人参皂苷Rg1对小鼠腹腔巨噬细胞的影响

目的研究人参皂苷Rg1对小鼠腹腔巨噬细胞的影响,并探讨其作用机制。方法无菌分离小鼠腹腔巨噬细胞,制备单细胞悬液,加入不同终浓度的人参皂苷Rg1 4 h后,细菌脂多糖LPS(10μg/ml)作用24 h,直径1μm的微球(1×1010/L)结合流式细胞术分析Rg1对小鼠腹腔巨噬细胞吞噬作用的影响;Griess试剂盒检测NO的释放;H2DCFDA染色检测ROS的含量;Fluo-4/AM染色检测Rg1对Ca2+超载的影响;Sytox R Green染色,荧光酶标仪检测Rg1对CHX、CTX诱导的细胞凋亡的影响。结果 10、20μmol/L的Rg1时能明显抑制LPS诱导的巨噬细胞NO和ROS的产生以及吞噬微球的能力;10μmol/L的Rg1能显著抑制Ion诱导的的巨噬细胞Ca2+的超载以及CHX、CTX诱导的细胞的凋亡。结论 Rg1具有显著的抗炎作用,并可抵抗多种因素诱导的细胞凋亡,为进一步开发为免疫治疗药物提供了依据。     

漆黄素对淋巴细胞和巨噬细胞分泌NO的免疫调节作用(英文)

目的探讨漆黄素(fisetin)在体外对小鼠T淋巴细胞增殖、活化、凋亡、ROS的释放和巨噬细胞NO释放功能的影响。方法 CFDA-SE标记技术结合流式细胞术检测漆黄素对T淋巴细胞增殖及增殖指数(PI)的影响。荧光抗体染色结合流式细胞术分析漆黄素对T淋巴细胞在Con A的刺激下CD25的表达水平的影响。DIOC染色检测细胞线粒体膜电势的变化情况,H2DCFDA染色检测细胞ROS释放的变化。Griess试剂盒用以检测巨噬细胞NO释放功能。结果漆黄素(2.5,5和10μmol/L)明显抑制T淋巴细胞增殖,增殖指数(PI)降低并具有剂量依赖性。漆黄素可明显抑制CD25的表达(P<0.01)及ROS的释放。DiOC6(3)染色分析显示,漆黄素可促进细胞的凋亡。巨噬细胞在LPS和IFN-γ的刺激下的NO释放量明显增加,而在漆黄素作用下NO释放量明显下降(P<0.01)。结论漆黄素是一种潜在的免疫调节剂。     

银杏内酯B对小鼠腹膜巨噬细胞体外增殖、吞噬及产生NO和ROS的影响

目的:银杏内酯B(GB)是已知的天然而强效的血小板激活因子(PAF)受体(PAFR)拮抗剂,本研究中GB对小鼠腹膜巨噬细胞的增殖、吞噬及产生NO和ROS的影响,初步探讨其潜在的免疫调节作用与机制,从而为临床应用提供可靠的实验依据。方法:分离纯化小鼠腹膜巨噬细胞(PM),以不同浓度的GB(1.25～20μmol/L)预孵后加以脂多糖(LPS)刺激并继续培养至相应时间点;以MTT法检测GB对PM活力及LPS诱导的增殖的影响;以荧光微球的摄入结合流式细胞术评价PM的吞噬作用;以Griess法检测GB对LPS诱导的NO释放的影响;以H2DCFDA标记结合流式细胞术检测PM的ROS水平。结果:LPS刺激后24 h时PM吞噬能力增强,ROS水平显著上升,并释放大量NO,至48 h时PM增殖明显,而经终浓度为5、10、20μmol/L GB在LPS刺激前对PM进行4 h预孵处理可以大幅下调PM对荧光微球的吞噬作用,显著降低PM的ROS水平及NO释放量,并能有效抑制PM的增殖。结论:GB能有效抑制LPS诱导的细胞增殖、吞噬作用、产生NO和ROS的水平,凭借GB对巨噬细胞的体外行为和功能的出色调节作用,理应将其作为天然的免疫抑制剂候选者进行更深入的研究。     

漆黄素对巨噬细胞和T淋巴细胞的免疫调节作用

目的探讨漆黄素(Fisetin,FIS)对小鼠巨噬细胞吞噬功能、NO的释放及对T淋巴细胞体外活化、增殖的影响。方法无菌制备小鼠巨噬细胞悬液及淋巴细胞悬液;荧光微球结合流式细胞术(FCM)分析FIS对巨噬细胞吞噬作用的影响;Griess试剂盒检测巨噬细胞NO的释放;双色荧光抗体染色结合FCM,检测CD3+T细胞CD69的表达水平;CFSE标记技术检测T细胞增殖的情况。结果 2.5μmol/L,5μmol/L,10μmol/LFIS均能明显抑制巨噬细胞的吞噬微球的能力;FIS能抑制LPS和IFN-γ刺激的巨噬细胞的NO的产生(P<0.05);FIS对ConA刺激的T细胞表达CD69有抑制作用,并能有效抑制ConA诱导的T细胞增殖(P<0.01),且均呈剂量依赖性。结论 FIS能显著抑制小鼠腹腔巨噬细胞的吞噬能力和分泌NO的能力,并能够抑制T细胞的活化和增殖,有望发展成为一种新的免疫抑制药物。     

淫羊藿甙对小鼠腹腔巨噬细胞体外增殖、吞噬和产生NO的影响

无菌分离小鼠腹腔巨噬细胞,制备单细胞悬液,加入不同终浓度的淫羊藿甙孵育4 h后,加入细菌脂多糖LPS(终浓度20 mg/L)48 h后以MTT法检测其增殖;加药孵育4 h后,分别加入直径1μm和2μm的微球(终浓度1×1010/L),5 h后利用流式细胞术(FCM)检测吞噬情况;加入LPS(终浓度20 mg/L)24 h后Griess试剂盒检测巨噬细胞NO的量。结果显示ICA在终浓度1.5、3.0μmol/L时能明显抑制LPS刺激的巨噬细胞增殖(P<0.01)和NO的产生,并促进其吞噬微球的功能。提示ICA可能抑制LPS信号转导从而抑制巨噬细胞增殖,并且通过抑制其活化,下调iNOS有关的炎症因子,使NO减少;对于未活化的巨噬细胞,ICA可能通过上调其吞噬功能,增强固有免疫系统来抵御病原体侵入。     

表没食子儿茶素没食子酸酯对IFN-γ/LPS刺激的小鼠髓源性巨噬细胞极化的影响

目的:探讨表没食子儿茶素没食子酸酯(EGCG)对IFN-γ/LPS刺激的小鼠髓源性巨噬细胞促炎细胞因子表达的影响。方法:取6~8周龄C57BL/6小鼠骨髓细胞,经100 ng/ml巨噬细胞集落刺激因子(M-CSF)体外诱导分化为骨髓巨噬细胞(BMDM),加入50 ng/ml干扰素γ(IFN-γ)和1μg/ml细菌脂多糖(LPS)刺激,并同时暴露于12.5~50μmol/L EGCG处理48 h,利用实时荧光定量PCR(qRT-PCR)和酶联免疫吸附试验(ELISA)检测巨噬细胞促炎细胞因子IL-1β、TNF-α和i NOS的mRNA表达水平和蛋白含量。结果:IFN-γ和LPS处理可刺激巨噬细胞IL-1β、TNF-α和i NOS表达显著上调,而EGCG则能抑制IFN-γ和LPS刺激的IL-1β、TNF-α和i NOS表达,且存在剂量依赖性。结论:EGCG能够减弱IFN-γ和LPS刺激的髓源性巨噬细胞的促炎作用。     

红景天苷对小鼠原代T淋巴细胞体外行为的影响

目的:分析红景天苷(Sal)对小鼠原代T淋巴细胞体外行为的影响。方法:无菌分离并培养BALB/c近交系小鼠淋巴结细胞,MTT比色法检测并分析Sal对小鼠T淋巴细胞活力的影响;荧光染料标记的单克隆抗体染色结合流式细胞术(FCM)检测刀豆蛋白A(Con A)诱导作用下Sal对T淋巴细胞活化抗原CD69表达水平的影响。羧基二乙酸荧光素琥珀酰亚胺酯(CFDA-SE)染色分析T淋巴细胞体外增殖情况。2',7'-二氯二氢荧光素二乙酸酯(H2DCFDA)荧光染色检测小鼠T淋巴细胞胞内活性氧簇(ROS)生成。Di OC6(3)染色检测Sal对T淋巴细胞胞内线粒体活性的影响以及地塞米松(DEX)诱导作用下Sal对T淋巴细胞线粒体膜电位的影响。制备小鼠胸腺T淋巴细胞悬液,FCM检测DEX诱导作用下胸腺T淋巴细胞的凋亡率。结果:终浓度为80、160和320μmol/L的Sal均能提高T淋巴细胞活化抗原CD69的表达水平(P0.05)。Sal能促进Con A诱导72 h条件下T淋巴细胞体外增殖(P0.01),且能减少T淋巴细胞胞内ROS生成量(P0.05),对T淋巴细胞线粒体膜电位有保护作用(P0.01)。Sal能明显降低DEX诱导条件下的胸腺T淋巴细胞凋亡率(P0.01)。结论:Sal能够促进T淋巴细胞的体外活化和增殖,减少T淋巴细胞内ROS的产生,维持细胞内线粒体膜电位稳定性,抑制DEX诱导条件下胸腺T淋巴细胞的凋亡。     

人参皂甙Rb1对小鼠腹腔巨噬细胞体外吞噬及细胞因子和NO分泌的影响

目的:研究人参皂甙Rb1(Ginsenoside Rb1)对小鼠腹腔巨噬细胞体外吞噬及细胞因子和一氧化氮(NO)分泌的影响。方法:分离制备小鼠腹腔巨噬细胞悬液;MTT法检测不同终浓度的人参皂甙Rb1对巨噬细胞的毒性;流式细胞术检测Rb1对巨噬细胞吞噬直径1μm微球的影响;用Griess试剂盒检测Rb1对巨噬细胞NO量的影响;使用流式液相蛋白定量检测技术Cytometric Bead Array(CBA)检测Rb1对LPS刺激巨噬细胞分泌细胞因子的影响。结果:终浓度为5、10、20μmol/L的Rb1对巨噬细胞的吞噬功能具有促进作用,明显抑制LPS诱导的吞噬作用(P<0.05);Rb1能抑制巨噬细胞NO的产生(P<0.01);Rb1能够调节LPS诱导的细胞因子的产生。结论:Rb1对巨噬细胞可能具有双向调节作用,通过抑制LPS信号的转导,调节巨噬细胞因子的分泌,抑制其活化,使NO减少;对于未活化的巨噬细胞,可能通过上调其吞噬功能,增强固有免疫系统来抵御病原体侵入。     

葛根素对脂多糖诱导N9小胶质细胞激活的抑制作用

目的:探索葛根素对脂多糖(LPS)诱导N9小胶质细胞激活的抑制作用,为其在神经退行性疾病的防治方面提供依据。方法:用MTT法检测脂多糖和葛根素对小胶质细胞的细胞毒性作用;用流式细胞术(FCM)检测葛根素对LPS诱导N9细胞内活性氧(ROS)产生的抑制作用;分别用Griess法和FCM检测细胞培养液和细胞内一氧化氮(NO)含量;用HE染色法观察LPS激活N9细胞,及葛根素恢复细胞静息状态下细胞的形态;以FCM检测细胞凋亡和细胞周期。结果:葛根素(100~200μmol/L)能使LPS激活的N9细胞,从阿米巴样的活化形状明显恢复至静息态的圆形;LPS激活的N9细胞能产生大量的NO到培养液中,葛根素(50~200μmol/L)能使NO的产生减少,其中,高浓度组(200μmol/L)与LPS模型组相比,NO从23.45±0.19μmol/L降至12.43±0.11μmol/L(P0.01)。胞内ROS检测表明:葛根素(200μmol/L)同样明显降低ROS的产生。葛根素对激活的N9细胞胞内ROS的产生具有抑制作用,高浓度组(200μmol/L)能使其恢复至对照组水平;此外,葛根素能显著抑制LPS诱导的细胞凋亡,并恢复LPS对N9细胞G0/G1期的阻滞作用。结论:葛根素具有抑制小胶质细胞激活的作用。     

脱水淫羊藿素对小鼠巨噬细胞免疫功能的影响

目的:研究脱水淫羊藿素(AHI)在体外对脂多糖(LPS)诱导的小鼠巨噬细胞免疫功能的影响。方法:分离制备小鼠骨髓来源巨噬细胞;CCK-8法检测不同终浓度的AHI对巨噬细胞的毒性;采用Griess试剂盒检测AHI对巨噬细胞产生NO的影响;流式细胞术(FCM)检测AHI对巨噬细胞吞噬E.coli颗粒的影响;利用FCM结合双色免疫荧光染色技术检测AHI对巨噬细胞早期活化标志CD69的表达情况;使用流式液相蛋白定量检测技术(CBA)检测AHI对LPS刺激巨噬细胞分泌细胞因子的影响。结果:终浓度为2.5、5、10μmol/L的AHI对活化的小鼠巨噬细胞均具有明显的免疫抑制作用,特别是5μmol/L AHI能明显抑制经LPS刺激的巨噬细胞早期活化,释放NO,吞噬E.coli颗粒,以及分泌IL-6、MCP-1、TNF和IL-12p70四种细胞因子。结论:AHI对LPS诱导的小鼠巨噬细胞的活化具有明显的抑制作用,是一种潜在的免疫抑制剂。     

连翘提取物对小鼠腹腔巨噬细胞体外吞噬和NO释放的影响

目的:分析连翘(FS)对小鼠腹腔巨噬细胞的体外吞噬和体外NO释放的影响.方法:无菌收集小鼠腹腔巨噬细胞和羧基荧光素乙酰乙酸琥珀酰亚胺酯(CFDA-SE)标记大肠杆菌DH5α,短期培养3 h后流式细胞术(FCM)分析FS对腹腔巨噬细胞体外吞噬的影响.用LPS体外刺激活化腹腔巨噬细胞,Griess Reagent试剂盒检测并分析FS对巨噬细胞体外释放NO的影响.结果:FCM分析显示,终浓度为40、80、160 mg/L的FS对小鼠腹腔巨噬细胞体外吞噬具有明显的促进作用(P<0.05).不同终质量浓度的FS对LPS诱导小鼠腹腔巨噬细胞体外NO的释放均有抑制作用(P<0.05).结论:FS可以促进小鼠腹腔巨噬细胞的体外吞噬和抑制NO体外的释放.     

转移程度不同的小鼠肝癌细胞株对腹腔巨噬细胞免疫功能的影响

目的 研究转移程度不同的小鼠肝癌细胞株对腹腔巨噬细胞功能的影响.方法 在两株转移程度不同的小鼠腹水型肝癌模型中,取荷瘤小鼠腹腔巨噬细胞,检测其在干扰素γ(IFN-γ)和脂多糖(LPS)刺激下产生一氧化碳(NO)和肿瘤坏死因子α(TNF-α)的水平,并检测其活化后的杀伤能力.进一步采用夹心酶联免疫吸附测定(ELISA)方法,研究不同荷瘤小鼠腹水中IFN-γ与转化生长因子β1(TGF-β1)的水平,并用相应抗体封闭TGF-β1后,检测活化巨噬细胞产生NO能力及杀伤活性.结果 经IFN-γ和LPS活化后,荷瘤小鼠腹腔巨噬细胞分泌NO和TNF-α能力明显低于正常巨噬细胞,杀伤能力下降.高转移性肝癌小鼠巨噬细胞分泌NO水平和杀伤能力均低于低转移性小鼠,但其分泌TNF-α量较高.此外,荷瘤小鼠腹水含较高水平IFN-γ与TGF-β1,不同转移程度荷瘤小鼠IFN-γ水平接近,但高转移性肝癌小鼠腹水含更多TGF-β1,而且TGF-β1的封闭可导致与肿瘤细胞共培养的巨噬细胞分泌NO的能力部分恢复.结论 肿瘤细胞可以通过分泌TGF-β1等抑制性因子下调巨噬细胞的活性和免疫功能.肿瘤的转移程度可能与其分泌免疫抑制因子的能力相关.     

Pre-exposure of murine macrophages to lipopolysaccharide inhibits the induction of nitric oxide synthase and reduces leishmanicidal activity

Murine macrophages produce nitric oxide (NO) from L-arginine on stimulation with lipopolysaccharide (LPS), alone or with interferon-γ (IFN-γ). The effect of incubation of macrophages with low concentrations of LPS on NO synthesis on subsequent stimulation was investigated, using a murine macrophage cell line, J774, and peritoneal macrophages from CBA mice. Cells which had been incubated with LPS produced significantly lower amounts of NO, and expressed lower levels of NO synthase activity, following stimulation with IFN-γ and LPS, or with a high concentration of LPS. This effect was not reversed by tumor necrosis factor-α. The ability of CBA macrophages to kill the intracellular parasite Leishmania major was markedly reduced by pre-incubation with LPS. Reduced NO production by macrophages previously exposed to LPS is a manifestation of endotoxin tolerance, and may represent an important means of regulation of NO synthesis and thus a survival mechanism for intracellular parasites.     

Mechanisms involved in apoptosis of human macrophages induced by lipopolysaccharide from Actinobacillus actinomycetemcomitans in the presence of cycloheximide

Actinobacillus actinomycetemcomitans is a major periodontopathic bacterium with multiple virulence factors, including lipopolysaccharide (LPS). Previous reports have demonstrated that LPS induced apoptosis in a murine macrophage-like cell line, J744.1, as well as in peritoneal macrophages from C3H/HeN mice in the presence of cycloheximide (CHX). However, the detailed molecular mechanisms involved in the apoptosis of macrophages induced by LPS and CHX are not well known. To clarify the possible role of LPS in the induction of macrophage apoptosis, we investigated cell death induced by LPS from A. actinomycetemcomitans and CHX in human macrophage-like U937 cells, which were differentiated by 12-O-tetradecanoylphorbol 13-acetate (TPA), and also assessed the molecular mechanisms involved in the process. We found that TPA-differentiated U937 cells usually showed resistance to LPS-induced apoptosis. However, in the presence of CHX, LPS induced release of cytochrome c without modifying steady-state levels of Bcl-2, Bcl-xL, Bax, and Bak. Treatment with LPS in the presence of CHX also led to activation of caspase-3 and apoptosis via, in part, the CD14/toll-like receptor 4 (TLR4). The induction of cytochrome c release may have been due to dephosphorylation of Akt and Bad, which were cooperatively induced by CHX and LPS. However, endogenous tumor necrosis factor alpha- and Fas-induced signals, extracellular signal-regulated kinase kinase/mitogen-activated protein kinases and I-kappa B alpha/nuclear factor-kappa B (NF-kappa B) were not required for caspase-3-dependent apoptosis. These results emphasize the possible important role of the mitochondrial apoptotic pathway leading to caspase-3 activation in LPS-induced apoptosis of human macrophages in the presence of CHX.     

Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major

Murine macrophages can be activated to produce nitric oxide (NO) and superoxide and these two radicals can react to form peroxynitrite, a powerful oxidant which may be involved in parasite killing. We now show that murine macrophages activated with zymosan and interferon-γ (ZYM/IFN-γ) produced both superoxide (peaking 1–2 h after stimulation, then rapidly declining) and NO (barely detectable at 6 h, peaking by 24 h). Macrophages activated with ZYM alone produced only superoxide, while stimulation with lipopolysaccharide (LPS) and IFN-γ induced NO but not superoxide. Cells stimulated with ZYM/IFN-γ or LPS/IFN-γ killed Leishmania major to a similar degree, an effect that was completely blocked by the addition of N-iminoethyl-L -ornithine. However, macrophages stimulated with ZYM alone were unable to kill L. major. S-nitroso-acetyl-penicillamine, which release NO, was highly leishmanicidal when added directly to the parasites. 3-morpholino-sydnonimine hydrochloride which releases both NO and superoxide simultaneously, was also efficient at killing L. major and this cytotoxicity was greatly enhanced by the addition of superoxide dismutase. Finally, authentic peroxynitrite failed to induce any cytotoxic effect, even at a high concentration. Thus macrophages can produce either NO, superoxide or both, depending on the stimulus. However, the killing of L. major is dependent only on the production of NO.     

α-MSH对脂多糖诱导小鼠腹腔巨噬细胞SOCS-3mRNA表达的影响

目的:观察α-黑色素细胞刺激素(α-MSH)对脂多糖(LPS)诱导小鼠腹腔巨噬细胞SOCS-3mRNA表达及NO释放的影响,以探讨α-MSH拮抗LPS的作用机制。方法:用半定量逆转录多聚酶链反应(RT-PCR)的方法检测LPS诱导体外培养的小鼠腹腔巨噬细胞SOCS-3mRNA表达水平和给予α-MSH后对SOCS-3mRNA表达的影响;用Griess试剂检测单独给予LPS与同时给予α-MSH和LPS作用巨噬细胞后对NO生成量的影响。结果:未受LPS刺激的小鼠腹腔巨噬细胞表达低水平的SOCS-3。LPS组SOCS-3的表达和NO的生成显著高于对照组,而同时给予α-MSH和LPS培养,巨噬细胞的SOCS-3的表达明显低于LPS组,NO的生成则几乎完全阻断。结论:LPS在启动炎症的过程中可能也激活了SOCS介导的负调节机制;但SOCS可能不参与α-MSH的抗LPS作用。     

Phagocytosis of Leishmania mexicana amastigotes by macrophages leads to a sustained suppression of IL-12 production

Healing of leishmaniases is dependent on activation of parasitized macrophages (Mϕ) by IFN-γ, which is secreted by Leishmania-specific Th1 cells. IL-12 enhances IFN-γ production by Th1 cells and is crucial for cure. The host cells of Leishmania sp., Mϕ, are a main source of IL-12 in vivo. We report that infection of quiescent murine Mϕ with L. mexicana or L. major amastigotes does not induce IL-12 production. Moreover, infection suppresses IL-12 secretion by Mϕ activated by LPS, by CD40 cross-linking or cognate interaction with Th1 cells. IL-12 secretion is also suppressed in Mϕ activated after phagocytosis of latex beads. Suppression is independent of engagement of CR3 or FcγR during phagocytosis, is not mediated by IL-10 and does not alter steady state IL-12p40 mRNA levels. In addition, suppression of IL-12 secretion does not depend on Mϕ activation concurrent to infection. In contrast, NO production was not inhibited. Thus, Mϕ effector functions are differentially affected and this may be a general effect of phagocytosis of non-activating particles. The possible implications of this effect on the infection are discussed.     

Apoptosis of Trypanosoma musculi co-cultured with LPS activated macrophages: enhanced expression of nitric oxide synthase INF-gamma and caspase

Trypanosoma musculi-macrophage co-cultures were studied to investigate the biological role of lipopolysaccharide (LPS) induced cytokines in controlling the proliferation of parasites in vitro. Macrophages, isolated by peritoneal lavage, sustained the growth and proliferation of the parasites. Macrophages activated with LPS were characterized by up-regulation of nitric oxide synthase (iNOS) and phagocytosis of fluorescent latex spheres. Activated macrophages showed marked inhibition of the association and proliferation of the parasites. The LPS treated macrophages produced cytokines, especially interferon gamma (INF-gamma), which was detected by Western blot. Trypanosomes, inhibited from association with macrophages, did not proliferate and instead formed clusters held together by their flagella. Cells in these clusters were apoptotic, as demonstrated by the Apoptag reaction and gel fragmentation assay. In addition, high levels of caspase 8 and caspase 3 were shown in floating trypanosome clusters. The results would suggest that INF-gamma and other cytokines released by activated macrophages, possibly functioning through the INF-gammaR1, Fas ligand, CD95 or other death ligands in the trypanosome plasma membrane initiates the apoptosis cascade in trypanosomes.