Anti-inflammatory effect of flavonoids isolated from Korea Citrus aurantium L. on lipopolysaccharide-induced mouse macrophage RAW 264.7 cells by blocking of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signalling pathways

Citrus fruits are an abundant source of various flavonoids, which have been used as a traditional herbal medicine in Korea and China. Most flavonoids are known to have anti-oxidant, anti-bacterial and analgesic properties. In this study, it was examined whether flavonoids (nobiletin, naringin and hesperidin) isolated from Korea Citrus aurantium L. inhibited the pro-inflammatory mediators including cytokines by blocking nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signalling in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The flavonoids suppressed mRNA and protein expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in LPS-induced macrophages. The molecular mechanism was associated with inhibition of the degradation/phosphorylation of I-κB-α and nuclear translocation of the NF-κB p-65 as well as phosphorylation of MAPK by flavonoids. These results suggest that flavonoids have anti-inflammatory effects by suppressing expression of COX-2, iNOS and cytokines by blocking the NF-κB and MAPK signalling in RAW 264.7 macrophages.     

蒲公英糖蛋白体外抗炎作用及对NF-κB信号通路的调控

探讨蒲公英糖蛋白(glycoprotein from Taraxacum,TG)对由脂多糖引起的RAW264.7细胞炎症的抗炎效果,并阐明其活性的基本分子机制。利用脂多糖刺激RAW264.7细胞,建立体外炎症模型,采用噻唑蓝比色法检测TG对RAW264.7细胞的增殖毒性,Griess试剂法检测了一氧化氮(nitric oxide,NO)的分泌情况,反转录聚合酶链式反应检测炎症细胞因子——白细胞介素-6(interleukin 6,IL-6)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)m RNA以及诱导型一氧化氮合酶(inducible nitric oxide synthase,i NOS)m RNA的表达水平,酶联免疫吸附测定法测定IL-6和TNF-α的分泌量,Western blot检测P-IκB-α蛋白的表达水平,用以研究TG对核转录因子-κB(nuclear factor-kappa B,NF-κB)信号转导通路的抑制作用。结果表明:TG能够显著甚至极显著地抑制NO的分泌,IL-6、TNF-α、i NOS的m RNA的表达,IL-6和TNF-α的分泌(P0.05、P0.01)。TG高度显著上调了IκB-α的蛋白表达(P0.001),并显著下调了P-IκB-α的蛋白表达(P0.05),且与TG质量浓度成正比。其中在TG质量浓度为250、500、1 000μg/m L时对TNF-α分泌量的抑制率分别为28.6%、65.4%、89.3%,对IL-6分泌量的抑制率分别为32.3%、54.1%、85.7%。TG间接抑制了NF-κB信号转导途径,有显著的体外抗炎效果且抗炎效果与TG的质量浓度呈剂量依赖性。     

Toona sinensis and its major bioactive compound gallic acid inhibit LPS-induced inflammation in nuclear factor-κB transgenic mice as evaluated by in vivo bioluminescence imaging

In the present study, we investigated the anti-inflammatory effects of a nutritious vegetable Toona sinensis (leaf extracts, TS) and its major bioactive compound gallic acid (GA) by analysing LPS-induced NF-κB activation in transgenic mice, using bioluminescence imaging. Mice were challenged intraperitoneally with LPS (1 mg/kg) and treated orally with TS or GA (100 or 5 mg/kg, respectively). In vivo and ex vivo imaging showed that LPS increased NF-κB luminescence in the abdominal region, which was significantly inhibited by TS or GA. Immunohistochemical and ELISA analyses confirmed that TS and GA inhibited LPS-induced NF-κB, interleukin-1β, and tumour necrosis factor-α expression. Microarray analysis revealed that biological pathways associated with metabolism and the immune responses were affected by TS or GA. Particularly, LPS-induced thioredoxin-like 4B (TXNL4B) 2 expression in the small intestine, and TXNL4B, iNOS, and COX-2 expression in RAW 264.7 cells were significantly inhibited by TS or GA. Thus, the anti-inflammatory potential of TS was mediated by the downregulation of NF-κB pathway.     

蛹虫草多糖调节小鼠巨噬细胞RAW264.7免疫活性的分子机制

多种真菌多糖近年来因其免疫调节活性成为保健食品领域研究的热点。本实验以食药用真菌蛹虫草提取物蛹虫草多糖（Cordyceps militaris polysaccharides,CMP）为研究材料,初步探究CMP对小鼠巨噬细胞RAW264.7免疫活性的调节机制。噻唑蓝实验结果显示CMP无细胞毒性,且100、200 μg/mL CMP可以明显增强RAW264.7细胞活性;中性红法、Griess法和酶联免疫吸附检测结果表明25～200 μg/mL的CMP以剂量依赖方式增强RAW264.7细胞吞噬活性并增加一氧化氮（nitric oxide,NO）和白介素-1β（interleukin-1β,IL-1β）分泌,肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）的分泌呈先升高后降低的趋势,在100 μg/mL时达到最高值;抑制剂中和实验结果显示当Toll样受体4（Toll-like receptors 4,TLR4）和甘露糖受体（mannose receptor,MR）受到抑制时,CMP诱导的RAW264.7免疫反应显著降低,这表明TLR4和MR均为CMP激活巨噬细胞的受体;此外,Western blot结果显示丝裂原活化蛋白激酶（mitogen-activated protein kinases,MAPK）信号通路也参与CMP诱导的RAW264.7细胞分泌NO、TNF-α和IL-1β。表明TLR4和MR/MAPK信号传导途径在CMP诱导巨噬细胞RAW264.7产生免疫应答时发挥了重要作用。     

Proteomic response of LAP-activated RAW 264.7 macrophages to the anti-inflammatory property of fungal ergosterol

Ergosterol is the principal fungal sterol in edible mushrooms and yeast lees in wine. In this study, the immunoregulatory action of ergosterol was evaluated by a proteomic approach using two-dimensional gel electrophoresis to identify proteins with modified expression in LPS-challenged RAW 264.7 macrophages treated with ergosterol. By comparing the proteome signatures, alterations of multiple proteins were observed, including NF-κB cascade-related proteins, Rho family GTPase protein, and cytoskeleton-related proteins. The down-regulation of NF-κB cascade-associated proteins coincided with the inhibition of TNF-α production and COX-2 expression in the ergosterol-treated RAW 264.7 cells. Western blotting analysis indicated that ergosterol inhibits LPS-induced inflammation in RAW 264.7 macrophages by suppressing the NF-κB signalling pathway.     

Anti-inflammatory effect of the hexane fraction from Orostachys japonicus in RAW 264.7 cells by suppression of NF-κB and PI3K-Akt signaling

We investigated the anti-inflammatory effect of the hexane fraction from Orostachys japonicus (OJH) in LPS (lipopolysaccharide)-stimulated RAW 264.7 cells. Pretreatment with OJH dose-dependently reduced the cellular NO (nitric oxide) concentration and also inhibited expression of iNOS (inducible nitric oxide synthase) protein and mRNA. By the prevention of IκBα (inhibitory factor kappa B alpha) phosphorylation and degradation, OJH inhibited LPS-induced NF-κB (nuclear factor-kappa B) activation. OJH had no effect on the LPS-induced phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), or p38, whereas it attenuated the phosphorylation of Akt in a dose-dependent manner. In addition, OJH suppressed the LPS-induced expression of LPS receptors CD14 and TLR4 (toll like receptor 4). These results suggest that OJH may interrupt LPS-induced pro-inflammatory cascades through inhibition of NF-κB and Akt activation.     

硒化低聚氨基多糖对RAW264.7细胞的免疫调节作用

本实验旨在研究硒化低聚氨基多糖对小鼠巨噬细胞RAW264.7免疫功能的影响。用噻唑蓝比色法分别考 察硒化低聚氨基多糖、Na2SeO3对巨噬细胞RAW264.7增殖能力的影响,并观察巨噬细胞形态的变化。通过中性红 法、酶联免疫吸附测定法及实时荧光定量反转录聚合酶链式反应法分别检测巨噬细胞的吞噬能力、肿瘤坏死因子 （tumor necrosis factor-α,TNF-α）、白介素（interleukin,IL）-6及IL-10细胞因子的分泌量及其mRNA表达水平。 结果表明：硒质量浓度为100～1 000 μg/L时,硒化低聚氨基多糖对细胞的相对增殖率无显著影响;而Na2SeO3的硒 质量浓度超过200 μg/L即对巨噬细胞RAW264.7产生细胞毒性作用。硒化低聚氨基多糖能增强巨噬细胞吞噬活性, 显著提高RAW264.7细胞TNF-α、IL-6及IL-10分泌量及其mRNA表达水平,且效果比Na2SeO3处理组、低聚氨基多糖 处理组及Na2SeO3＋低聚氨基多糖复合添加组好。实验结果提示硒化低聚氨基多糖对小鼠巨噬细胞RAW264.7具有一 定的免疫调节作用。     

羊栖菜多酚通过核转录因子-κB/丝裂原活化蛋白激酶通路缓解脂多糖诱导的RAW264.7细胞炎症反应

目的：研究羊栖菜多酚对脂多糖（lipopolysaccharide,LPS）诱导小鼠单核巨噬细胞白血病细胞RAW264.7细胞炎症反应的影响。方法：噻唑蓝法测定细胞活力;Griess法测定一氧化氮（NO）水平;实时荧光定量聚合酶链式反应测定白细胞介素（interleukin,IL）-6、IL-1β、肿瘤坏死因子（tumor necrosis factor,TNF）-α、环氧合酶2（cyclooxygenase-2,COX-2）和诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）的基因相对表达量;流式细胞术测定细胞吞噬能力;蛋白免疫印迹法测定信号通路丝裂原活化蛋白激酶（mitogen-activated protein kinases,MAPKs）和核转录因子（nuclear factor,NF）-κB信号通路关键蛋白表达水平。结果：羊栖菜多酚对RAW264.7细胞的安全质量浓度范围为0～160 μg/mL。与LPS组相比,羊栖菜多酚剂量依赖性降低巨噬细胞吞噬能力并抑制NO的生成。同时,羊栖菜多酚下调促炎细胞因子（IL-1β、IL-6、TNF-α）和炎症诱导酶（iNOS、COX-2）的mRNA水平,且作用效果与给药剂量及LPS刺激时间相关。这些炎性介质的表达与羊栖菜多酚抑制p38 MAPK和NF-κB p65的激活有关。结论：羊栖菜多酚可通过减弱p38 MAPK和NF-κB p65信号通路的激活水平,抑制下游炎症介质的转录表达,从而缓解LPS诱导的巨噬细胞炎症反应。     

Blueberries reduce pro-inflammatory cytokine TNF-α and IL-6 production in mouse macrophages by inhibiting NF-κB activation and the MAPK pathway

Blueberries (BB) have been reported to attenuate atherosclerosis in apoE-deficient (ApoE(-/-) ) mice. The aim of this study was to evaluate the effects of BB in reducing pro-inflammatory cytokine production in mouse macrophages. ApoE(-/-) mice were fed AIN-93G diet (CD) or CD formulated to contain 1% freeze-dried BB for 5 wk. TNF-α and IL-6 were lower in serum of BB-fed mice and TNF-α expression in aorta was down-regulated with BB feeding. Protein level and mRNA expression of TNF-α and IL-6 were significantly lower in the peritoneal macrophages from mice fed BB without or with LPS or oxLDL stimulation. RAW264.7 macrophages were treated with polyphenol-enriched extracts made from the sera of rats fed CD (SEC) or CD containing 10% BB (SEB). SEB significantly inhibited LPS-induced mRNA expression and protein levels of TNF-α and IL-6. Furthermore, SEB inhibited the phosphorylation of IκB, NF-κB p65, MAPK p38 and JNK. All of these are important signaling pathways involved in the production of TNF-α and IL-6.     

水溶性豆渣酸性多糖对RAW264.7细胞的免疫调节作用及其机制

研究水溶性豆渣多糖(water-soluble soybean polysaccharides,SSPS)对RAW264.7细胞的免疫调节作用.首先,以NO浓度为指标,明确SSPS、水溶性豆渣酸性多糖A1(soluble soybean acidic polysaccharide A1,SSPS-A1)和水溶性豆渣酸性...     

毛蕊花糖对巨噬细胞RAW264.7免疫调节作用的研究

目的：明确毛蕊花糖对巨噬细胞RAW264.7免疫功能的调节作用。方法：分别以质量浓度25、50、100、200、400μg/mL的毛蕊花糖对RAW264.7巨噬细胞进行培养,以10μg/mL的脂多糖(LPS)为阳性对照组、不加任何样品的培养基为空白对照组。通过噻唑蓝(MTT)实验、吞噬中性红实验、NO释放量测定及酶联免疫吸附法测定细胞因子(IL-6、IL-1β、IFN-α、IFN-γ)的分泌量,评价毛蕊花糖的免疫调节作用。结果：质量浓度为25～400μg/mL的毛蕊花糖对巨噬细胞RAW264.7作用24h后,可显著提高巨噬细胞的增殖活性、吞噬活性、NO释放量以及细胞因子的分泌水平,并且毛蕊花糖溶液质量浓度为200μg/mL时效果最好。结论：毛蕊花糖对小鼠腹腔巨噬细胞系RAW264.7具有免疫调节活性,是一种良好的免疫增强剂。     

醋蛋液对巨噬细胞RAW264.7免疫调节活性的影响

采用细胞计数试剂盒（CCK）-8法检测不同剂量的醋蛋液对小鼠巨噬细胞RAW264.7免疫调节活性的影响,倒置显微镜及荧光显微镜下分别观察不同分组条件下对RAW264.7细胞形态的影响及细胞核的损伤程度,探讨醋蛋液对RAW264.7细胞分泌免疫因子的影响及其免疫调节活性的机理。结果表明,当醋蛋液浓度为40 μL/mL时,RAW264.7细胞活性极显著升高为（145.3±23.02）%（P＜0.01）。醋蛋液高剂量组（20 μL/mL）巨噬细胞体积变大并逐渐伸出伪足,与正常细胞有明显差异,但未达到炎症相关形态学特征;醋蛋液高剂量组（20 μL/mL）可以促进巨噬细胞分泌白介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）和一氧化氮（NO）分泌,可以有效缓解巨噬细胞细胞核损伤、细胞膜电位降低造成的细胞损伤及细胞凋亡;醋蛋液高剂量组（20 μL/mL）可以上调蛋白激酶B（AKT）以及核因子-κB（NF-κB）的表达量,进而激活巨噬细胞,调节机体免疫力。     

Evaluation of the anti-inflammatory effects of phloretin and phlorizin in lipopolysaccharide-stimulated mouse macrophages

Many reports suggest that phloretin and phlorizin have antioxidant properties and can inhibit glucose transportation, the anti-inflammatory effects and mechanism of phloretin and phlorizin remain unclear. This study aims to evaluate the anti-inflammatory effects of phloretin and phlorizin in LPS-stimulated murine RAW264.7 macrophages. RAW264.7 cells were pretreated with various concentrations of phloretin or phlorizin (3–100 μM) and cell inflammatory responses were induced with LPS. Pretreated with 10 μM phloretin significantly inhibited the levels of NO, PGE₂, IL-6, TNF-α, iNOS and COX-2. Furthermore, it was demonstrated that phloretin suppressed the nuclear translocation of NF-κB subunit p65 proteins, and decreased phosphorylation in MAPK pathways. Surprisingly, phlorizin did not suppress the inflammatory response in LPS-stimulated RAW264.7 cells. These results suggest that phloretin has an anti-inflammatory effect that reduces levels of proinflammatory cytokines and mediators in RAW264.7 cells.     

Regulation of virus-induced inflammatory response by β-carotene in RAW264.7 cells

Carotenoids are effective antioxidants, which can quench singlet oxygen, suppress lipid peroxidation, and prevent oxidative damage. Both Pseudorabies virus (PRV) and human Herpes simplex virus (HSV) are DNA viruses, and their pathogenesis and immunobiology are similar. However, PRV does not infect humans. Therefore, PRV was used to infect murine macrophages (RAW264.7 cells), to mimic HSV-induced inflammation. Meanwhile, the influence of β-carotene on PRV-induced inflammation was also investigated. Results indicated that β-carotene inhibited (p < 0.05) NO, IL-1β, IL-6, and MCP-1 production in PRV-infected RAW264.7 cells. β-Carotene also suppressed (p < 0.05) NF-κB (p50 and p65), phosphorylation of extracellular-signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) expression. It could be concluded that the anti-inflammatory effect of β-carotene is mainly through a suppression of cytokine expression in PRV-induced inflammation, which results from NF-κB inactivation. β-Carotene can be considered a potential anti-inflammatory agent for DNA-virus infection.