7,8-Dihydroxyflavone exhibits anti-inflammatory properties by downregulating the NF-κB and MAPK signaling pathways in lipopolysaccharide-treated RAW264.7 cells

7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, has received considerable attention as a selective tyrosine kinase receptor B agonist. Several studies have indicated that 7,8-DHF has neurotrophic and antioxidant activities. However, little is known about the cellular and molecular mechanisms underlying the anti-inflammatory activity of 7,8-DHF. Therefore, we investigated whether 7,8-DHF affects the expression of inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Our results indicated that 7,8-DHF significantly attenuated secretion of LPS-induced inflammatory mediators nitric oxide (NO), prostaglandin E? (PGE?) and interleukin-1β (IL-1β) in RAW264.7 cells. Additionally, LPS-induced expression of inducible NO synthase (iNOS), cyclooxygenase (COX)-2 and IL-1β was decreased by pre-treatment with 7,8-DHF. Our results also showed that 7,8-DHF reduces LPS-induced nuclear factor-κB (NF-κB) activity via the suppression of the nuclear translocation of NF-κB p65 and the degradation of inhibitor κB (lκB). In addition, 7,8-DHF inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) such as extracellular-signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). These results suggest that the anti-inflammatory property of 7,8-DHF is related to the downregulation of iNOS, COX-2 and IL-1β, due to NF-κB inhibition as well as to the negative regulation of MAPK activation in RAW264.7 cells. Thus, 7,8-DHF may be a novel therapeutic agent for the prevention of various inflammatory diseases.     

Anti-inflammatory activity of ethylacetate fraction of Cliona celata

We evaluated the ability of the ethylacetate fraction of marine sponge, Cliona celata (ECC), harvested from Korean seaside to regulate the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 cells. ECC dose-dependently inhibited both the expression of iNOS protein and mRNA, resulting in decreased production of nitric oxide (NO), with an IC(50) of 80.5 μg/mL. To investigate action mechanism by which ECC inhibits NO production and iNOS expression, we examined the activation of IκB in LPS-stimulated RAW264.7 cells. ECC clearly inhibited translocation of nuclear factor-κB (NF-κB) p65 subunits from cytosol to nucleus, which correlated with its inhibitory effects on IκB-α phosphorylation and degradation. Furthermore, ECC potently suppressed both the reporter gene expression and DNA-binding activity of NF-κB, which was associated with decreased p65 protein levels in the nucleus. Here, we show for the first time that ECC inhibits NF-κB activation through the inhibition of IκB degradation.     

Inhibition of LPS-induced inflammatory biomarkers by ethyl acetate fraction of Patrinia scabiosaefolia through suppression of NF-κB activation in RAW 264.7 cells

Patrinia scabiosaefolia (PS) has been used for curing various types of inflammatory-related disorders. However, the precise mechanism of the anti-inflammatory activity of PS remains unclear. Here, we investigated the anti-inflammatory effects of several fractions isolated from the PS in RAW 264.7 macrophages. The results indicated that the ethyl acetate fraction of PS (EAPS) concentration highly suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) and IL-6 productions without a cytotoxic effect on RAW 264.7 cells. EAPS inhibited the expressions of LPS-induced iNOS and COX-2 protein and their mRNA in a dose-dependent manner. Particularly, EAPS suppressed the level of nuclear factor-κB (NF-κB) activity, which was linked with the suppression of LPS-induced phosphorylation of p65 at serine 276 and p65 translocation into nuclei, but not MAPK signaling. In addition, treatment with EAPS inhibited the production of TNF-α in LPS-injected mice and suppressed the production of IL-6 and TNF-α in LPS-stimulated splenocytes from BALB/c mice. Therefore, we demonstrate here that Patrinia scabiosaefolia potentially inhibits the biomarkers related to inflammation through the blocking of NF-κB p65 activation, and it may be a potential therapeutic candidate for the treatment of inflammatory diseases.     

Spironolactone inhibits production of proinflammatory mediators in response to lipopolysaccharide via inactivation of nuclear factor-κB

The effect of spironolactone (SPIR) on lipopolysaccharide (LPS)-induced production of proinflammatory mediators was examined using RAW 264.7 macrophage-like cells and mouse peritoneal macrophages. SPIR significantly inhibited LPS-induced production of nitric oxide (NO), tumor necrosis factor-α and prostaglandin E2. The inhibition was not mediated by cell death. SPIR reduced the expression of an inducible NO synthase mRNA in response to LPS. SPIR significantly inhibited phosphorylation of p65 nuclear factor (NF)-κB in response to LPS. Furthermore, SPIR inhibited phosphorylation of IκB kinase (IKK) as an upstream molecule of NF-κB in response to LPS. LPS did not induce the production of aldosterone in RAW 264.7 cells. Taken together, SPIR is suggested to inhibit LPS-induced proinflammatory mediators via inactivation of IKK/NF-κB in LPS signaling.     

Dichloromethane Fraction of Laminaria japonica Ethanolic Extract Inhibits Lipopolysaccharide-Induced Nitric Oxide Synthase and Cyclooxygenase-2 Expression in RAW 264.7 Cells via NF-κB Pathway

Strong anti-inflammatory activity has been found in Laminaria japonica dichloromethane fraction (LDF); however, the molecular mechanisms underlying its anti-inflammatory activity are not reported. Our results indicated that LDF inhibited LPS-induced nitric oxide and prostaglandin E(2) production in a dose-dependent manner and suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in RAW 264.7 cells. Also, levels of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1β and IL-6 were remarkably reduced by LDF in LPS-treated RAW 264.7 cells. LDF greatly inhibited promoter activity of nuclear factor-κB (NF-κB) and translocation of NF-κB subunits by prevention of the degradation of inhibitor κB-α in LPS-treated RAW 264.7 cells (p?相似文献   

Inhibition of LPS-induced inflammatory biomarkers by ethyl acetate fraction of Patrinia scabiosaefolia through suppression of NF-κB activation in RAW 264.7 cells

Patrinia scabiosaefolia (PS) has been used for curing various types of inflammatory-related disorders. However, the precise mechanism of the anti-inflammatory activity of PS remains unclear. Here, we investigated the anti-inflammatory effects of several fractions isolated from the PS in RAW 264.7 macrophages. The results indicated that the ethyl acetate fraction of PS (EAPS) concentration highly suppressed lipopolysaccharide (LPS)-induced nitric oxide (NO) and IL-6 productions without a cytotoxic effect on RAW 264.7 cells. EAPS inhibited the expressions of LPS-induced iNOS and COX-2 protein and their mRNA in a dose-dependent manner. Particularly, EAPS suppressed the level of nuclear factor-κB (NF-κB) activity, which was linked with the suppression of LPS-induced phosphorylation of p65 at serine 276 and p65 translocation into nuclei, but not MAPK signaling. In addition, treatment with EAPS inhibited the production of TNF-α in LPS-injected mice and suppressed the production of IL-6 and TNF-α in LPS-stimulated splenocytes from BALB/c mice. Therefore, we demonstrate here that Patrinia scabiosaefolia potentially inhibits the biomarkers related to inflammation through the blocking of NF-κB p65 activation, and it may be a potential therapeutic candidate for the treatment of inflammatory diseases.     

Roburic Acid Suppresses NO and IL-6 Production <Emphasis Type="Italic">via</Emphasis> Targeting NF-κB and MAPK Pathway in RAW264.7 Cells

In the present study, we investigated the anti-inflammatory effect of roburic acid on production of nitric oxide (NO) and interlukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. We found that roburic acid reduced production of NO and IL-6, and the expression of inducible nitric oxide synthases (iNOS). Meanwhile, phosphorylation of inhibitor of κBα (IκBα) and IκB kinase α/β (IKKα/β), as well as translocation of nuclear factor-κB (NF-κB) to the nucleus, was suppressed by roburic acid treatment. In addition, phosphorylation of mitogen-activated protein kinase (MAPKs) including p38 and c-Jun-NH₂-terminal kinase (JNK) was inhibited. Roburic acid exhibited inhibitory activities on production of NO and IL-6 via blocking IKK/IκB/NF-κB and MAPKs pathway, suggesting the potential application as a drug candidate for therapy of inflammatory diseases.     

EstA protein,a novel virulence factor of Streptococcus pneumoniae, induces nitric oxide and pro-inflammatory cytokine production in RAW 264.7 macrophages through NF-κB/MAPK

In the present study we characterized the molecular mechanism by which esterase A (EstA) protein, a novel virulence factor of Streptococcus pneumoniae induces inflammation. Stimulation of RAW 264.7 macrophages with purified EstA protein induced the expression of inducible nitrogen oxide synthase (iNOS) mRNA and nitrogen oxide (NO) production in a concentration-dependent manner. Inhibitors of iNOS, NF-κB, p38 and ERK 1/2 MAPK pathways significantly decreased (50–78%) EstA-induced NO production. Similarly, EstA induced TNF-α, IL-1β and IL-6 mRNA expression in RAW 264.7 macrophages in a dose-dependent manner, and pre-treatment of the cell cultures with specific NF-κB, p38 and ERK 1/2 MAPK pathway inhibitors significantly decreased EstA-induced TNF-α, IL-1β and IL-6 protein production. Furthermore, immunoblot analysis revealed the degradation of the inhibitory kappa B (IKB-α) in response to EstA stimulation. Taken together, our data suggests that EstA protein is a novel inducer of NO and pro-inflammatory cytokines by activating the NF-κB, p38 and ERK 1/2 MAPK pathways during inflammatory responses. Future studies on the upstream protein kinases of the MAPK/NF-κB pathways and the kinetics of cytokine production will provide further details into the mechanism of EstA-induced inflammatory response.     

β-sitosteryl-3-O-β-glucopyranoside isolated from the bark of Sorbus commixta ameliorates pro-inflammatory mediators in RAW 264.7 macrophages

The bark of Sorbus commixta has been used in Asian traditional medicine for treatment of cough, asthma, bronchial disorders, gastritis and dropsy. However, the anti-inflammatory effect of β-sitosteryl-3- O -β-glucopyranoside, a major compound of the bark of S. commixta, is poorly understood. In this study, we investigated the anti-inflammatory effect and the underlying molecular mechanisms of β-sitosteryl-3-O-β-glucopyranoside in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Prostaglandin E₂ (PGE₂) and cytokines released from cells were measured using EIA assay kit. The expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, Tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) was measured by real-time polymerase chain reaction (RT-PCR) and/or Western blot analysis. β-sitosteryl-3-O-β-glucopyranoside inhibited the production of nitric oxide (NO) and PGE₂ along with the expression of iNOS and COX-2 in LPS-stimulated RAW264.7 cells. In addition, β-sitosteryl-3-O-β-glucopyranoside reduced the release of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6. Moreover, β-sitosteryl-3-O-β-glucopyranoside inhibited the NF-κB activation induced by LPS, which was associated with the abrogation of IκBα degradation and subsequent decreases in nuclear p65 levels. The result suggested that the β-sitosteryl-3-O-β-glucopyranoside inhibited NO and pro-inflammatory productions by down-regulating the gene expression of pro-inflammatory mediators via the negative regulation of the NF-кB pathway in LPS-stimulated RAW 264.7 cells.     

The inhibition of lipopolysaccharide-induced tumor necrosis factor-α and nitric oxide production by Clostridium perfringens α-toxin and its relation to α-toxin-induced intracellular ceramide generation

The effect of Clostridium perfringens α-toxin on production of tumor necrosis factor (TNF)-α and nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was studied. The pretreatment of wild type α-toxin, but not the inactive mutant, significantly decreased LPS-induced TNF-α and NO production. α-Toxin inhibited the expression of TNF-α and an inducible type of NO synthase protein and mRNA. Furthermore, it inhibited the phosphorylation of IκB-α and p65 NF-κB subunit, and the NF-κB luciferase reporter gene activity in LPS-stimulated cells. The pretreatment of α-toxin increased the level of intracellular ceramide. Taken together, Clostridium perfringens α-toxin pretreatment was suggested to inhibit LPS-induced TNF-α and NO production through the inhibition of NF-κB activation. The relationship between α-toxin-induced intracellular ceramide generation and the NF-κB inhibition is discussed.     

Licochalcone A isolated from licorice suppresses lipopolysaccharide-stimulated inflammatory reactions in RAW264.7 cells and endotoxin shock in mice

Licochalcone A (LicA), a major phenolic constituent of the licorice species Glycyrrhiza inflata, exhibits various biological properties, including chemopreventive, anti-bacterial, and anti-spasmodic activity. We report that LicA inhibits inflammatory reactions in macrophages and protects mice from endotoxin shock. Our in vitro experiments showed that LicA suppressed not only the generation of nitric oxide (NO) and prostaglandin (PG)E(2), but also the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 induced by lipopolysaccharide (LPS) in RAW264.7 cells. Similarly, LicA inhibited the production of inflammatory cytokines induced by LPS in RAW264.7 cells, including IL-1 beta and IL-6. In an animal model, LicA protected BALB/c mice from LPS-induced endotoxin shock, possibly through inhibiting the production of inflammatory cytokines and NO. Collectively, LicA inhibited the production of inflammatory mediators and may be a potential target for treatment of various inflammatory diseases.     

The Essential Oil Isolated from Artemisia capillaris Prevents LPS-Induced Production of NO and PGE2 by Inhibiting MAPK-Mediated Pathways in RAW 264.7 Macrophages

Artemisia capillaris (A. capillaris) is used in traditional Korean herbal medicine for its believedanti-inflammatory activities. Previous studies have suggested that the essential oil of A. capillaris contains the active components responsible for its pharmacological effect, even though the mechanism for its action is unclear. This study examined the inhibitory effects of the essential oil of A. capillaris on the lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). The essential oil significantly inhibited the production of NO in the LPS-stimulated RAW 264.7 macrophages, which was mediated by the down-regulation of inducible NO synthase (iNOS) expression but not by its direct cytotoxic activity. The essential oil also blocked the secretion of PGE₂ and the expression of cyclooxygenase-2 (COX-2) in the LPS-stimulated cells. Western blot analysis showed that the essential oil inhibited the phosphorylation of IκB-α, nuclear translocation of p65, and subsequent activation of NF-κB. In addition, the essential oil suppressed the LPS-stimulated activation of mitogen-activated protein kinases (MAPKs) as well as the AP-1 DNA-binding activity. Moreover, MAPK inhibitors significantly reduced the LPS-induced production of NO and PGE₂. Collectively, we suggest that the oil inhibits the expression and production of inflammatory mediators by blocking the MAPK-mediated pathways and inhibiting the activation of NF-κB and AP-1.     

Forsythin inhibits lipopolysaccharide-induced inflammation by suppressing JAK-STAT and p38 MAPK signalings and ROS production

Objective

Forsythin (FOR) is an active ingredient extracted from the fruit of the medicinal plant Forsythia suspensa (Thunb.) Vahl. Here, we investigated the effect of FOR on LPS-induced inflammatory response and the underlying molecular mechanisms in RAW264.7 macrophages.

Materials and methods

RAW264.7 cells were pre-treated with or without FOR and then stimulated with or without LPS. The productions of TNF-α, IL-1β, IL-6, PGE₂ and NO were determined by ELISA and nitrite analysis, respectively. The expressions of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were measured by Western blotting and RT-PCR analysis. The activations of signaling molecules were detected by Western blotting using phosphorylation specific antibodies. Reactive oxygen species (ROS) production was determined by ROS assay.

Results

LPS-induced productions of IL-1β, IL-6, TNF-α, NO and PGE₂ were inhibited by FOR in a dose-dependent manner. FOR also suppressed the LPS-elevated expressions of iNOS and COX-2. Further investigations revealed that FOR significantly inhibited the LPS-induced activations of JAK-STATs and p38 MAPKs, but not of IKKα/β in LPS-stimulated RAW264.7 cells. Additionally, FOR interfered with both JAK-STATs and p38 MAPKs signaling pathways to modulate the expressions of IL-1β, IL-6, TNF-α, iNOS and COX-2. Furthermore, FOR reduced the LPS-induced ROS accumulation, validating that FOR serves as an antioxidant.

Conclusions

Our data suggested that FOR exerts anti-inflammatory action, at least in part, via suppressing LPS-induced activation of JAK-STATs and p38 MAPKs signalings and production of ROS in macrophage cells.     

Spirodela polyrhiza (L.) Sch. ethanolic extract inhibits LPS-induced inflammation in RAW264.7 cells

Objective: Spirodela polyrhiza (L.) Sch. is widely used in Korean traditional medicine. No previous work has investigated in detail the anti-inflammatory activities of S. polyrhiza or assessed in vitro their potential underlying mechanism(s). We assessed the effects of S. polyrhiza ethanolic extract (SPEE) on the production of inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW264.7 cells and investigated some potential underlying mechanisms. Additionally, we performed simultaneous determination of seven flavonoids in S. polyrhiza by high-performance liquid chromatography (HPLC)-photodiode array (PDA). Materials and methods: RAW264.7 cells were subjected to 5, 10, 20, and 50 μg/mL of SPEE for 1 h then treated with LPS for 24 h. Production of namely nitric oxide (NO), prostaglandin E(2) and cytokine levels were measured by the Griess reagent and ELISA, respectively. To investigate the underlying mechanisms of the anti-inflammatory activities of SPEE, expression of NO synthase (iNOS), cyclooxygenase-2 (COX-2), and nuclear factor-kappa B (NF-κB) proteins were evaluated by western blot analysis. HPLC analysis was performed using a Gemini C(18) column at 40°C and PDA detection at 340 nm. Results: SPEE treatment significantly inhibited the LPS-induced production of NO, prostaglandin E(2), interleukin-6, and tumor necrosis factor-α and inhibited the expression of iNOS and COX-2 via attenuation of NF-κB p65 expression. The contents of the seven flavonoids in S. polyrhiza range from 0.25 to 8.77 mg/g. Conclusions: These results indicate that the anti-inflammatory activity of SPEE may be NF-κB p65 signaling. Also, the method will help to improve quality control of S. polyrhiza.     

醛肽类蛋白酶体抑制剂对LPS诱导小鼠巨噬细胞炎症介质表达的影响

目的： 探讨醛肽类蛋白酶体抑制剂MG132对脂多糖（LPS）诱导小鼠巨噬细胞Raw264.7 核转录因子-κB（NF-κB)活化、炎性因子一氧化氮(NO)和肿瘤坏死因子α(TNF-α)分泌以及诱导型一氧化氮合酶(iNOS)表达的影响。方法： 利用报告基因检测法分析转染细胞（pNiFty-SEAP/HEK293）中NF-κB的活性;采用DAF-2DA荧光探针法检测Raw264.7细胞内NO的产生;蛋白免疫印迹法探讨细胞中iNOS和IκB-α蛋白表达情况;酶联免疫吸附法测定Raw264.7细胞上清中TNF-α的含量。结果： 预先加入MG132能够显著抑制LPS诱导的TNF-α分泌,其抑制率由5 μmol/L时的36.7%升高到10 μmol/L时的60.4%。反映NO含量的荧光强度值随着MG132给药浓度的增加而降低,其抑制率从2 μmol/L时的29.5%达到10 μmol/L的55.9%。预刺激后MG132可使胞浆中iNOS蛋白表达减少,IκB-α蛋白却明显增加,NF-κB的活性随着给药浓度的升高而不断降低。结论: MG132能够抑制LPS诱导的TNF-α和NO的产生,减少iNOS表达,具有抗炎作用。其作用机制可能与IκB降解受阻,导致NF-κB活性降低有关。     

Short-Chain Fatty Acids Suppress Lipopolysaccharide-Induced Production of Nitric Oxide and Proinflammatory Cytokines Through Inhibition of NF-κB Pathway in RAW264.7 Cells

Short-chain fatty acids (SCFAs) produced by the colonic bacterial fermentation of dietary fiber contribute a significant proportion of daily energy requirement. Furthermore, these compounds are modulators of macrophage function and potential targets for the development of new drugs. The aims of this study were to evaluate the effects of three types of SCFAs (sodium acetate (NaAc), sodium propionate (NaP), and sodium butyrate (NaB)) on the production of NO and inducible nitric oxide synthase (iNOS) and proinflammatory and antiinflammatory cytokines (tumor necrosis factor-α (TNF-α) and interleukin (IL-1, IL-6, and IL-10)) and to observe the effect of NaAc on inhibiting lipopolysaccharide (LPS)-induced NF-κB activation in LPS-stimulated RAW264.7 cells. The results show that three types of SCFAs (acetate, propionate, and butyrate) reduced the production of proinflammatory factors, including TNF-α, IL-1β, IL-6, and NO, and inhibited the vitality of iNOS. Meanwhile, SCFAs enhanced the production of antiinflammatory cytokine IL-10 in lower concentrations (1-1,200?μmol/L). Like NaB, NaAC inhibited LPS-induced NF-κB activation. These results may hold promise on the role that SCFAs have on the prevention and treatment of various inflammatory conditions.