猪苓多糖对巨噬细胞一氧化氮生成和细胞内还原型谷胱甘肽的影响

本文观察了猪苓多糖 (PPS )对小鼠腹腔巨噬细胞一氧化氮 (NO )生成和细胞内还原型谷胱甘肽 (GSH )的影响。结果显示 :(1)PPS对小鼠腹腔巨噬细胞NO生成具有明显的促进作用 ;(2 )细胞内GSH浓度随NO生成增加而减少 ;(3)这些作用能被NO生成抑制剂L NMMA所抑制。结果表明PPS能促进小鼠腹腔巨噬细胞NO生成 ,并同时消耗细胞内GSH。提示细胞内GSH可能起到调节巨噬细胞NO生成和保护宿主细胞免受NO介导的细胞毒作用。     

一氧化氮在巨噬细胞的细胞毒效应中的作用

目的:探讨一氧化氮(NO)对巨噬细胞细胞毒效应的影响。方法:将20只昆明种小鼠分为两组,一组皮下接种S180横纹肌肉瘤细胞,另一组作为正常对照组。分别取两组小鼠腹腔灌洗液中的巨噬细胞与K562肿瘤细胞共同培养,检测在培养液中加入左旋精氨酸(L-Arg)和G-单甲基左旋精氨酸(L-NAME)后巨噬细胞杀伤率的改变。结果:正常小鼠腹腔灌洗液中的巨噬细胞在L-Arg和L-NAME存在的情况下细胞毒效应没有改变;荷瘤小鼠腹腔灌洗液中的巨噬细胞在L-Arg组和空白组中的细胞毒效应明显增强。结论:NO是巨噬细胞细胞毒作用的一个效应分子。     

香菇多糖对小鼠腹腔巨噬细胞一氧化氮生成的影响及其机理

为进一步探讨香菇多糖（Lentinan,LTN）的免疫调节和抗肿瘤作用机理,本实验研究了LTN对小鼠腹腔巨噬细胞一氧化氮（NO）生成和诱导型一氧化氮合酶（iNOS）的影响。结果显示,LTN能明显促进小鼠腹腔巨噬细胞NO生成,并且同干扰素-γ（IFN－γ）具有协同促进作用。结果还表明,LTN的这一促进作用能被RNA转录抑制剂放线菌素D、蛋白质合成抑制剂放线菌酮和一氧化氮合酶（NOS）抑制剂L－NMA所抑制。提示LTN的免疫调节和抗肿瘤作用机理可能与其诱导巨噬细胞iNOS合成、促进NO生成有关。     

双歧杆菌对裸鼠腹腔巨噬细胞产生细胞因子以及细胞毒活性的影响

目的探讨青春型双歧杆菌对巨噬细胞Μ功能的调节作用。方法将双歧杆菌注射于裸鼠腹腔 ,用小鼠胸腺细胞增殖法、L929细胞体外杀伤法及Griess试剂 ,分别测定裸鼠腹腔Μ产生的IL 1 ,TNF α及NO的水平 ;同时观察了Μ体外杀瘤活性的变化。结果双歧杆菌注射组裸鼠腹腔Μ产生的IL 1 ,TNF α及NO的水平分别为 :0.55±0.024(A值) ,(165±41)103U/L及(53±6)μmol/L ;对照组分别为 :0.34±0.021(A值) ,(12±6)103 U/L及(31±13)μmol/L。三组数据各自比较均有显著性差异(P<0.01)。同时 ,体外杀瘤活性也明显增强(P<0.01)。结论青春型双歧杆菌能激活Μ ,增强其杀瘤活性 ,并使之分泌大量的细胞毒性效应分子。     

灵芝孢子粉碱提多糖对小鼠巨噬细胞的免疫调节作用

目的 :研究灵芝孢子粉碱提多糖 (LZSBS)对小鼠巨噬细胞的激活作用。方法 :用灵芝孢子粉碱提多糖刺激体外培养的小鼠巨噬细胞 ,用ELISA法检测巨噬细胞分泌至培养基中的TNF α和IL 1β的含量 ;用Griess法检测培养上清中NO的含量。小鼠巨噬细胞对乳胶颗粒的吞噬率用显微镜计数。结果 :经灵芝孢子粉碱提多糖刺激后 ,小鼠巨噬细胞变大 ,颜色加深 ,并能显著刺激巨噬细胞分泌TNF α和IL 1β ,并产生大量的NO。小鼠巨噬细胞对乳胶颗粒的吞噬功能也明显的增强。结论 :灵芝孢子粉碱提多糖对小鼠巨噬细胞具有明显的激活作用     

黄芪多糖通过NF-κB诱导巨噬细胞产生NO和TNF-α

目的 通过体外试验研究黄芪多糖(Astragalus mongholicus polysaccharides,ASP)激活巨噬细胞产生NO和TNF-α的分子机制和细胞内信号转导机制.方法 黄芪多糖刺激RAW264.7细胞,用Western blot方法 检测细胞核内核转录因子-κB(NF-κB)的变化.用Griess还原法观察黄芪多糖对巨噬细胞释放NO的作用的影响以及NF-κB抑制剂对黄芪多糖诱导巨噬细胞释放NO作用和分泌TNF-α的影响.ELISA法检测黄芪多糖对巨噬细胞分泌肿瘤坏死因子-α(TNF-α)的变化.结果 100μg/ml黄芪多糖刺激RAW264.7细胞,4 h后可引起细胞核内NF-κB含量显著增加,6 h达到顶峰.16 h后可显著诱导NO[(18.9±1.5)μmol/L;P<0.01]释放和TNF-α分泌[(81.2±16.7)pg/ml,P<0.01]的增加,以及诱导型一氧化氮合酶(iNOS)[(23.54±2.41)U/mg蛋白质,P<0.01]活性的增加.NF-κB抑制剂吡咯烷二硫代氨基甲酸盐(PDTC)可明显抑制黄芪多糖诱导RAW264.7生成NO和分泌TNF-α.结论 NF-κB在黄芪多糖诱导巨噬细胞生成NO和TNF-α过程中发挥重要作用.     

NF-κB "decoy"寡核苷酸对LPS诱导巨噬细胞产生NO及活性氧的影响

目的 :探讨NF -κB“decoy”寡核苷酸 (ODNs)对脂多糖 (LPS)诱导小鼠巨噬细胞株J774 1产生一氧化氮 (NO)及活性氧的影响。方法 :通过体外细胞培养技术 ,观察转染NF -κB“decoy”ODNs对LPS刺激巨噬细胞产生NO和活性氧 (ROS)的影响 ,以及诱导型一氧化氮合酶 (iNOS)活力的变化。结果 :在LPS刺激的巨噬细胞中转染NF -κB“decoy”ODNs减少NO和ROS的产生 ,降低iNOS的活力。结论 :NF -κB“decoy”ODNs可以对抗LPS诱导巨噬细胞产生的ROS和NO ,可能与其特异性竞争抑制活化NF -κB结合位点有关。     

磨菇多糖对小鼠S180肉瘤抗瘤效应的初步研究

研究了磨菇多糖（Ｌｅｎｔｉｎａｎ）抑制肿瘤生长和免疫调节作用，结果表明：磨菇多糖对小鼠肉瘤Ｓ１８０有较强的抑瘤作用，在体内可明显促进小鼠脾淋巴细胞转化率，增强小鼠ＮＫ细胞活性和腹腔巨噬细胞的杀伤活性，提示磨菇多糖可刺激机体免疫系统，激发机体的抗肿瘤机制，具有明显的抗肿瘤效应。     

TNF-α对小鼠腹腔渗出细胞抗真菌活性的影响

目的：研究TNF—α对小鼠腹腔渗出细胞的激活和杀菌活性的效应。方法：以IL-12、IL-18、IFN—γ、TNF-α及抗体诱导小鼠腹腔渗出细胞和巨噬细胞，应用ELISA法测定细胞因子变化，Griess反应法测定培养上清液中NO的含量，检测巨噬细胞内新型隐球菌菌落数。结果：IL-12和IL-18联合应用协同诱导腹腔渗出细胞产生TNF—α，且能被IFN—γ抗体所抑制。IFN—γ、TNF-α协同诱导巨噬细胞产生NO和增强其杀伤新型隐球菌活性。结论：TNF—α在细胞因子相互调节诱导增强巨噬细胞杀真菌活性中起重要作用。     

一氧化氮抑制剂对腹膜透析大鼠腹膜形态及表面层的影响

目的:探讨高糖透析液对大鼠腹膜巨噬细胞一氧化氮(NO)的产生、诱导型一氧化氮合酶(iNOS)的表达和腹膜透析通透性的影响以及NO抑制剂的保护作用。方法: 培养生理盐水、1.5％和4.25％葡萄糖透析液透析下的大鼠腹腔内的巨噬细胞, 检测细胞培养上清液NO的浓度和巨噬细胞iNOS的表达, 并观察1.5％及4.25％葡萄糖透析液和NO抑制剂透析下大鼠的液体超滤量和腹膜形态的变化。结果:大鼠腹腔内巨噬细胞NO的产生随透析液中葡萄糖含量增加而增加;腹腔巨噬细胞iNOS的表达, 葡萄糖透析组明显高于生理盐水组;NO抑制剂对光镜下腹膜形态无影响, 但明显减轻4.25%葡萄糖透析液对超滤量和腹膜表面层的减少作用。结论:一氧化氮抑制剂可明显改善高糖透析大鼠的腹膜通透性, 并减轻高糖对腹膜表面层的损伤。     

Activated peritoneal macrophages inhibit the proliferation of rat ascites hepatoma AH-130 cells via the production of tumor necrosis factor-<Emphasis Type="Italic">α</Emphasis> and nitric oxide

OBJECTIVE: To study the effect of peritoneal macrophages on tumor cell proliferation, we cultured ascites hepatoma AH-130 cells with unstimulated, or lipopolysaccharide (LPS)- or interleukin (IL)-2-stimulated rat peritoneal macrophages, and examined the proliferation of AH-130 cells. MATERIALS AND METHODS: Rat peritoneal macrophages isolated from male Wistar rats were co-cultured with AH-130 cells in the absence or presence of LPS or IL-2. After incubation, proliferation of AH-130 cells was analyzed using flow cytometry. In addition, the levels of tumor necrosis factor (TNF)-alpha and nitric oxide (NOx, nitrate + nitrite) in the culture supernatants were measured. Furthermore, anti-TNF-alpha antibody (10 microg/ml) and nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine (L-NMMA, 100 microM) were added to the coculture, and their effect on AH-130 cell proliferation was examined. RESULTS: When AH-130 cells were co-cultured with unstimulated peritoneal macrophages, proliferation of AH-130 cells was not affected. In contrast, when AH-130 cells were cocultured with peritoneal macrophages in the presence of LPS (0.1-20 microg/ml) or IL-2 (1-200 U/ml), proliferation of AH130 cells was dose-dependently suppressed by LPS or IL-2. Moreover, LPS- or IL-2-stimulation increased the levels of TNF-alpha and NOx in the supernatants of AH-130 cell and macrophage co-culture, although LPS and IL-2 did not induce TNF-alpha and NOx production by AH-130 cells incubated without macrophages. Interestingly, anti-TNF-alpha antibody and L-NMMA significantly inhibited the suppression of AH-130 cell proliferation by LPS- or IL-2-stimulated macrophages (p < 0.05). Furthermore, exogenously added recombinant rat TNF-alpha (0.26-1300 ng/ml) or NO donor (GSNO, S-nitroso-L-glutathione) (0.1 - 10 mM) dose-dependently suppressed the proliferation of AH-130 cells in the absence of macrophages. CONCLUSION: Together these observations suggest that when peritoneal macrophages are activated by LPS and IL-2, they suppress the proliferation of ascites hepatoma AH-130 cells via the production of TNF-alpha and nitric oxide.     

Role of IFN-gamma on dissociation between nitric oxide and TNF/IL-6 production by murine peritoneal cells after restimulation with bacterial lipopolysaccharide

Murine peritoneal exudate cells (PEC) pre-exposed to bacterial lipopolysaccharide (LPS) show augmented nitric oxide (NO) production by LPS restimulation, in contrast to LPS tolerance with reduced production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6). Significant amounts of interferon-gamma (IFN-gamma) were detected in the PEC cultures on LPS stimulation, and anti-IFN-gamma antibody suppressed the LPS-induced NO, but not TNF-alpha and IL-6, production. Addition of anti-IFN-gamma antibody to the cultures in the LPS pre-exposure step strongly suppressed the augmented NO production on LPS restimulation. Anti-IL-12 antibody, which suppressed the LPS-induced IFN-gamma production, also suppressed the augmented NO production, as did anti-IFN-gamma antibody. Taken together, we propose the following mechanisms: (1) T and NK cells in PEC produce IFN-gamma by the action of IL-12, which is derived from LPS-stimulated macrophages, and (2) the de novo-produced IFN-gamma activates macrophages to augment NO production on LPS restimulation.     

The nitric oxide-producing properties of Ulmi radicis cortex extract.

We investigated the effect of Ulmi radicis cortex extract (UrCE) on the production of nitric oxide (NO). Stimulation of mouse peritoneal macrophages with UrCE after the treatment of recombinant interferon-gamma (rIFN-gamma) resulted in the increased NO synthesis. UrCE had no effect on NO synthesis by itself. When UrCE was used in combination with rIFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of UrCE on NO synthesis was shown 6 h after treatment with rIFN-gamma. NO production was inhibited by NG-monomethyl-L-arginine. The increased production of NO from rIFN-gamma plus UrCE-stimulated cells was decreased by the treatment of protein kinase C inhibitor such as staurosporin. In addition, synergy between rIFN-gamma and UrCE was mainly dependent on UrCE-induced tumor necrosis factor-alpha (TNF-alpha) secretion. All the preparations of UrCE were endotoxin free. These results suggest that the capacity or UrCE to increase NO production from rIFN-gamma-primed mouse peritoneal macrophages is the result of UrCE-induced TNF-alpha secretion.     

Modulation of murine peritoneal macrophage function by chronic exposure to arsenate in drinking water

Exposure of humans to arsenic is associated with various adverse health effects including immunotoxicity and elevated risk of cancer development. Specific mechanisms of these effects are not well understood. In the present study we investigated some functional parameters of peritoneal macrophages isolated from mice exposed for 12 weeks to sodium arsenate in drinking water at 0.5, 5, and 50 mgAs/l. The experimental conditions were matched with the environmental conditions of arsenic exposure in humans. To characterize function of the macrophages, we assessed their ability to release nitric oxide (NO), reactive oxygen species (ROS), and tumor necrosis factor-alpha (TNF-alpha) in response to common stimulants. To this end the isolated cells were stimulated with lipopolysaccharide (1 microg/ml) to assess NO and TNF-alpha production (the WEHI-164 bioassay) or with phorbol myristate acetate (5 microg/ml) to assess superoxide production (NBT reduction test). As a result, in mice exposed to 0.5, 5, and 50 mgAs/l we observed decreased production of NO (9 +/- 2, 8 +/- 2, 11 +/- 5 microM NO2-, respectively, versus 27 +/- 7 microM in control) and superoxide (41.3 +/- 18.2%, 52.8 +/- 15.1% and 55.9 +/- 12.9%, respectively, less than in control). Despite reduced NO production, expression of iNOS mRNA in RT-PCR, showed similar levels in exposed and control animals. We did not see any significant influence of the exposure on TNF-alpha release and mRNA expression. The potential consequences of decreased production of NO and superoxide by peritoneal macrophages as observed in exposed mice may suggest impaired response of the cells against infection or developing tumor cells.     

Production of nitric oxide by murine peritoneal macrophages in vitro on treatment with prolactin and growth hormone: involvement of protein tyrosine kinases, Ca(++), and MAP kinase signal transduction pathways

Prolactin (PRL) and growth hormone (GH) (somatotropin) have been known to possess immunomodulatory properties. In the present studies we have investigated the production of nitric oxide (NO) and TNF-alpha by murine peritoneal macrophages in vitro on treatment with PRL and GH and the signal transduction mechanism involved. It is observed that significantly enhanced production of NO is induced in macrophages on treatment with PRL and GH. It is further observed that protein tyrosine kinases, MAP kinases and Ca(++) channeling are involved in NO production by macrophages on in vitro treatment with PRL and GH. GH and PRL induced nitric oxide did not have any effect on the expression and production of TNF-alpha. PRL or GH induced TNF-alpha production by murine macrophages was insensitive in the presence of competitive inhibitor of NOS, L-NMMA. Similarly, there is no autocrine or paracrine effect of TNF-alpha on GH or PRL induced NO production and iNOS expression.     

Tumor necrosis factor-alpha: central regulatory cytokine in the induction of macrophage antimicrobial activities

Expression of activated macrophage resistance to infection requires the cooperative interaction of interferon-gamma (IFN-gamma) and either interleukin-2 (IL-2), interleukin-4 or granulocyte/macrophage-colony-stimulating factor: no single cytokine is effective. For IFN-gamma and IL-2, the effector activity can be suppressed by the presence of anti-tumor necrosis factor-alpha (TNF-alpha) antibodies in the reaction mixture. IFN-gamma and IL-2, only in combination, induce TNF-alpha-specific mRNA and secretion of this cytokine by macrophages. Development of intracellular killing activity by activated macrophages also requires the autocrine effects of TNF-alpha. IFN-gamma provides the first signal for the production of nitric oxide (NO), the effector molecule for intracellular destruction of parasites. When IFN-gamma-treated cells are infected with pathogens, they are stimulated to make TNF-alpha. Expression of intracellular killing, as well as production of NO, is inhibited by anti-TNF-alpha antibody.     

红景天苷对小鼠腹腔巨噬细胞体外增殖、凋亡、吞噬、ROS和NO产生的影响

目的:研究红景天苷(Sal)对小鼠腹腔巨噬细胞体外增殖、凋亡、吞噬、胞内活性氧簇(ROS)及分泌一氧化氮(NO)的影响,初步探讨其对小鼠腹腔巨噬细胞的免疫调节作用。方法:无菌分离小鼠腹腔巨噬细胞,并制备单细胞悬液,以不同终浓度(80μmol/L、160μmol/L及320μmol/L)的Sal和巨噬细胞共培养4 h,再以脂多糖(LPS)和γ-干扰素(IFN-γ)进行共刺激。利用MTT比色法检测Sal对巨噬细胞体外增殖的影响。用放线菌酮(CHX)诱导巨噬细胞凋亡,用Sytox G reen染色结合荧光酶标仪检测Sal对CHX诱导巨噬细胞凋亡的影响。用流式细胞术(FCM)检测Sal对巨噬细胞吞噬功能的影响。用2-7-二氯氢化荧光素乙二脂(H2DCFDA)染色法结合荧光酶标仪检测Sal对胞内ROS产生的影响;用G riess反应检测Sal对巨噬细胞分泌NO的影响。结果:MTT比色法检测显示,终浓度为80、160、320μmol/L的Sal均可显著促进LPS+IFN-γ刺激巨噬细胞增殖(P<0.05)。荧光酶标仪检测Syto xG reen染色法的结果显示,160μmol/L的Sal可抑制CHX诱导的巨噬细胞凋亡(P<0.01)。FCM结果显示,各浓度的Sal均能促进单纯药物组和实验药物组LPS+IFN-γ刺激巨噬细胞的吞噬功能(P<0.05)。用荧光酶标仪检测DH2DCFDA染色结果表明,各浓度的Sal对LPS+IFN-γ刺激的巨噬细胞胞内ROS的产生均具有显著的抑制作用(P<0.01)。Griess反应检测NO含量的结果显示,各浓度的Sal对LPS+IFN-γ刺激巨噬细胞产生NO均具有促进作用(P<0.05)。结论:Sal对LPS和IFN-γ刺激的巨噬细胞增殖具有显著的促进作用,对CHX诱导的巨噬细胞凋亡具有显著的抑制作用,对静息态和活化态的巨噬细胞的吞噬功能均有增强作用,并能减少LPS和IFN-γ活化的巨噬细胞胞内ROS的产生;但能促进LPS和IFN-γ活化的巨噬细胞NO的分泌。     

Differential effects of interleukin-4 and interleukin-10 on nitric oxide production by murine macrophages

OBJECTIVE: To study the effect of interleukin (IL)-4 and IL-10 on nitric oxide (NO) production by macrophages. MATERIALS AND METHODS: Elicited or resident peritoneal macrophages (PMO) and a macrophage cell line Raw264.7 were primed by IL-4 or IL-10 for 6 hours, and were further incubated in the presence of interferon (IFN)-gamma and/or lipopolysaccharide (LPS) for 48 hours. NO2- accumulation in the supernatant of cultured cells was used as an indicator of NO production and was determined by the standard Griess reaction adapted for microplates. The amount of tumor necrosis factor (TNF)-alpha in the culture supernatants was determined with a commercially available ELISA kit. The absorbance was measured at 450 nm with a microplate photometer. RESULTS: IL-4 inhibited NO production by murine macrophages of different sources and the macrophage cell line Raw264.7. In contrast, different macrophage populations showed differential responses to IL-10. After stimulation with LPS or IFN-gamma, IL-10 suppressed NO production by elicited PMO but enhanced NO production by resident PMO or by Raw264.7. Both IL-4 and IL-10 inhibited the production of TNF-alpha, which has been shown to play a crucial role in NO production. In the presence or the absence of blocking antibody to TNF-alpha, IL-10 always enhanced NO production by resident PMO. This result suggests that the inhibition of TNF-alpha production and the enhancement of NO production by resident PMO stimulated with IL-10 are independent, coexisting events. CONCLUSIONS: Factors other than TNF-alpha have been suspected to influence NO production by macrophages, and this study indicates that IL-10 may be a candidate cytokine for resident PMO.