and prostaglandin E2. ECa 233 also inhibitedCOX-2, phosphorylation of ERK1/2 and the activation of NF-κB. Moreover, the formation of reactive oxygen species (ROS) was decreased in response to LPS-inflamed keratinocytes. Conclusions: ECa 233 inhibits LPS-stimulated production of inflammatory mediators in keratinocytes via suppressing ERK1/2 andNF-κB pathways. The suppressive effect of ECa 233 may be related to an inhibition of ROS production.

SIRT7 Regulates Lipopolysaccharide-Induced Inflammatory Injury by Suppressing the NF-κB Signaling Pathway.

Mastitis has severely affected the cattle industry worldwide and has resulted in decreased dairy production and cattle reproduction. Although prevention and treatment methods have been implemented for decades, cattle mastitis is still an intractable disease. Sirtuin 7 (SIRT7) is an NAD+-dependent deacetylase that is involved in various biological processes, including ribosomal RNA synthesis and protein synthesis, DNA damage response, metabolism, and tumorigenesis. However, whether SIRT7 participates in inflammation remains unknown. Our results revealed that SIRT7 is downregulated in tissue samples from mastitic cattle. Therefore, we isolated dairy cow mammary epithelial cells (DCMECs) from breast tissues and developed an in vitro model of lipopolysaccharide- (LPS-) induced inflammation to examine SIRT7 function and its potential role in inflammation. We showed that SIRT7 was significantly downregulated in LPS-treated DCMECs. SIRT7 knockdown significantly increased the LPS-stimulated production of inflammatory mediators, like reactive oxygen and nitric oxide, and upregulated TAB1 and TLR4. In addition, SIRT7 knockdown significantly increased the phosphorylation of TAK1 and NF-κBp65 in LPS-treated DCMECs. Moreover, SIRT7 knockdown promoted the translocation of NF-κBp-p65 to the cell nucleus and then increased the secretion of inflammatory cytokines (IL-1β and IL-6). In contrast, SIRT7 overexpression had the opposite effects when compared to SIRT7 knockdown in LPS-treated DCMECs. In addition, SIRT7 overexpression attenuated LPS-induced DCMEC apoptosis. Taken together, our results indicate that SIRT7 can suppress LPS-induced inflammation and apoptosis via the NF-κB signaling pathway. Therefore, SIRT7 may be considered as a potential pharmacological target for clinical mastitis therapy.

Design, synthesis and investigation of potential anti-inflammatory activity of O-alkyl and O-benzyl hesperetin derivatives

Hesperetin has been known to exert several activities such as anti-oxidant, antitumor and anti-inflammatory. To find hesperetin derivatives showing better activity, sixteen novel hesperetin derivatives were designed and synthesized. The new obtained compounds were investigated for their anti-inflammatory activity by inhibiting interleukin-1β (IL-1β), interleukin-6 (IL-6) and production of nitric oxide (NO) in mouse RAW264.7 macrophages, and the structure-activity relationship of them was discussed. Among them, the compound 1l, 2c demonstrated more effective inhibitory activity of IL-1β and IL-6, meanwhile, the compound 1l showed the best inhibition of NO production. The results of NO inhibition study were basically accord with the molecular docking results of inducible nitric oxide synthase (iNOS). Furthermore, the expression of LPS-induced iNOS and components of NF-κB signaling pathway were reduced by compound 1l. Our results suggest that the inhibitory effect of compound 1l on LPS-stimulated inflammatory mediator production in RAW 264.7 cells is associated with the suppression of NF-κB signaling pathway and inhibition of iNOS protein and iNOS activity. From in vivo study, it was also observed that compound 1l had hepato-protective and anti-inflammatory effects in CCl4-induced acute liver injury mouse models.

Raphanus sativus L. seeds prevent LPS-stimulated inflammatory response through negative regulation of the p38 MAPK-NF-κB pathway.

The seeds of Raphanus sativus L. (RSL) have long been used as anti-inflammatory traditional medicine. However, scientific bases for the purported potential of the medicine and the associated mechanisms were barely defined. This study investigated the effects of RSL seeds on lipopolysaccharide (LPS)-stimulated inflammatory responses in vitro and in vivo. Treatment with 100 μg/ml ethyl acetate fraction (REF), which was isolated from water extract of the seeds, significantly inhibited LPS-stimulated production of nitric oxide (P < 0.05), interleukin-6 (P < 0.001), and tumor necrosis factor (TNF)-α (P < 0.001) in RAW264.7 cells. Oral supplementation with 30 mg/kg REF protected mice by 90% against LPS-induced septic death and prevented the increases of serum TNF-α and interferon-γ levels in LPS-injected mice. When REF was divided into four sub-fractions (REF-F1-F4), REF-F3 showed the greatest activity to suppress LPS-stimulated production of inflammatory mediators. We subsequently isolated an active fraction from the REF-F3 and identified sinapic acid as the main constituent. The addition of 50 μg/ml active fraction markedly inhibited LPS-stimulated production of inflammatory mediators by suppressing p38 MAPK and nuclear factor-κB activation. Furthermore, supplementation with the active fraction (10 mg/kg) improved the survival rate of LPS-injected mice by 80% of the untreated control. Additional experiments revealed that sinapic acid was the active component responsible for the anti-inflammatory potential of RSL seeds. Collectively, our current results suggest that both RSL seeds and sinapic acid may be attractive materials for treating inflammatory disorders caused by endotoxins.

Anti-inflammatory Effects of (-)-Epicatechin in Lipopolysaccharide-Stimulated Raw 264.7 Macrophages

Purpose: To investigate the protective effects of (-)-epicatechin on lipopolysaccharide (LPS)-induced inflammation in Raw 264.7 murine macrophages and the possible underlying mechanisms. Methods: The effects of epicatechin on LPS-stimulated production of inflammatory mediators in Raw 264.7 cells were evaluated by enzyme-linked immunosorbent assay. Results: Epicatechin in doses of 5, 25 and 50 µM remarkably (p < 0.05) inhibited the production of proinflammatory mediators including nitric oxide (NO) and prostaglandin E2 (PGE 2), as well as the production of pro-inflammatory cytokines including tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 in LPS-induced Raw 264.7 macrophages. Conclusion: The results suggest that epicatechin can inhibit inflammatory response and may be a potential therapeutic candidate for the treatment of chronic inflammatory diseases.

L-ascorbate attenuates the endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation and NF-κB translocation in cortical neurons/glia Cocultures.

In response to acute insults to the central nervous system, such as pathogen invasion or neuronal injuries, glial cells become activated and secrete inflammatory mediators such as nitric oxide (NO), cytokines, and chemokines. This neuroinflammation plays a crucial role in the pathophysiology of chronic neurodegenerative diseases. Endogenous ascorbate levels are significantly decreased among patients with septic encephalopathy. Using the bacterial endotoxin lipopolysaccharide (LPS) to induce neuroinflammation in primary neuron/glia cocultures, we investigated how L-ascorbate (vitamin C; Vit. C) affected neuroinflammation. LPS (100 ng/ml) induced the expression of inducible NO synthase (iNOS) and the production of NO, interleukin (IL)-6, and macrophage inflammatory protein-2 (MIP-2/CXCL2) in a time-dependent manner; however, cotreatment with Vit. C (5 or 10 mM) attenuated the LPS-induced iNOS expression and production of NO, IL-6, and MIP-2 production. The morphological features revealed after immunocytochemical staining confirmed that Vit. C suppressed LPS-induced astrocytic and microglial activation. Because Vit. C can be transported into neurons and glia via the sodium-dependent Vit. C transporter-2, we examined how Vit. C affected LPS-activated intracellular signaling in neuron/glia cocultures. The results indicated the increased activation (caused by phosphorylation) of mitogen-activated protein kinases (MAPKs), such as p38 at 30 min and extracellular signal-regulated kinases (ERKs) at 180 min after LPS treatment. The inhibition of p38 and ERK MAPK suppressed the LPS-induced production of inflammatory mediators. Vit. C also inhibited the LPS-induced activation of p38 and ERK. Combined treatments of Vit. C and the inhibitors of p38 and ERK yielded no additional inhibition compared with using the inhibitors alone, suggesting that Vit. C functions through the same signaling pathway (i.e., MAPK) as these inhibitors. Vit. C also reduced LPS-induced IκB-α degradation and NF-κB translocation. Thus, Vit. C suppressed the LPS-stimulated production of inflammatory mediators in neuron/glia cocultures by inhibiting the MAPK and NF-κB signaling pathways.

Development of a transgenic mouse for hepatitis C virus core protein-induced liver disease

Development of a transgenic mouse for hepatitis C virus core protein-induced liver disease

Synthesis and anti-inflammatory evaluations of β-lapachone derivatives

β-Lapachone (β-LAPA), a natural product from the lapacho tree in South America, is a potential chemotherapeutic agent that exhibit a wide variety of pharmacological effects such as anti-virus, anti-parasitic, anti-cancer, and anti-inflammatory activities. In order to discover novel anti-inflammatory agents, we have synthesized a series of β-LAPA derivatives for evaluation. Among them, 4-(4-methoxyphenoxy)naphthalene-1,2-dione (6b) was found to be able to inhibit NO and TNF-α released in LPS-induced Raw 264.7 cells. Inhibition of iNOS and COX-2 was also observed in compound 6b treated cells. Mechanism studies indicated that 6b exhibited anti-inflammatory properties by suppressing the release of pro-inflammatory factors through down-regulating NF-κB activation. In addition, it suppressed NF-κB translocation by inhibiting the phosphorylation of p38 kinase. Our results also indicate that the inhibitory effect of 6b on LPS-stimulated inflammatory mediator production in Raw 264.7 cell is associated with the suppression of the NF-κB and MAPK signaling pathways. A low cytotoxicity (IC50=31.70μM) and the potent anti-inflammatory activity exhibited by compound 6b make this compound a potential lead for developing new anti-inflammatory agents. Further structural optimization of compound 6b is on-going.

Anti-inflammatory effects of egg white combined with chalcanthite in lipopolysaccharide-stimulated BV2 microglia through the inhibition of NF-κB, MAPK and PI3K/Akt signaling pathways

Egg white-chalcanthite (EWCC) is a mixture of egg white and chalcanthite prepared by roasting chalcanthite (which is a natural mineral mainly composed of CuSO4•5H2O) to the point of dehydration, pulverizing the dehydrated chalcanthite and then mixing the pulverized chalcanthite to react with egg white to trigger a reaction. When egg white-chalcanthite is prepared in this manner, the toxicity of chalcanthite is neutralized by the egg white, so that the toxicity is reduced or removed and the pharmaceutical properties are increased. However, the cellular and molecular mechanisms underlying the pharmacological activity of EWCC remain poorly understood. In this study, we investigated the inhibitory effects of EWCC on the production of lipopolysaccharide (LPS)-induced pro-inflammatory mediators in BV2 microglia. Our data indicated that the EWCC treatment significantly inhibited the excessive production of nitric oxide and prostaglandin E2 in LPS-stimulated BV2 microglia in a concentration-dependent manner without causing cytotoxicity. It also attenuated the expression of inducible nitric oxide synthase, cyclooxygenase-2 and pro-inflammatory cytokines, including interleukin-1β and tumor necrosis factor-α. Moreover, EWCC exhibited anti-inflammatory properties by the suppression of nuclear factor‑κB (NF-κB) activation by blocking IκB-α degradation, downregulation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt pathways. Our results indicate that the inhibitory effects of EWCC on LPS-stimulated inflammatory mediator production in BV2 microglia are associated with the suppression of the NF-κB, MAPK and PI3K/Akt signaling pathways. These findings suggest that EWCC may offer a substantial therapeutic potential for the treatment of neurodegenerative diseases that are accompanied by microglial activation.

3,4,5-Tricaffeoylquinic Acid Inhibits the Lipopolysaccharide-Stimulated Production of Inflammatory Mediators in Keratinocytes

Background and Purpose: Microbial product lipopolysaccharide (LPS) has been shown to be involved in the pathogenesis of inflammatory skin diseases. Caffeoyl derivatives have demonstrated anti-inflammatory and antioxidant effects. However, the effect of 3,4,5-tricaffeoylquinic acid (3,4,5-triCQA) on the production of microbial product-induced inflammatory mediators in keratinocytes has not yet been studied. Experimental Approach: Using human keratinocytes, we investigated the effect of 3,4,5-triCQA on the LPS-stimulated production of inflammatory mediators in relation to the nuclear factor (NF)-ĸB, Akt and ERK pathways. Results: 3,4,5-triCQA inhibited the LPS-induced expression of Toll-like receptor 4, and the production of cytokines and chemokines in keratinocytes. 3,4,5-triCQA, Bay 11-7085, Aĸt inhibitor and ERK inhibitor each attenuated the LPS-induced production of inflammatory mediators by inhibiting the NF-ĸB, Akt and ERK pathways. Conclusions and Implications: 3,4,5-triCQA may attenuate the LPS-stimulated production of inflammatory mediators in keratinocytes by suppressing the Toll-like receptor 4 expression-mediated activation of the Akt, ERK and NF-ĸB pathways. 3,4,5-triCQA may exert a preventive effect against microbial product-induced inflammatory skin diseases.

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

Ethnopharmacological relevanceThe 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 methodsThe 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. ResultsWEPT 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. ConclusionsThese 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 effects of cordycepin via suppression of inflammatory mediators in BV2 microglial cells

Cordyceps militaris, a traditional medicinal mushroom, produces the bioactive compound cordycepin (3'-deoxyadenosine). Although cordycepin has been shown to have pharmacological, immunological stimulating, anti-cancer, and anti-inflammatory activities, its activities and cellular mechanisms during microglial activation have yet to be elucidated. Thus, we evaluated the anti-inflammatory effect of cordycepin on the production of inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV2 microglia. We also investigated the effects of cordycepin on LPS-induced nuclear factor-kappaB (NF-κB) activation and on phosphorylation of mitogen-activated protein kinases (MAPKs). After LPS stimulation, nitric oxide (NO), prostaglandin E 2 (PGE 2), and pro-inflammatory cytokine production was detected in BV2 microglia. However, we found that cordycepin significantly inhibited the excessive production of NO, PGE 2, and pro-inflammatory cytokines in a concentration-dependent manner without causing cytotoxicity. In addition, cordycepin suppressed NF-κB translocation by blocking IkappaB-α (IκB-α) degradation and inhibited the phosphorylation of Akt, ERK-1/2, JNK, and p38 kinase. Our results indicate that the inhibitory effect of cordycepin on LPS-stimulated inflammatory mediator production in BV2 microglia is associated with the suppression of the NF-κB, Akt, and MAPK signaling pathways. Therefore, cordycepin may be useful in treating neurodegenerative diseases by inhibiting inflammatory mediator production in activated microglia.

MAP kinases differentially regulate the expression of macrophage hyperactivity after thermal injury.

Thermal injury increases the capacity of macrophages (Mphi) to produce various inflammatory mediators, (i.e., Mphi hyperactivity), which is believed to be involved in the development of subsequent immunosuppression, sepsis, and multiple organ failure. The signal transduction pathways involved in the expression of Mphi hyperactivity post-burn, however, remain to be clearly elucidated. To study this C57BL/6 female mice were subjected to a 25% TBSA burn and splenic Mphis were isolated 7 days later. LPS-stimulated inflammatory mediator production and MAPK expression (P38 ERK 1/2 and JNK) were determined. Burn injury increased LPS-induced P38 MAPK, suppressed JNK activation and ERK 1/2 activation was unaltered. These changes in MAPK activation were paralleled by the increased production of PGE(2), TNF-alpha, IL-1beta, IL-6, and IL-10. Differential sensitivity to the inhibition of the MAPK pathways was observed with regard to the mediator evaluated and the presence or absence of burn injury. In general cytokine production in the burn group was in part resistant to the inhibition of a single MAPK pathway as compared with shams. Thus, burn injury increases cross-talk between the MAPKs pathways, suggesting that alterations MAPK activation and signal transduction contribute to the development Mphi hyperactivity post-injury.

Glossogyne tenuifolia Acts to Inhibit Inflammatory Mediator Production in a Macrophage Cell Line by Downregulating LPS-Induced NF-κB

Glossogyne tenuifolia (hsiang-ju) (GT) is a traditional antipyretic herb used in Chinese medicine; however, no information is available to explain its action. The objective of this research was to elucidate the molecular pharmacological activity and the effective components in the ethanol extract of GT. We found that GT had potent anti-inflammatory effects on the lipopolysaccharide (LPS)-activated murine macrophages, RAW264.7. GT downregulated LPS-induced expression of inducible nitric oxide synthase (iNOS) by blocking its transcription. GT also caused a dose-dependent inhibition of the release of prostaglandin E₂ by repressing the promoter activity of the inducible cyclooxygenase (COX-2) gene. Moreover, GT exerted a dose-dependent inhibition of the LPS-stimulated release of the proinflammatory cytokines, TNF-α, IL-1β, IL-6, and IL-12. To determine the mechanism by which GT inhibits LPS signaling, we focused on nuclear factor-ĸB (NF-ĸB) activation. Western blot analysis revealed that GT abolished LPS-induced inhibitor-ĸB phosphorylation. The electrophoretic mobility shift assay demonstrated that GT abolished LPS-mediated ĸB DNA binding activity. Moreover, macrophages were transfected with a vector coding for the luciferase reporter gene under the control of NF-ĸB cis-acting elements, and the transfected macrophages showed that the LPS-stimulated luciferase activity was GT-sensitive. These results suggest that GT attenuates inflammatory mediator synthesis of activated macrophages through an NF-ĸB-dependent pathway. The active components of GT were identified as oleanolic acid and luteolin-7-glucoside. Both of these compounds inhibited LPS-stimulated inflammatory mediator production and NF-ĸB activation. We conclude that GT inhibits NF-ĸB-mediated gene expression and downregulates inflammatory mediator production in murine macrophages.

Glossogyne tenuifolia acts to inhibit inflammatory mediator production in a macrophage cell line by downregulating LPS-induced NF-kappa B.

Glossogyne tenuifolia (hsiang-ju) (GT) is a traditional antipyretic herb used in Chinese medicine; however, no information is available to explain its action. The objective of this research was to elucidate the molecular pharmacological activity and the effective components in the ethanol extract of GT. We found that GT had potent anti-inflammatory effects on the lipopolysaccharide (LPS)-activated murine macrophages, RAW264.7. GT downregulated LPS-induced expression of inducible nitric oxide synthase (iNOS) by blocking its transcription. GT also caused a dose-dependent inhibition of the release of prostaglandin E(2) by repressing the promoter activity of the inducible cyclooxygenase (COX-2) gene. Moreover, GT exerted a dose-dependent inhibition of the LPS-stimulated release of the proinflammatory cytokines, TNF-alpha, IL-1 beta, IL-6, and IL-12. To determine the mechanism by which GT inhibits LPS signaling, we focused on nuclear factor-kappa B (NF-kappa B) activation. Western blot analysis revealed that GT abolished LPS-induced inhibitor-kappa B phosphorylation. The electrophoretic mobility shift assay demonstrated that GT abolished LPS-mediated kappa B DNA binding activity. Moreover, macrophages were transfected with a vector coding for the luciferase reporter gene under the control of NF-kappa B cis-acting elements, and the transfected macrophages showed that the LPS-stimulated luciferase activity was GT-sensitive. These results suggest that GT attenuates inflammatory mediator synthesis of activated macrophages through an NF-kappa B-dependent pathway. The active components of GT were identified as oleanolic acid and luteolin-7-glucoside. Both of these compounds inhibited LPS-stimulated inflammatory mediator production and NF-kappa B activation. We conclude that GT inhibits NF-kappa B-mediated gene expression and downregulates inflammatory mediator production in murine macrophages.