LPS致敏巨噬细胞产生O_2~及其信号机制

目的 :探讨LPS诱导的巨噬细胞 (MΦ)致敏及其信号机制。方法 :巨噬细胞样细胞株RAW 2 6 4 .7以10 μg/LLPS预处理 1h后洗去 ,再以 1mg/LPMA、1μmmol/LfMLP、10 0 μg/LLPS、1g/LZyms和 15mg/LMDP攻击 1h ,检测上清中超氧阴离子 (O 2 )的产生 ,并用荧光显微成像系统检测细胞内游离钙离子浓度 ( [Ca2 +]i) ,探讨 [Ca2 +]i与LPS致敏MΦ产生O 2 的关系。结果 :LPS预处理后以上述刺激剂攻击 1h均能不同程度致敏O 2 的产生 ,且致敏程度在一定范围内与LPS剂量和作用时间相关 ;细胞内静息态 [Ca2 +]i也呈LPS剂量和时间依赖性升高 ,均显著相关。且LPS预处理后用PMA攻击 ,细胞内瞬时 [Ca2 +]i升高幅度明显高于LPS未处理组。用A2 3187使细胞内 [Ca2 +]i升高可代替LPS致敏O 2 的产生 ,而预先用BAPTA和EGTA降低细胞内 [Ca2 +]i则可阻断LPS致敏O 2 的作用。结论 :LPS可致敏MΦ产生O 2 ;LPS预处理后细胞内静态 [Ca2 +]i的升高是LPS诱导O 2 致敏的重要原因。     

LPS致敏巨噬细胞产生及其信号机制

目的探讨LPS诱导的巨噬细胞(Mφ)致敏及其信号机制.方法巨噬细胞样细胞株RAW 264.7以10μg/L LPS预处理1 h后洗去,再以1 mg/LPMA、1μmmol/LfMLP、100μg/L LPS、1 g/LZyms和15mg/L MDP攻击1h,检测上清中超氧阴离子(O-2)的产生,并用荧光显微成像系统检测细胞内游离钙离子浓度([Ca2+]i),探讨[Ca2+]i与LPS致敏Mφ产生O-2的关系.结果LPS预处理后以上述刺激剂攻击1 h均能不同程度致敏O-2的产生,且致敏程度在一定范围内与LPS剂量和作用时间相关;细胞内静息态[Ca2+]i也呈LPS剂量和时间依赖性升高,均显著相关.且LPS预处理后用PMA攻击,细胞内瞬时[Ca2+]i升高幅度明显高于LPS未处理组.用A23187使细胞内[Ca2+]i升高可代替LPS致敏O-2的产生,而预先用BAPTA和EGTA降低细胞内[Ca2+]i则可阻断LPS致敏O-2的作用.结论LPS可致敏Mφ产生O-2;LPS预处理后细胞内静态[Ca2+]i的升高是LPS诱导O-2致敏的重要原因.     

巨噬细胞LPS相关模式识别受体的研究进展

脓毒症(sepsis)是由各种致病微生物或其毒素引起的全身炎症反应综合征(systemic inflammatory response syndrome, SIRS),是严重感染、重度创伤、大手术后和休克常见的并发症,进一步发展可导致脓毒性休克、急性呼吸窘迫综合征(acute respiratory distress syndrome, ARDS)和多器官功能障碍综合征(multi-organ dysfunction syndrome,MODS)等致命性并发症.     

LBP对LPS激活巨噬细胞内p38信号通路的影响 

目的:探讨脂多糖结合蛋白(LBP)对脂多糖(LPS)激活肺泡巨噬细胞内p38信号通路的调节作用。方法:经硫酸铵盐析、Bio-Rex70阳离子交换层析和MonoQ阴离子交换层析, 从大鼠急性期血清中分离纯化LBP。分别用0.01mg/L和1mg/L的LPS刺激肺泡巨噬细胞, 并加入不同浓度LBP(0mg/L、0.01mg/L、0.1mg/L、1mg/L和10mg/L), 观察肺泡巨噬细胞中p38蛋白激酶的磷酸化程度。结果:纯化的大鼠LBP在SDS-PAGE的60kD处呈现单一条带, 并可增强LPS与单核细胞的结合。当LPS浓度为0.01mg/L时, 1mg/L以下的LBP可明显增敏LPS对肺泡巨噬细胞内p38信号通路的激活, 并且这种增敏作用随LBP浓度的增加而增强。但LBP为10mg/L时, LBP对LPS的增敏作用反而有所减弱;当LPS为1mg/L时, LBP对LPS激活肺泡巨噬细胞内p38信号通路无调节作用。结论:LBP对低浓度LPS(0.01mg/L)激活肺泡巨噬细胞内p38信号通路有明显的调节作用;而高浓度LPS(1mg/L)不需LBP的增敏作用, 可能通过LBP非依赖途径直接激活p38信号通路。     

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。     

LPS致大鼠肺泡巨噬细胞NF-κB促进TNF-α分泌

目的: 观察脂多糖（LPS）致大鼠肺泡巨噬细胞（AMs）中核因子NF-κB活性及其调控肿瘤坏死因子-α（TNF-α）分泌的作用。 方法: LPS作用大鼠AMs后，用电泳迁移率改变分析法（EMSA）测NF-κB活性；用特异的反义寡核苷酸阻断NF-κB亚基（p65）后，Western blotting检测p65表达变化；ELISA法检测细胞上清中TNF-α的含量。 结果: 于LPS作用AMs后4 h，NF-κB活性达到峰值，24 h仍维持在高水平；作用后4 h上清中TNF-α的含量达到峰值。反义寡核苷酸阻断NF-κB亚基（p65）表达后，LPS致AMs上清中TNF-α的含量显著低于未阻断组（P<0.01）。结论: LPS致大鼠AMs中NF-κB正向调控TNF-α分泌。     

小鼠腹腔巨噬细胞基态游离钙离子浓度的不均一性及其和细胞反应性的关系

目的:在单细胞水平上研究小鼠腹腔巨噬细胞(PM)基态游离钙离子浓度([Ca²⁺]_i)的不均一性及其和细胞反应性的关系。方法:用荧光指示剂Fura-2/AM结合荧光显微镜成像系统检测单个PM基态的及用激动剂刺激细胞后的[Ca²⁺]_i;同时结合NBT染色法定量检测单个PM产超氧阴离子(O₂^-)水平。结果:对7只正常小鼠共392个PM基态[Ca²⁺]_i的研究表明小鼠PM的基态[Ca²⁺]_i呈正态分布[(54±24)nmol/L,n=392],但波动范围较大(从10nmol/L到高于100nmol/L),以[Ca²⁺]_i在40-60nmol/L的细胞数量最多(约占50%)。用PMA、fMLP刺激后PM[Ca²⁺]_i升高,且受刺激后[Ca²⁺]_i升高的峰值和基态[Ca²⁺]_i之间呈正相关(PMA刺激组:r=052,P<0.01,n=58;fMLP刺激组:r=0.59,P<0.01,n=44。此两组实验均以不同的小鼠重复3次,其它两只小鼠的结果与上同。下面的表述方法同此)。另外小鼠PM的基态[Ca²⁺]_i与其受PMA刺激后产生O₂^-的量也呈显著正相关(r=0.42,P<0.01,n=43,重复4次)。结论:小鼠PM的基态[Ca²⁺]_i是不均一的,且基态[Ca²⁺]_i的高低和该细胞对致炎因子的反应性密切相关。     

脂多糖活化的大鼠肺泡巨噬细胞TNF-α产生的新机制

目的：探讨脂多糖（LPS）活化的肺泡巨噬细胞中低氧诱导因子－1α（HIF-1α）的表达及其在肺泡巨噬细胞产生肿瘤坏死因子α（TNF-α）中的作用。 方法： 应用HIF-1α 诱骗法（HIF-1α decoy）抑制其作用，并用Western blotting、半定量RT-PCR、酶联免疫吸附法（ELISA）分别检测HIF-1α蛋白、mRNA的表达和TNF-α的产生。 结果： HIF-1α蛋白含量在LPS组（1.95±0.57）和HIF-1α decoy组（1.89±0.59）均明显高于对照组（0.41±0.14，P＜0.05）；HIF-1α mRNA的表达在对照组（0.7838±0.3183）、LPS组（0.7622±0.3387）和HIF-1α decoy组（0.8155±0.3594）之间无显著差异（P>0.05）；LPS刺激24 h后，大鼠肺泡巨噬细胞TNF-α的产生明显高于对照组（61 ng/L vs 156 ng/L, P<0.05），抑制HIF-1α后TNF-α的产生明显低于LPS刺激组（90 ng/L vs 156 ng/L, P<0.05），但 HIF-1α decoy组TNF-α的产生仍然高于对照组（61 ng/L vs 94 ng/L, P<0.05）。 结论： 大鼠肺泡巨噬细胞在LPS的刺激下HIF-1α蛋白的稳定性增强使其表达明显上调，并且促进TNF-α的产生，提示HIF-1α在肺部慢性炎症性疾病如COPD的发病机制中可能有重要作用。     

CCK-8抑制LPS诱导的大鼠肺间质巨噬细胞TLR4及IL-1β的表达

目的观察八肽胆囊收缩素(cholecystokinin octapeptide,CCK-8)对外源性脂多糖(lipopolysaccharide,LPS)激活大鼠肺间质巨噬细胞(pulmonary interstitial macrophages,PIMs)Toll样受体4(Toll likereceptor4,TLR4)及IL-1β表达的影响,探讨CCK-8的抗炎作用机制。方法分离培养大鼠PIMs,经LPS、CCK-8及溶剂单独或共同孵育不同时间后,采用Northern blot、ELISA、RT-PCR技术检测TLR4 mRNA、IL-1β及IL-1β mRNA表达的变化。结果LPS(1mg／L)刺激可使PIMs中TLR4 mRNA表达明显增强;随着LPS孵育时间的延长,细胞中的IL-1β含量逐渐增多,24h达高峰,IL-1β mRNA表达水平同时明显升高;CCK-8可剂量依赖性抑制LPS诱导的大鼠PIMs TLR4 mRNA、IL-1β及IL-1β mRNA的表达。结论CCK-8对LPS激活的PIMs TLR4及IL-1β表达有负性调节作用,是CCK-8抗炎作用机制之一。     

Spontaneous cytokine production and its effect on induced production

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Multiparameter flow cytometry is often used to examine cell-specific cytokine production after in vitro phorbol 12-myristate 13-acetate and ionomycin induction, with brefeldin A or other agents added to inhibit protein secretion. Spontaneous ex vivo production reportedly rarely occurs. We examined the spontaneous production of interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) by peripheral-blood B lymphocytes, T cells, CD8(-) T cells, CD8(+) T cells, CD3(-) CD16/56(+) lymphocytes (natural killer [NK] cells), CD3(+) CD16/56(+) lymphocytes (natural T [NT] cells), and/or monocytes of 316 acutely ill hospitalized persons and 62 healthy adults in Malawi, Africa. We also evaluated the relationship between spontaneous and induced cytokine production. In patients, spontaneous TNF-alpha production occurred most frequently, followed in descending order by IFN-gamma, IL-8, IL-4, IL-10, IL-6, and IL-2. Various cells of 60 patients spontaneously produced TNF-alpha; for 12 of these patients, TNF-alpha was the only cytokine produced spontaneously. Spontaneous cytokine production was most frequent in the immunoregulatory cells, NK and NT. For IL-2, IL-4, IL-6, IL-8, and IL-10, spontaneous cytokine production was associated with greater induced production. For TNF-alpha and IFN-gamma, the relationships varied by cell type. For healthy adults, IL-6 was the cytokine most often produced spontaneously. Spontaneous cytokine production was not unusual in these acutely ill and healthy persons living in an area where human immunodeficiency virus, mycobacterial, malaria, and assorted parasitic infections are endemic. In such populations, spontaneous, as well as induced, cell-specific cytokine production should be measured and evaluated in relation to various disease states.     

Ginger extract inhibits LPS induced macrophage activation and function

Background  

Macrophages play a dual role in host defence. They act as the first line of defence by mounting an inflammatory response to antigen exposure and also act as antigen presenting cells and initiate the adaptive immune response. They are also the primary infiltrating cells at the site of inflammation. Inhibition of macrophage activation is one of the possible approaches towards modulating inflammation. Both conventional and alternative approaches are being studied in this regard. Ginger, an herbal product with broad anti inflammatory actions, is used as an alternative medicine in a number of inflammatory conditions like rheumatic disorders. In the present study we examined the effect of ginger extract on macrophage activation in the presence of LPS stimulation.     

Stimulatory effect of LPS and feedback effect of PGE2 on IL-27 production

Interleukin (IL)-27 is a new member of the IL-6/IL-12 family, composed of two subunits, the Epstein-Barr virus-induced gene 3 (EBI3) and p28 chains (p28), and produced by activated monocytes and dendritic cells. IL-27 plays an important role in the regulation of differentiation of naive T helper cells and has diverse effects on innate immune cells. However, the pro-inflammatory mechanisms of IL-27 are still not well known. In this study, we investigated the effect of lipopolysaccharide (LPS) on the production of IL-27. We found that LPS-stimulated IL-27 production was in a dose-dependent and time-dependent manner in THP-1 cells. We have also shown that IL-27 induced PGE2 production and COX-2 gene expression at the level of mRNA as well as protein. Moreover, we found feed back effect of PGE2 on the production of IL-27 in THP-1 cells. The results suggest that PGE2 significantly inhibits LPS-induced IL-27 production, without affecting basal IL-27 expression. Further experiment suggests that PGE2 and LPS regulate IL-27 through NF-κB pathway. Our findings may have wide implication for IL-27 in inflammatory diseases.     

染料木黄酮的抗巨噬细胞泡沫化效应及其分子机制研究

目的 研究染料木黄酮对动脉粥样硬化过程中巨噬细胞泡沫化的抑制作用,并探讨其抗泡沫化的分子作用机制.方法 采用体外ox-LDL诱导小鼠单核巨噬细胞RAW264.7产生泡沫化,通过油红O染色观察细胞内脂质聚集程度判定细胞的泡沫化程度以及药物效应;利用报告基因细胞检测方法评价染料木黄酮对代谢性核受体过氧化物酶体增殖物激活受体PPAR和肝X受体LXR的转录调控功能的激动作用;利用实时定量PCR检测染料木黄酮对巨噬细胞中ox-LDL摄取以及胆固醇逆转运相关基因的mRNA水平.结果 ox-LDL处理的空白组小鼠RAW264.7单核巨噬细胞内聚积大量的脂质,而经过10 ug/ml染料木黄酮处理后的RAW264.7细胞内脂滴量明显减少,细胞泡沫化程度被大幅度降低.瞬时转染的细胞报告基因的转录激活效应研究结果说明,染料木黄酮对核受体PPAR和LXR均有较高的转录激活效应,其EC50值分别为3.5～9.2 μg/ml和1.6 ～ 3.3 μg/ml.定量PCR研究结果显示,染料木黄酮可以有效地使巨噬细胞中的胆固醇逆转运基因LXRα、LXRβ、ABCA1、ABCGl、和SR-B1的表达上调,而作为主要摄取ox-LDL受体的CD36基因的表达无明显变化.结论 染料木黄酮可以有效抑制动脉粥样硬化过程中巨噬细胞的泡沫化,而该作用机制可能是通过激活PPAR和LXR通路,上调胆固醇逆转运蛋白的ABCA1、ABCG1、LXRα、LXRβ、SR-B1基因的表达,增强了巨噬细胞的胆固醇外排,从而防止动脉粥样硬化的发生和发展.     

Comparison of mechanisms of IL-3 induced histamine release and IL-3 priming effect on human basophils

We have investigated the mechanisms by which interleukin-3 (IL-3) induces histamine release and primes basophils for increased histamine release in response to anti-IgE- and N-formyl-methionyl-leucyl-phenylalanine (fMLP). The responsiveness of basophils from atopic donors was variable, only 5/11 subjects showing release of > 10%, to IL-3 in the range 0.1-100 ng/ml. IL-3-induced histamine release required both extracellular Ca2+ and cell membrane IgE, removal of membrane IgE by lactate stripping or desensitization of basophils by incubation with anti-IgE in a Ca2+-free medium blocking IL-3-induced histamine release. IL-3 also primed basophils for histamine release by anti-IgE and fMLP in the same concentration range as it evoked histamine release. When IL-3 and either anti-IgE or fMLP were combined, the result was additive or supra-additive depending on the basophil donor. Unlike IL-3-evoked histamine release, IL-3 priming of basophils for fMLP-induced histamine release was shown to be independent of the presence of both cell surface IgE and of extracellular calcium. The protein kinase C (PKC) inhibitor, staurosporine (10 nM), inhibited anti-IgE induced histamine release, but neither IL-3 induced histamine release nor IL-3 priming of IgE- and fMLP-induced histamine release. Pertussis toxin (1.0 microg/ml) inhibited fMLP-induced histamine release but not anti-IgE-induced histamine release, IL-3-evoked histamine release or IL-3 priming. These results indicate that IL-3 modulates mediator release from human basophils by two mechanisms; a direct release of histamine which involves cell surface IgE and the influx of extracellular calcium but which is unlikely to proceed by the same mechanism as cross-linkage of IgE, and a priming effect which is independent of IgE and extracellular Ca2+ and which enhances the secretory effects of a wide range of unrelated secretagogues.     

肌肽对脂多糖诱导脑微血管内皮细胞凋亡的保护作用及机制探讨

目的　探讨肌肽对脂多糖（lipopolysaccharides,LPS）引起脑微血管内皮细胞（HBMEC）凋亡的保护作用及机制。 方法　用倒置显微镜观察细胞形态变化;MTT法检测细胞存活率;流式细胞仪检测细胞内的活性氧（ROS）含量及细胞凋亡情况;Western blot法检测NF-κB P65、白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）蛋白表达。 结果　与对照组相比,LPS组细胞存活率略降低（P>0.05）,早期凋亡率明显增加,同时ROS含量、核内NF-κB P65及胞浆IL-1β和TNF-α的含量均显著增加（P<0.05）。与LPS组相比,LPS+肌肽组（10 mmol/L、20 mmol/L、40 mmol/L）凋亡率有明显降低,ROS含量、核内NF-κB P65蛋白量及胞浆IL-1β和TNF-α的含量均不同程度显著降低（P<0.05）。 结论　肌肽可能通过抑制LPS诱导ROS释放,避免NF-κB P65过度激活,进而减少IL-1β和TNF-α分泌,对脑微血管内皮细胞发挥保护作用。     

脂多糖对白细胞介素27的调节作用及其与环氧化酶2及前列腺素E2之间的相关性研究

目的 探讨脂多糖(LPS)在THP-1细胞对白细胞介素27(IL-27)的调节作用,阐明IL-27与环氧化酶2(COX-2)及前列腺素E2(PGE2)之间的关系.方法 采用不同浓度的LPS和IL-27刺激THP-1细胞,并在不同时间采用酶联免疫吸附试验(ELISA)检测细胞上清中IL-27和PGE2的含量,同时采用反转录PCR和Western blot检测COX-2的mRNA和蛋白表达的变化.结果 LPS在THP-1细胞能够诱导IL-27的产生,并呈时间和剂量依赖关系;IL-27能够在mRNA和蛋白水平诱导COX-2的表达,同时诱导PGE2的产生,其诱导作用呈时间和剂量依赖效应.结论 LPS能够在细胞水平刺激IL-27的分泌,同时IL-27能够诱导COX-2的表达和PGE2的产生.