激活素A抑制脂多糖活化巨噬细胞的作用机制

目的探讨激活素A(Activin A)抑制脂多糖(Lipopolysaccharides,LPS)活化巨噬细胞的作用机制。方法取小鼠巨噬细胞系RAW264.7细胞,分别添加Activin A(5 ng/ml)、LPS(1μg/ml)和Activin A(5 ng/ml)+LPS(1μg/ml),同时设以含单纯2.5%胎牛血清的DMEM培养液培养的细胞作为对照孔,培养24 h后,采用还原酶法检测细胞分泌一氧化氮(Nitric oxide,NO)的水平,流式细胞术分析TLR2和TLR4蛋白的表达水平,RT-PCR分析细胞ActRⅡA和ActRⅡB基因mRNA的转录水平。结果 Activin A和LPS单独作用均促进RAW264.7细胞分泌NO,但二者联合使用时,Activin A可抑制LPS刺激RAW264.7细胞的NO分泌;Activin A能抑制LPS上调RAW264.7细胞TLR4蛋白的表达,但对TLR2蛋白的表达无影响;LPS可促进RAW264.7细胞ActRⅡA基因mRNA的转录水平,但对ActRⅡB基因mRNA的转录水平无影响。结论 Activin A通过调控TLR4途径抑制LPS的作用,LPS可能通过促进ActRⅡA的表达进一步增强Activin A的负反馈调节作用。     

81种药用植物提取物影响小鼠巨噬细胞RAW264.7活化的研究

目的通过对81种药用植物提取物影响巨噬细胞活化的研究,获得相关活性数据,为后续研究开发和利用提供试验数据和物质基础。方法将提取物样品作用于RAW264.7细胞一定时间后,用ELISA法测定细胞分泌的TNF-α,以判断样品对细胞是否有活化作用;将LPS和81种提取物样品作用于RAW264.7细胞一定时间后,用ELISA法测定细胞分泌的TNF-α,以判断样品是否对LPS诱导的细胞活化有抑制作用。结果对81种药用植物提取物的研究结果表明,有34种植物提取物样品能够使RAW264.7细胞TNF-α表达上调,使其增加TNF-α的分泌;有2种植物提取物样品能够使LPS诱导的RAW264.7细胞TNF-α表达下调,使其减少TNF-α的分泌。结论通过对81种药用植物提取物的筛选研究表明,既有对巨噬细胞有活化作用的样品,也有能够抑制LPS诱导的巨噬细胞活化的样品,为进一步的研究,提供了相关活性数据和物质基础。     

hCAP-18/LL-37基因转染对RAW264.7细胞活化功能的影响

目的探讨抗菌肽hCAP-18/LL-37基因转染对巨噬细胞RAW264.7活化功能的影响。方法将重组质粒pcD-NA4/Myc-His-hCAP-18/LL-37瞬时转染RAW264.7细胞,MTT法检测细胞的增殖活性;中性红吞噬试验检测细胞的吞噬能力;RT-PCR法分析细胞活化相关分子CD80、CD86、TLR4及细胞因子IL-1β、TNF-αmRNA的转录。结果 hCAP-18/LL-37基因转染可促进经脂多糖(Lipopolysaccharide,LPS)处理的RAW264.7细胞的增殖活性与吞噬能力(P<0.05);可上调RAW264.7细胞CD80、CD86、TLR4、IL-1β、TNF-α基因mRNA的转录水平。结论 hCAP-18/LL-37基因转染可通过促进RAW264.7细胞增殖活性、吞噬能力及活化相关分子表达,调控巨噬细胞的活化功能。     

绿豆肽对RAW264.7巨噬细胞的免疫调节作用

目的探讨绿豆肽对RAW264. 7巨噬细胞的免疫调节作用。方法在体外培养的RAW264. 7巨噬细胞中,分别加入10、100、200和400μg/mL的绿豆肽,检测RAW264. 7巨噬细胞的增殖能力、吞噬能力、SOD酶活力、NO含量和细胞因子分泌量,同时检测绿豆肽对经脂多糖(lipo-polysaccharide,LPS)诱导的RAW264. 7巨噬细胞中细胞因子分泌和LC3、p62蛋白表达水平的影响。结果与空白对照组比较,不同浓度绿豆肽组RAW264. 7巨噬细胞的增殖率及吞噬率显著提高(P 0. 05),400和200μg/mL组的SOD酶活力、NO含量及分泌TNF-α、IL-1β、IL-6水平均显著升高(P 0. 05)。各浓度绿豆肽均可显著抑制LPS诱导RAW264. 7巨噬细胞的TNF-α、IL-1β和IL-6分泌量(P 0. 05),浓度为400μg/mL时,上述细胞因子的分泌量与空白对照组差异无统计学意义(P 0. 05)。不同浓度绿豆肽均可降低LPS诱导RAW264. 7巨噬细胞胞内LC3的含量,上调p62蛋白的表达量。结论绿豆肽可激活巨噬细胞、增加自身代谢酶活力、释放细胞因子,通过抗炎作用及抑制细胞自噬,从而提高宿主细胞的免疫功能。     

激活素A对小鼠巨噬细胞RAW264.7吞噬活性的促进作用

目的探讨激活素A对巨噬细胞吞噬活性的促进作用及其机制。方法分别通过中性红试验、鸡红细胞法和流式细胞术,检测激活素A对巨噬细胞系RAW264.7细胞的吞饮活性、吞噬活性以及对MHCⅠ、Ⅱ类分子和CD68表达的影响。结果激活素A可明显促进RAW264.7细胞的吞饮及吞噬活性,对MHCI、II类分子表达没有影响,但可明显上调巨噬细胞表面分子CD68的表达。结论激活素A可促进巨噬细胞系RAW264.7细胞吞噬活性,这与其促进巨噬细胞成熟有关。     

脂多糖刺激对小鼠腹腔巨噬细胞HMGB1表达的影响

目的观察脂多糖(LPS)刺激对小鼠腹腔巨噬细胞高迁移率族蛋白B1(HMGB1)表达的影响。方法取正常Swiss小鼠腹腔巨噬细胞,以LPS分别刺激4、8、12、18、24和36h后,RT-PCR法检测小鼠腹腔巨噬细胞HMGB1基因mRNA的转录水平,Western blot法检测HMGB1蛋白的表达水平,观察LPS刺激与HMGB1释放的时效关系。结果LPS刺激后4～18h,细胞内HMGB1基因mRNA的转录水平无明显变化,24h开始明显增强,刺激24h和36h与对照组相比,差异有统计学意义,HMGB1蛋白在LPS刺激后18h开始表达明显增加,并一直持续至36h。结论LPS可促进小鼠腹腔巨噬细胞HMGB1的表达,并具有时间依赖性。     

补充生物活性肽QRPR对巨噬细胞RAW264.7抵御脂多糖刺激的作用

目的研究补充生物活性肽QRPR(Gln-Arg-Pro-AH)对巨噬细胞RAW264.7抵御脂多糖(lipopolysaccharides,LPS)刺激的作用。方法用不同浓度的LPS刺激RAW264.7细胞不同时间,ELISA法测定细胞培养上清中细胞因子白介素-6(interleukin-6,IL-6)及肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)的浓度,确定LPS刺激RAW264.7细胞的最适浓度和时间,建立RAW264.7细胞抵御LPS刺激模型。采用ELISA法测定QRPR肽对RAW264.7细胞抵御LPS刺激时IL-6和TNF-α表达量的影响。MTS法检测QRPR肽对RAW264.7细胞的毒性作用。结果100 ng/m L LPS诱导16 h为刺激RAW264.7细胞验证QRPR肽调节作用的最佳浓度和时间。QRPR肽可以抑制RAW264.7细胞受LPS刺激后IL-6和TNF-α的产生,QRPR肽浓度为250μmol/L时,其降低LPS刺激产生IL-6和TNF-α的能力最强。QRPR肽本身对RAW264.7细胞的生长既无促进作用,也无毒性和抑制作用。结论补充活性肽QRPR对巨噬细胞RAW264.7抵御LPS刺激具有积极的调节作用。     

激活素A对巨噬细胞系RAW264.7细胞吞饮活性及TLR4表达的双向调节作用

目的探讨激活素A(ActivinA)对巨噬细胞系RAW264.7细胞活性的调节作用及其可能的机制。方法取对数生长期的小鼠巨噬细胞系RAW264.7细胞,加入1μg/ml脂多糖(LPS),继续培养8h,采用ELISA法检测细胞分泌ActivinA水平;分别加入ActivinA、LPS和ActivinA+LPS,中性红染料法检测细胞吞饮活性;流式细胞术分析细胞表面分子MHCⅠ、MHCⅡ及Toll样受体4(TLR4)的表达水平。结果LPS呈时间依赖性刺激RAW264.7细胞分泌ActivinA;ActivinA可明显促进静息RAW264.7细胞的吞饮活性,而对MHCⅠ、MHCⅡ及TLR4的表达水平无明显影响;ActivinA和LPS共同作用,ActivinA明显抑制了LPS活化的RAW264.7细胞的吞饮活性,并下调TLR4的表达。结论ActivinA可能以自分泌/旁分泌形式参与巨噬细胞活性调节,其抑制LPS作用与TLR4表达有关。     

金柑结合多酚的制备及其对RAW264.7巨噬细胞免疫调节活性

以萃取游离多酚后的金柑果渣为材料,采用高压脉冲电场(PEF)进行处理,提取其中的结合多酚(NEPP).在单因素(场强、脉冲数和液料比)实验基础上,以金柑NEPP含量为响应值,通过响应面法对金柑NEPP提取条件进行优化;之后应用RAW 264.7巨噬细胞模型,分别采用MTT法、中性红比色法以及Griess法,测定金柑NE...     

玉米须缓解脂多糖诱导的小鼠急性肺损伤

目的:探讨玉米须缓解脂多糖(LPS)诱导的小鼠急性肺损伤的保护作用。方法:将60只小鼠随机分为正常组、模型组、地塞米松组(0. 005g/kg. d)、玉米须低、中、高剂量组(2. 5、5、10 g/kg. d),每组10只。灌胃给药,每天1次,连续给药10 d,末次给药2 h后除正常组小鼠气管滴注等量生理盐水外,其他各组小鼠气管滴注脂多糖(10 mg/kg),12 h后眼球采血,处死小鼠,取新鲜肺组织,ELISA检测血清中TNF-α和IL-1β水平以及肺组织中MPO活性,计算肺组织湿/干重比值,HE检查肺组织的病理变化。结果:与正常组比较,模型组小鼠肺组织湿/干比值、血清中TNF-α和IL-1β水平、肺组织中MPO活性均显著性升高(P 0. 05,P 0. 01)。与模型组比较,地塞米松组、玉米须中剂量和高剂量组小鼠肺组织湿/干比值、血清中TNF-α和IL-1β水平、肺组织中MPO活性均显著性降低(P 0. 05,P 0. 01),并改善了肺组织损伤。结论:玉米须能够对脂多糖诱导的小鼠急性肺损伤有一定的保护作用,其机制可能与抑制体内炎性介质释放有关。     

小鼠MIF基因的原核表达及纯化

目的克隆小鼠巨噬细胞移动抑制因子(MIF)基因,在原核系统中表达并进行初步纯化,为研究MIF的功能奠定基础。方法提取小鼠肺组织总RNA,采用RT-PCR技术扩增MIF基因,克隆入pMD18-T载体,酶切鉴定及测序后,再定向插入原核表达载体pET-28a(+)中,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE及Western blot鉴定后,进行纯化。结果克隆得到的目的基因片段大小与预期相符,测序结果与GenBank中报道的序列完全一致。重组MIF蛋白相对分子质量约为15000,以包涵体形式存在,表达量约占菌体总蛋白的40%,且具有良好的反应原性。经初步纯化后,纯度达95%以上。结论已成功克隆了小鼠MIF基因,并在原核系统中表达了重组蛋白,纯化的蛋白纯度较高,可用于后续试验。     

Brucella BtpB Manipulates Apoptosis and Autophagic Flux in RAW264.7 Cells

Brucella transfers effectors into host cells, manipulating cellular processes to its advantage; however, the mechanism by which effectors regulate cellular processes during infection is poorly understood. A growing number of studies have shown that apoptosis and autophagy are critical mechanisms for target cells to cope with pathogens and maintain cellular homeostasis. BtpB is a Brucella type IV secretion system effector with a complex mechanism for manipulating host infection. Here, we show that the ectopic expression of BtpB promoted DNA fragmentation. In contrast, an isogenic mutant strain, ΔbtpB, inhibited apoptosis compared to the wild-type strain B. suis S2 in RAW264.7 cells. In addition, BtpB inhibited autophagy, as determined by LC3-II protein levels, the number of LC3 puncta, and p62 degradation. We also found that BtpB reduced autophagolysosome formation and blocked the complete autophagic flux. Moreover, our results revealed that the autophagy inhibitor, chloroquine, reduces Brucella’s intracellular survival. Overall, our data unveil new mechanisms of virulence implicating the effector BtpB in regulating host intracellular infection.     

金柑结合多酚的制备及其对RAW 264.7巨噬细胞免疫调节活性的研究

以萃取游离多酚后的金柑残渣为材料，采用高压脉冲电场（High intensity pulsed electric field，PEF）进行处理，提取其中的结合多酚（Non-extractable polyphenol，NEPP）。在单因素（场强、脉冲数和液料比等）实验考察的基础上，以金柑NEPP含量为响应值，通过响应面法对金柑NEPP提取条件进行优化；之后应用RAW 264.7巨噬细胞模型，分别采用MTT 法、中性红比色法以及Griess法，测定金柑NEPP处理后的RAW 264.7细胞生长情况、吞噬活性和释放NO能力，以评价其免疫活性。结果表明，金柑NEPP的提取工艺回归模型显著，拟合性良好，并预测金柑NEPP的最大理论得率为1.756 mg/g DW；优化后的PEF提取条件为：脉冲数450次，场强11.86 kv/cm，液料比0.341: 1 mL/mg。在优化条件下，金柑NEPP的得率（1.5 mg/g DW）接近预测值，表明优化后的PEF法提取工艺可用于金柑NEPP的提取。当金柑NEPP浓度为100 μg/mL时，对RAW 264.7巨噬细胞无毒性作用，可以显著地抑制巨噬细胞NO的分泌水平，并提高其吞噬活性     

Conjugated Linoleic Acid Isomers Reduce Cholesterol Accumulation in Acetylated LDL-Induced Mouse RAW264.7 Macrophage-Derived Foam Cells

Synthetic activators of peroxisome proliferator-activated receptors (PPAR)-alpha and -gamma are capable of reducing macrophage foam cell cholesterol accumulation through the activation of genes involved in cholesterol homeostasis. Since conjugated linoleic acids (CLA) were also demonstrated to activate PPARalpha and PPARgamma in vivo and in vitro, we tested the hypothesis that CLA are also capable of reducing macrophage foam cell cholesterol accumulation. Thus, mouse RAW264.7 macrophage-derived foam cells were treated with CLA isomers, c9t11-CLA and t10c12-CLA, and linoleic acid (LA), as reference fatty acid, and analyzed for the concentrations of free and esterified cholesterol, cholesterol efflux and expression of genes involved in cholesterol homeostasis (CD36, ABCA1, LXRalpha, NPC-1, and NPC-2). Treatment with c9t11-CLA and t10c12-CLA, but not LA, lowered cholesterol accumulation, stimulated acceptor-dependent cholesterol efflux, and increased relative mRNA concentrations of CD36, ABCA1, LXRalpha, NPC-1, and NPC-2 (P < 0.05). In conclusion, the present study showed that CLA isomers reduce cholesterol accumulation in RAW264.7 macrophage-derived foam cells presumably by enhancing lipid acceptor-dependent cholesterol efflux.     

Danshensu Promotes Cholesterol Efflux in RAW264.7 Macrophages

Contemporary research suggests that macrophage foam cell and cholesterol efflux defect play pivotal role in atherogenesis. We reported on the heretofore unknown therapeutic effect of Danshensu (DSS) in reducing intracellular cholesterol level and unraveled the mechanism of DSS promotes cholesterol efflux. Oxidized low‐density lipoprotein stimulation of Raw264.7 cells into foam cells, which were treated with DSS and co‐treated with Simvastatin and Rosiglitazone. PPARγ, ABCA1, ABCG1, SR‐BI, CD36, and LXR‐α mRNA were quantified by Real‐Time PCR. Western blotting was used to determine protein expression of PPARγ, ABCA1 and CD36. Cellular cholesterol handling was studied by measurement of intracellular lipid droplets concentration and cholesterol efflux. DSS significantly reduced scavenger receptor CD36 and its orthologue SR‐BI. In addition, DSS stimulated the upregulation of cellular cholesterol exporters ABCA1 and ABCG1 to reduce intracellular lipid accumulation. DSS can reduce lipid deposition in Raw264.7 foam cells by balancing CD36 and ABCA1 protein expression.     

Lysophosphatidylcholine Exhibits Selective Cytotoxicity,Accompanied by ROS Formation,in RAW 264.7 Macrophages

Lysophosphatidylcholine (lysoPtdCho) is a component of oxidized low density lipoprotein, and is involved in the pathogenesis of atherosclerosis and inflammation. We studied the effects of lysoPtdCho on cytotoxicity, reactive oxygen species (ROS) production, activation of the extracellular signal-regulated kinase (ERK), mitogen-activated protein kinases and pro-inflammatory gene expression in RAW 264.7 murine macrophage cells. When cells were exposed to lysoPtdCho with various acyl chains in a culture medium containing 10% fetal bovine serum, only 1-linoleoyl (C18:2) lysoPtdCho showed a remarkable cytotoxicity, reaching the highest level at 24 h, and elicited ROS production, suggesting that oxidative stress might be implicated in the cytotoxicity of 1-linoleoyl (C18:2) lysoPtdCho. Presumably in support of this, antioxidants such as magnolol or trolox prevented 1-linoleoyl (C18:2) lysoPtdCho-induced cytotoxicity as well as ROS production, although only partially. Furthermore, the phosphorylation of ERK 1/2 and the expression of pro-inflammatory cytokines such as IL-1β, CCL2 and CCL5 were augmented by 1-linoleoyl (C18:2) lysoPtdCho. Meanwhile, there was no structural importance of the acyl chain for the cytotoxic action of lysoPtdCho during 10 min incubation in serum-free media. Taken together, it is suggested that in a serum-containing medium, 1-linoleoyl (C18:2) lysoPtdCho can cause a significant cytotoxicity through ROS production, probably accompanied by activation of ERK and induction of related inflammatory cytokines, in RAW 264.7 cells.     

Protective Effect of Membrane-Free Stem Cells against Lipopolysaccharide and Interferon-Gamma-Stimulated Inflammatory Responses in RAW 264.7 Macrophages

Recently, adipose-derived stem cells (ADSCs) are considered to be ideal for application in cell therapy or tissue regeneration, mainly due to their wide availability and easy access. In this study, we examined the anti-inflammatory effects of membrane-free stem cell extract (MFSC-Ex) derived from ADSCs against lipopolysaccharide (LPS)/interferon-gamma (IFN-γ) on RAW 264.7 macrophage cells. Exposure of RAW macrophages to LPS and IFN-γ stimuli induced high levels of nitric oxide (NO), cyclooxygenase-2 (COX-2), and prostaglandin E₂ (PGE₂) production. However, pretreatment with MFSC-Ex inhibited LPS/IFN-γ-induced these pro-inflammatory mediators. To clarify the molecular mechanisms underlying the anti-inflammatory property of MFSC-Ex, we analyzed nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) protein expressions by Western blotting. Our study showed that treatment of MFSC-Ex significantly down-regulated inducible nitric oxide synthase (iNOS) and COX-2 protein expressions. Furthermore, phosphorylation of extracellular signal-regulated kinase (ERK) and p38 was also blocked by treatment with MFSC-Ex, indicating that inhibitory effect of MFSC-Ex on MAPK signaling cascade may attribute to inactivation of NF-κB. From these findings, we suggest that MFSC-Ex exert anti-inflammatory activities, which suppressed LPS/IFN-γ-induced production of NO, COX-2 and PGE₂ by regulation of NF-κB and MAPK signaling pathway in RAW 264.7 macrophages. In conclusion, MFSC-Ex might provide a new therapeutic opportunity to treatment of inflammatory-related diseases.     

Antimicrobial Peptides Mediate Apoptosis by Changing Mitochondrial Membrane Permeability

Changes in mitochondrial membrane permeability are closely associated with mitochondria-mediated apoptosis. Antimicrobial peptides (AMPs), which have been found to enter cells to exert physiological effects, cause damage to the mitochondria. This paper reviews the molecular mechanisms of AMP-mediated apoptosis by changing the permeability of the mitochondrial membrane through three pathways: the outer mitochondrial membrane (OMM), inner mitochondrial membrane (IMM), and mitochondrial permeability transition pore (MPTP). The roles of AMPs in inducing changes in membrane permeability and apoptosis are also discussed. Combined with recent research results, the possible application prospects of AMPs are proposed to provide a theoretical reference for the development of AMPs as therapeutic agents for human diseases.