Viili多糖对巨噬细胞RAW264.7激活及分泌细胞因子的影响

探讨viili胞外多糖(Viili exopolysaccharides,VEPS)对小鼠巨噬细胞RAW264.7激活和增殖的影响。噻唑蓝(MTT)比色法检测细胞的生长与增殖;中性红吞噬实验检测吞噬活性;Griess试剂盒检测培养上清液中NO分泌量,ELISA法检测VEPS不同浓度及不同作用时间培养上清中IL-6,IL-1β含量;扫描电子显微镜观察VEPS对细胞形态的影响;碘化丙啶(PI)染色检测VEPS对细胞周期的影响。结果显示,VEPS对RAW264.7细胞的增殖、吞噬能力、分泌NO、IL-6、IL-1β等都有显著的促进作用,VEPS为100μg/ml时促进作用最明显,呈剂量相关,作用72h时细胞因子分泌量达到最大,72h后下降。VEPS激活巨噬细胞并使其变得扁平伸展且形成伪足。VEPS促进G1期细胞增多,提高细胞的增殖能力。VEPS免疫调节作用与其激活RAW264.7细胞,促进NO、IL-6、IL-1β等分泌有关,且VEPS与LPS对RAW264.7细胞有相似的作用规律。以上结果证明VEPS能激活巨噬细胞,也可能最终激活淋巴细胞,达到增强非特异性和特异性免疫的作用。     

Chamomile: an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking RelA/p65 activity

Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we investigated the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and explored its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β, IL-6 and TNFα-induced NO levels in RAW 264.7 macrophages. Chamomile caused reduction in LPS-induced iNOS mRNA and protein expression. In RAW 264.7 macrophages, LPS-induced DNA binding activity of RelA/p65 was significantly inhibited by chamomile, an effect that was mediated through the inhibition of IKKβ, the upstream kinase regulating NF-κB/Rel activity, and degradation of inhibitory factor-κB. These results demonstrate that chamomile inhibits NO production and iNOS gene expression by inhibiting RelA/p65 activation and supports the utilization of chamomile as an effective anti-inflammatory agent.     

Anti-inflammatory effects of galangin on lipopolysaccharide-activated macrophages via ERK and NF-κB pathway regulation

Inflammation is the major symptom of the innate immune response to microbial infection. Macrophages, immune response-related cells, play a role in the inflammatory response. Galangin is a member of the flavonols and is found in Alpinia officinarum, galangal root and propolis. Previous studies have demonstrated that galangin has antioxidant, anticancer, and antineoplastic activities. However, the anti-inflammatory effects of galangin are still unknown. In this study, we investigated the anti-inflammatory effects of galangin on RAW 264.7 murine macrophages. Galagin was not cytotoxic to RAW 264.7 cells, and nitric oxide (NO) production induced by lipopolysaccharide (LPS)-stimulated macrophages was significantly decreased by the addition of 50?μM galangin. Moreover, galangin treatment reduced mRNA levels of cytokines, including IL-1β and IL-6, and proinflammatory genes, such as iNOS in LPS-activated macrophages in a dose-dependent manner. Galangin treatment also decreased the protein expression levels of iNOS in activated macrophages. Galangin was found to elicit anti-inflammatory effects by inhibiting ERK and NF-κB-p65 phosphorylation. In addition, galangin-inhibited IL-1β production in LPS-activated macrophages. These results suggest that galangin elicits anti-inflammatory effects on LPS-activated macrophages via the inhibition of ERK, NF-κB-p65 and proinflammatory gene expression.     

Aire 对巨噬细胞极化影响的研究

目的:研究自身免疫调节因子(Aire)对巨噬细胞极化的影响。方法:分别用LPS、IL-4 以及LPS 联合免疫复合物刺激小鼠单核巨噬细胞系RAW264郾7 细胞、稳定表达GFP-Aire 的RAW264.7 细胞(A33-3) 细胞和稳定表达GFP 的RAW264.7 细胞(C1-6),使其向M1(LPS)、M2a(IL-4)和M2b(LPS 联合免疫复合物)型巨噬细胞极化。通过Real-time PCR 检测各组细胞中M1 型巨噬细胞特征分子IL-1、iNOS 和IL-6,M2a 型特征分子Arg-1 和M2b 型特征分子IL-10 的表达水平,研究Aire 对各种类型巨噬细胞极化的影响。结果:LPS 在0.5 g/ ml 浓度时,RAW264.7 细胞中M1 型巨噬细胞产物IL-1 、iNOS和IL-6 基因表达量最高;而IL-4 以及LPS 联合免疫复合物的刺激作用有显著的剂量依赖性,都在浓度最高时RAW264.7 细胞中Arg1(M2a)和IL-10(M2b)基因表达量最高。LPS 刺激后,A33-3 细胞中IL-1 和iNOS 表达水平明显高于C1-6 细胞,IL-6 则相反;IL-4 及LPS 联合免疫复合物刺激后,A33-3 细胞中Arg1 和IL-10 的表达水平明显低于C1-6 细胞。结论:Aire 可能促进巨噬细胞向M1 极化,同时抑制其向M2a 和M2b 极化。     

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.     

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.     

CpG ODN activates NO and iNOS production in mouse macrophage cell line (RAW 264.7)

Synthetic CpG containing oligodeoxynucleotide (CpG ODN) is recognized for its ability to activate cells to produce several cytokines, such as IL-12 and TNF-alpha. In the present study we have demonstrated that CpG ODN 1826, known for its immunostimulatory activity in the mouse system could, by itself, induce nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production from mouse macrophage cell line (RAW 264.7). Neutralizing antibody against TNF-alpha was not able to inhibit NO or iNOS production from the CpG ODN 1826-activated macrophages, suggesting that although the TNF-alpha was also produced by CpG ODN-activated macrophages, the production of iNOS was not mediated through TNF-alpha. Although both CpG ODN 1826 and lipopolysaccharide (LPS) were able to stimulate NO and iNOS production, the exposure time required for maximum production of NO and iNOS for the CpG ODN 1826-activated macrophages was significantly longer than those activated with LPS. These results were due probably to a delay of NF-kappaB translocation, as indicated by the delay of IkappaBalpha degradation. Moreover, the fact that chloroquine abolished NO and iNOS production from the cells treated with CpG ODN 1826 but not from those treated with LPS suggested that the induction of NO and iNOS production from the cells stimulated with CpG ODN (1826) also required endosomal maturation/acidification.     

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   

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.     

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.     

Chikusetsusaponin V inhibits inflammatory responses via NF-κB and MAPK signaling pathways in LPS-induced RAW 264.7 macrophages

Excessive activation of macrophages is implicated in various inflammation resulted injuries. Saponins from Panax japonicus (SPJ) have been shown to possess anti-inflammatory activities. However, whether Chikusetsusaponin V (CsV), the most abundant component of SPJ, can exert anti-inflammatory activities is unknown. The present study was aimed to investigate the anti-inflammatory effects of CsV in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells and the underlying mechanisms. Our data showed that CsV dose-dependently inhibited NO, iNOS, TNF-α and IL-1β expressions in LPS-stimulated RAW264.7 cells. Increased protein levels of nuclear NF-κB and elevated phosphorylation levels of ERK and JNK in LPS-stimulated RAW 264.7 cells were also found downregulated by CsV treatment. Furthermore, the increase of CD14 and TLR4 mRNA expression due to LPS stimulation were significantly reversed by CsV treatment. These results suggested that CsV attenuated LPS-induced inflammatory responses partly via TLR4/CD14-mediated NF-κB and MAPK pathways.     

Bidirectional Immunomodulatory Activities of Polysaccharides Purified From Pleurotus nebrodensis

A novel Pleurotus nebrodensis polysaccharide (PN50G) was purified, characterized, and cultured with RAW264.7 macrophages to evaluate its bidirectional immunostimulating characteristics. PN50G was purified by using Sepharose 4B gel filtration; the molecular weight of PN50G was distributed at 2,000 kDa. The total protein and carbohydrate constituent ratios in PN50G were 2.6?±?0.9 % and 92.4?±?6.1 % (w/w), respectively, which suggests that PN50G may be a major proteo-polysaccharide component. The phagocytosis of macrophages was improved significantly, and remarkable changes were observed in the morphology of PN50G-treated cells. Compared with the control group, the productions of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and inducible nitric oxide synthase (iNOS) in the macrophages as well as the messenger RNA expressions were strongly induced by PN50G. Pro-/anti-inflammatory (IL-6/IL-10, TNF-α/IL-10, NO/IL-10) cytokine secretion ratios by lipopolysaccharide-stimulated RAW264.7 macrophages were significantly decreased by PN50G treatments in a dose-dependent manner under an excessive immune experimental model. This study suggests that purified PN50G can improve immunity and suppress immune overactivity in LPS-induced macrophages in a preventive manner to coordinate innate immunity and inflammatory responses.     

草木犀石油醚提取物对小鼠巨噬细胞促炎介质的影响

目的 研究草木犀石油醚提取物在体外的抗炎作用.方法 采用小鼠巨噬细胞系RAW 264.7建立炎症细胞模型,加入10 μg/L的LPS培养液和不同浓度的草木犀石油醚提取物进行干预.ELISA法检测上清液中TNF-α, IL-1β, IL-6和NO的分泌量;实时荧光定量RT-PCR检测TNF-α, iNOS 和 COX-2的 mRNA表达;Western 印迹法检测COX-2 蛋白的表达.结果 草木犀提取物干预后细胞所分泌的炎性介质(TNF-α, IL-1β, IL-6和NO)与模型组相比均显著降低(P＜0.01),并存在剂量依赖关系;RT-PCR结果显示干预后细胞TNF-α,iNOS和COX-2的mRNA表达水平显著降低(P＜0.01),也存在剂量依赖关系;Western印迹结果显示草木犀石油醚提取物及地塞米松干预后COX-2蛋白水平明显降低(P＜0.01).结论 草木犀的石油醚提取物通过下调LPS诱导的巨噬细胞表达炎性介质而发挥其体外抗炎作用, 且其下调作用呈剂量依赖性.     

Expression of inducible nitric oxide synthase by stimulated macrophages correlates with their antihistoplasma activity.

The antihistoplasma activity of recombinant murine gamma interferon (rMuIFN-gamma)-treated macrophages of the RAW 264.7 cell line depends on the generation of nitric oxide (NO.) from L-arginine. Macrophages of the P388D1 cell line treated with rMuIFN-gamma do not produce NO. or inhibit the intracellular growth of Histoplasma capsulatum. NO. is generated by the inducible enzyme nitric oxide synthase (iNOS) formed by stimulated macrophages. Northern (RNA) blot analysis of RAW 264.7 cells revealed the expression of iNOS mRNA after exposure to rMuIFN-gamma. In contrast, rMuIFN-gamma-treated P388D1 cells did not produce detectable levels of iNOS. These data suggest that the failure of P388D1 cells to generate NO. and to restrict the intracellular growth of H. capsulatum is due to a lack of expression of iNOS following treatment with rMuIFN-gamma.     

NOD8对LPS诱导巨噬细胞释放NO、TNF-α和IL-1β的影响

 目的： 探讨NOD8对脂多糖（LPS）诱导巨噬细胞释放一氧化氮（NO）、肿瘤坏死因子α（TNF-α）和白细胞介素1β（IL-1β）的影响。方法： pEGFP-C2及pEGFP-NOD8重组质粒分别转染小鼠巨噬细胞RAW264.7,以LPS刺激RAW264.7细胞0、6、12、24 h后,采用Griess reagent法测定观察细胞分泌的NO水平;ELISA法检测IL-1β 和 TNF-α 的含量;荧光法测定活化的caspase-1水平; Western blotting检测NOD8蛋白表达及NF-κB  p65亚基的核转位情况。结果： (1)与转染pEGFP-C2空质粒组比较,转染pEGFP-NOD8质粒组NOD8蛋白表达明显增加。(2) LPS刺激6、12、24 h后,RAW264.7细胞释放NO、IL-1β及TNF-α均明显增加;而在pEGFP-NOD8+LPS组RAW264.7细胞, NO于12、24 h 的释放显著降低,IL-1β于6、12、24 h的释放也明显降低,TNF-α的释放则无明显变化。（3）在LPS刺激6、12、24 h后, RAW264.7细胞caspase-1活化水平均明显升高,胞浆NF-κB p65亚基表达明显减少,表明p65核转位增加;而pEGFP-NOD8+LPS组可显著抑制caspase-1的活化以及NF-κB p65亚基的核转位,差异有统计学意义。结论： NOD8可抑制LPS诱导的巨噬细胞NO与IL-1β释放,其作用机制可能与NOD8抑制caspase-1及NF-κB 的活化有关。     

N‐Palmitoylation of the Radioprotective Domain of Interleukin‐1 Affords Inhibition of LPS‐Induced Nitric Oxide Generation

Interleukin‐1β (IL‐1β), a cytokine involved in homeostatic processes such as the immune system and inflammatory reactions, is a potent inducer of nitric oxide. The nonapeptide of human IL‐1β (VQGEESNDK, position 163–171, specific radioprotective domain–SRD) has been shown to retain radioprotective, immunostimulatory, and adjuvant activities of the native molecule without any inflammatory and pyrogenic properties. Unlike the parent IL‐1, SRD did not induce nitric oxide (NO) in control or irradiated RAW 264.7 cells nor did it affect inducible nitric oxide synthase (iNOS) as shown by ELISA based mRNA assay (Quantikine). A lipophillic derivative of the SRD (a palmitoyl residue at the amino terminus of the SRD) was synthesized (palmitoyl specific radioprotective domain, P‐SRD) to find out if this structural derivatization would restore the NO‐inducing ability of IL‐1. Surprisingly, P‐SRD not only did not induce NO, but significantly inhibited lipopolysaccharide (LPS) stimulated nitric oxide (NO) production. Quantikine studies indicated that P‐SRD also inhibited iNOS in LPS stimulated macrophage cells, suggesting that decrease in NO production in the presence of P‐SRD was the result of iNOS mRNA inhibition. These results indicate that N‐palmitoylation of SRD may effectively ameliorate potentially fatal symptoms of LPS‐induced endotoxemic hypotensive shock associated with IL‐1 without inflammatory and pyrogenic toxic side effects.