Inhibition of NF-κB-dependent cytokine and inducible nitric oxide synthesis by the macrocyclic ellagitannin oenothein B in TLR-stimulated RAW 264.7 macrophages

Immunomodulatory effects of oenothein B (1), a macrocyclic ellagitannin from various Onagraceae species, have been described previously. However, the mechanisms underlying the anti-inflammatory activity of 1 have not been fully clarified. The effects of 1 were investigated on inducible nitric oxide synthase, TLR-dependent and TLR-independent signal transduction cascades, and cytokine expression using murine macrophages (RAW 264.7). Compound 1 (10-60 μg/mL) reduced NO production, iNOS mRNA, and iNOS protein levels in a dose-dependent manner, without inhibition of iNOS enzymatic activity. It reduced the binding of the NF-κB p50 subunit to the biotinylated-consensus sequence and decreased nuclear p65 translocation. Gallic acid as a subunit of the macrocyclic ellagitannin 1 showed a far lower inhibitory activity. Nitric oxide production was reduced by 1 after stimulation using TLR2 (Pam2CSK4) and TLR4 (Kdo2) agonists, but this compound did not inhibit inducible nitric oxide synthesis after stimulation using interferon-gamma. IL-1beta, IL-6, and TNF-alpha mRNA synthesis was clearly reduced by the addition of 1. Oenothein B (1) inhibits iNOS after stimulation with LPS, TLR2, and TLR4 agonists via inhibition of TLR/NF-κB-dependent inducible nitric oxide and cytokine synthesis independent from IFN-gamma/JAK/STAT pathways. The full molecular structure of this macrocyclic ellagitannin seems to be required for its immunomodulatory actions.     

雷公藤内酯醇对结肠癌细胞COX-2和iNOS表达的抑制作用

 目的研究雷公藤内酯醇(TP)抑制结肠癌SW114细胞株环氧化酶-2(COX-2)、诱导型一氧化氮合酶(iNOS)及其产物PGE2和NO的表达,探讨TP的抗肿瘤机制。方法不同浓度的TP作用于结肠癌细胞株24h,通过RT-PCR,Western印迹杂交,ELISA检测环氧化酶-2(COX-2)、诱导型一氧化氮合酶(iNOS)及其产物,同时提取各组细胞蛋白质,TransAM测定核转录因子NF-κB活性。结果TP对结肠癌细胞株COX-2和iNOS的表达及其产物PGE2和NO的合成有抑制作用,而且也抑制SW114细胞的NF-κB活性,这些作用与剂量呈依赖关系。结论TP对SW114细胞COX-2和iNOS的表达及其产物PGE2和NO的合成有抑制作用,这种作用可能通过增强TP对核转录因子NF-κB活性的抑制作用而实现,揭示了TP抗肿瘤的部分依据。     

Suppression of inducible nitric oxide synthase expression by furfuran lignans from flower buds of Magnolia fargesii in BV‐2 microglial cells

Activated microglia produces diverse neurotoxic factors such as nitric oxide (NO) and tumor necrosis factor‐α that serve as apoptotic inducers resulting in various neurodegenerative diseases. The inhibition of microglia‐derived NO production by inducible nitric oxide synthase (iNOS) has been reported to be beneficial in retarding neurodegenerative disorders. Three active lignans have been isolated from the flower buds of Magnolia fargesii by the bioassay‐guided fractionation using lipopolysaccharide (LPS)‐activated BV‐2 microglial cell culture system. The structures of them were identified as kobusin (1), aschantin (2) and fargesin (3) by the analyses of spectroscopic data. They inhibited the production of NO by activated microglia. Their IC₅₀ values were 21.8 ± 3.7, 14.8 ± 2.5 and 10.4 ± 2.8 μg/mL, respectively. They suppressed LPS‐induced NF‐κB activation and the expression of iNOS protein and mRNA. Furthermore, they showed scavenging activity of neurotoxic peroxynitrite that can be produced by NO and superoxide anion. These results imply that lignans from Magnolia fargesii might be beneficial for the treatment of neuro‐inflammatory diseases through the inhibition of iNOS expression and peroxynitrite scavenging potential. Copyright © 2009 John Wiley & Sons, Ltd.     

Heme oxygenase-1 mediates the anti-inflammatory effect of mushroom Phellinus linteus in LPS-stimulated RAW264.7 macrophages

This work aimed to elucidate the anti-inflammatory mechanism of the n-BuOH subfraction (PL) prepared from fruiting bodies of Phellinus linteus. PL induced heme oxygenase-1 (HO-1) of the RAW264.7 macrophages in concentration- and time-dependent manner. It suppressed induction of inducible nitric oxide synthase (iNOS) and subsequent production of nitric oxide (NO) through down-regulation of iNOS promoter activity in lipopolysaccharide (LPS)-stimulated macrophages. Zn(II) protoporphyrin IX (ZnPP), a specific inhibitor of HO-1, partly blocked suppression by PL on iNOS promoter activity and NO production, which were elevated in LPS-stimulated macrophages. LPS was able to enhance NO production via reactive oxygen species (ROS) generation, c-Jun NH(2)-terminal kinase (JNK) and c-Jun induction. ZnPP prevented PL from down-regulating ROS generation and JNK activation in LPS-stimulated macrophages. Taken together, PL shows its anti-inflammatory activity via mediation of HO-1 in an in vitro inflammation model.     

"脾虚"大鼠胃黏膜组织一氧化氮含量及补中益气汤的作用

目的：研究“脾虚”大鼠胃黏膜组织一氧化氮（NO）含量的变化及补中益气汤的作用。方法：采用Griess试剂法和紫外分光光度法检测胃黏膜组织的一氧化氮代谢产物（NO2^-和NO3^-）和过氧化亚硝酸（ONOO^-）。结果：大黄“脾虚”大鼠胃黏膜组织中NO3^-、iNOS及ONOO^-水平均低于正常大鼠，经补中益气汤治疗后能恢复到接近正常水平。结论：“脾虚”大鼠胃黏膜组织一氧化氮（NO）含量低于正常，补中益气汤能升高“脾虚”大鼠胃黏膜组织中NO的含量。     

Sclareol exhibits anti-inflammatory activity in both lipopolysaccharide-stimulated macrophages and the λ-carrageenan-induced paw edema model

Sclareol (1) is a natural fragrance compound used widely in the cosmetic and food industries. Lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and the λ-carrageenan-induced edema mouse paw model were applied to examine the anti-inflammatory potential of 1 and its possible molecular mechanisms. The experimental results obtained demonstrated that this compound inhibited cell growth, nitric oxide (NO) production, and the expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins in LPS-stimulated macrophages. Compound 1 also reduced paw edema, the tissue content of NO, tumor necrosis factor-alpha (TNF-α), malondialdehyde (MDA), iNOS and COX-2 protein expression, and neutrophil infiltration within the tissues after λ-carrageenan stimulation. The present study suggests that the anti-inflammatory mechanisms of 1 might be related to a decrease of inflammatory cytokines and an increase of antioxidant enzyme activity.     

芪麝丸体外抗炎的机制

目的:考察芪麝丸体外对小鼠RAW264.7巨噬细胞炎症模型的抗炎机制.方法:干扰素γ(1×104 U·mL-1)和脂多糖(100μg·L-1)协同诱导小鼠RAW264.7巨噬细胞(1×105/mL)24 h造成炎症模型;Griess反应测定细胞上清液中一氧化氮(NO)产量;Western blot检测诱导型一氧化氮合酶(iNOS)、环氧合酶(COX-2)的蛋白表达及丝裂原活化蛋白激酶(MAPK)信号转导通路的活化情况.结果:芪麝丸(250,500,1000 mg·L-1)呈剂量依赖性抑制细胞上清液中NO含量,且无细胞毒性.较模型组,经芪麝丸500,1000 mg·L-1剂量处理后,iNOS蛋白表达从(1.00±0.06)下调至(0.61±0.07)和(0.02±0.15),(P＜0.01),COX-2蛋白表达从(0.56±0.03)下调至(0.42±0.02),(0.30±0.03),(P＜0.01).胞外信号调节激酶(ERK)磷酸化水平从(1.04±0.04)下调至(0.79±0.06),(0.73 ±0.10),(P＜0.01).p38磷酸化水平从(0.51±0.06)下调至(0.39 ±0.07),(0.29±0.15),(P＜0.05),c-Jun氨基末端激酶(JNK)磷酸化水平从(1.05±0.03)下调至(0.65 ±0.02),(0.66±0.033),(P＜0.01).结论:芪麝丸部分通过抑制MAPK信号转导通路活化过程中关键蛋白胞外信号调节激酶(ERK)、JNK和p38丝裂原活化蛋白激酶(p38)磷酸化,下调iNOS基因和蛋白的表达从而减少NO的产量,同时下调COX-2蛋白表达,而发挥其抗炎效果.     

前荷叶碱对人脐静脉内皮细胞一氧化氮及一氧化氮合酶的影响

目的:观察前荷叶碱(Pronuciferine)对人脐静脉内皮细胞(HUVECs)分泌一氧化氮(NO)和一氧化氮合酶(NOS)的影响,探讨其对HUVECs血管活性分子分泌的调节作用.方法:从莲子心中提取前荷叶碱,体外培养HUVECs,用不同浓度(10μmol/L、1μmol/L、0.1μmol/L、0.01μmol/L)的前荷叶碱分别作用于HUVECs,观察细胞形态,MTT法观察前荷叶碱对HUVECs活性的影响,比色法测定各组的NO、总NOS(tNOS)、诱导型NOS(iNOS).结果:和对照组比较,前荷叶碱对内皮细胞形态和活性无明显影响,能增加HUVECs释放NO,及tNOS生成(p＜0.01),但对iNOS影响不大.结论:前荷叶碱可能通过增加tNOS生成而提高HUVECs释放NO,从而保护内皮功能.     

Ethanol extract of propolis inhibits nitric oxide synthase gene expression and enzyme activity

Propolis obtained from honeybee hives has been used in Oriental folk medicine as an anti-inflammatory, anti-carcinogenic, or immunomodulatory agent. However, the molecular basis for anti-inflammatory properties of propolis has not yet been established. Since nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been known to be involved in inflammatory and autoimmune-mediated tissue destruction, modulation of NO synthesis or action represents a new approach to the treatment of inflammatory and autoimmune diseases. The present study, therefore, examined effects of ethanol extract of propolis (EEP) on iNOS expression and activity of iNOS enzyme itself. Treatment of RAW 264.7 cells with EEP significantly inhibited NO production and iNOS protein expression induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-γ). EEP also inhibited iNOS mRNA expression and nuclear factor-kappa B (NF-κB) binding activity in a concentration-dependent manner. Furthermore, transfection of RAW 264.7 cells with iNOS promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene, revealed that EEP inhibited the iNOS promoter activity induced by LPS plus IFN-γ through the NF-κB sites of the iNOS promoter. In addition, EEP directly interfered with the catalytic activity of murine recombinant iNOS enzyme. These results suggest that EEP may exert its anti-inflammatory effect by inhibiting the iNOS gene expression via action on the NF-κB sites in the iNOS promoter and by directly inhibiting the catalytic activity of iNOS.     

Anti-inflammatory effect of Citrus Unshiu peel in LPS-stimulated RAW 264.7 macrophage cells

Citrus Unshiu peel (CUP) has been traditionally used in East Asia as a drug for the treatment of vomiting and dyspepsia. However, its effects on inflammation remain unknown. In this study, we investigated the effects of CUP on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The research focused on determining whether CUP could inhibit the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 and the activation of nuclear factor (NF)-κB, mitogen-activated protein kinases (MAPKs), as well as the secretion of nitric oxide (NO), prostaglandin (PG) E(2), tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in LPS-stimulated RAW 264.7 cells. We found that CUP represses LPS-induced iNOS and COX-2 gene expression as well as NO, PGE(2), TNF-α and IL-6 production. Additionally, CUP inhibited the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal kinase (JNK) MAPK, and suppressed IκBα degradation and nuclear translocation of NF-κB. Collectively, our results indicate that CUP inhibits the production of various inflammatory mediators via blockade of MAPK phosphorylation pursuant to the inhibition of IκBα degradation and the nuclear translocation of NF-κB. These findings are the first to clarify the mechanism underlying the anti-inflammatory effect exerted by CUP in RAW 264.7 macrophage cells stimulated by inflammatory agents.     

Tetramethylpyrazine inhibits production of nitric oxide and inducible nitric oxide synthase in lipopolysaccharide-induced N9 microglial cells through blockade of MAPK and PI3K/Akt signaling pathways,and suppression of intracellular reactive oxygen species

Aim of the study

To determine the inhibitory effect of tetramethylpyrazine (TMP) on lipopolysaccharide (LPS)-induced over-production of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in N9 microglial cells.

Materials and methods

N9 cells were pretreated with vehicle or TMP and then exposed to LPS for the time indicated. Cell viability was determined by methylthiazoyltetrazolium (MTT) assay. Nitrite assay was performed by Griess reaction. Expression of iNOS mRNA was examined by RT-PCR. Protein levels of iNOS, p38 mitogen-activated protein kinase (MAPK), ERK1/2, JNK, phosphatidylinositol 3-kinase (PI3K) and Akt were determined by western blot analysis. Formation of reactive oxygen species (ROS) was evaluated by fluorescence image system.

Results

TMP inhibited LPS-induced over-production of NO and iNOS in N9 cells. TMP also inhibited the NF-κB translocation from cytoplasm into nucleus of N9 cells. In addition, TMP showed blocking effect on the phosphorylation of p38 MAPK, ERK1/2, JNK and Akt, but not PI3K. Further, TMP suppressed the formation of intracellular ROS in LPS-induced N9 cells.

Conclusions

TMP inhibited production of NO and iNOS in LPS-induced N9 cells through blocking MAPK and PI3K/Akt activation and suppressing ROS production.     

Fermentation improves anti-inflammatory effect of sipjeondaebotang on LPS-stimulated RAW 264.7 cells

Sipjeondaebotang (SJ) has been used as a traditional drug in east-Asian countries. In this study, to provide insight into the biological effects of SJ and SJ fermented by Lactobacillus, we investigated their effects on lipopolysaccharide (LPS)-mediated inflammation in macrophages. The investigation was focused on whether SJ and fermented SJ could inhibit the production of pro-inflammatory mediators such as prostaglandin (PG) E(2) and nitric oxide (NO) as well as the expressions of cyclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB in LPS-stimulated RAW 264.7 cells. We found that SJ modestly inhibited LPS-induced PGE(2), NO and TNF-α production as well as the expressions of COX-2 and iNOS. Interestingly, fermentation significantly increased its inhibitory effect on the expression of all pro-inflammatory mediators. Furthermore, fermented SJ exhibited increased inhibition of p38 MAPK and c-Jun NH(2)-terminal kinase (JNK) MAPK phosphorylation as well as NF-κB p65 translocation by reduced IκBα degradation compared with either untreated controls or unfermented SJ. High performance liquid chromatography (HPLC) analysis showed fermentation by Lactobacillus increases liquiritigenin and cinnamyl alcohol contained in SJ, which are known for their anti-inflammatory activities. Finally, SJ fermented by Lactobacillus exerted potent anti-inflammatory activity by inhibiting MAPK and NF-κB signaling in RAW 264.7 cells.     

六味地黄丸对动脉粥样硬化小鼠iNOS表达的影响

目的:研究六味地黄丸对动脉粥样硬化(AS)小鼠一氧化氮(NO)及诱导型一氧化氮合酶(iNOS)表达的影响。方法:喂饲高脂饲料复制小鼠AS模型;Griess法检测血浆中NO水平;蛋白质免疫印迹法检测iNOS的表达。结果:AS小鼠9周时开始出现NO显著增加,六味地黄丸能显著抑制NO水平,并降低iNOS的表达。结论:六味地黄丸可能通过抑制iNOS的表达降低AS小鼠NO水平,防治AS。     

Effects of bee venom on the pro-inflammatory responses in RAW264.7 macrophage cell line

The purpose of this study is to elucidate the molecular mechanism of anti-inflammatory effect of bee venom (BV), which has been used for the treatment of various inflammatory diseases in oriental medicine. With this aim, we examined the effects of BV on the nitric oxide (NO) production by lipopolysaccharide (LPS) or sodium nitroprusside in RAW264.7 macrophages. We further investigated the effects of BV on the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinase (MAPK) with RT-PCR in LPS-stimulated RAW264.7 cells. BV suppressed the NO production and decreased the levels of iNOS, COX-2, NF-kappaB and MAPK mRNA in a dose-dependent manner. These results suggest that BV has an anti-inflammatory effect by inhibiting iNOS and COX-2 expression, possibly through suppression of NF-kappaB and MAPK expression.