金属离子刺激单核-巨噬细胞核因子κB受体活化子的研究

目的:探讨Cr3+、Co2+对体外培养的小鼠单核-巨噬细胞(RAW264.7)的细胞毒性以及刺激单核-巨噬细胞表达核因子κB受体活化子(RANK)在人工关节术后引起骨溶解的分子生物学机制.方法:在体外培养单核-巨噬细胞(RAW264.7).用Co2+、Cr3+分别干预单核-巨噬细胞,不同时间用噻唑蓝(MTT)比色法检测其细胞活性.将细胞分为5组,A组为单纯单核-巨噬细胞,B组为单核-巨噬细胞+500 mg/L Cr2+,C组为单核-巨噬细胞+500 mg/L Cr3+ +20 μmol/L SP600125,D组为单核-巨噬细胞+10mg/LCo2+,E组为单核-巨噬细胞+10mg/LCo2++20 μmol/LSP600125.用半定量逆转录-聚合酶链反应(RT-PCR)在24 h、48 h检测各组细胞的RANK mRNA的表达量.结果:与A组相比,B组和D组单核-巨噬细胞的细胞活力明显下降.在24 h、48 h时,Co2+和Cr3+均能刺激单核-巨噬细胞使其细胞RANK mRNA表达量增强(P<0.05),且SP600125可抑制Cr3+、Co2+刺激的单核-巨噬细胞RANK mRNA表达(P<0.05).结论:金属离子对单核-巨噬细胞有细胞毒性,且能够诱导单核-巨噬细胞RANK mRNA的表达,此外JNK通路参与金属离子刺激单核-巨噬细胞表面RANK的表达.     

氨基胍对脂多糖诱导的大鼠肺表面活性物质和肺泡巨噬细胞的影响

目的观察氨基胍(aminogunidine,AG)对脂多糖(1ipopolysaccharide,LPS)诱导的大鼠肺表面活性物质(pulmonary surfactant,PS)时程性变化的影响和对肺泡巨噬细胞(alveolar macrophage,AM)的影响,探讨AG对肺损伤组织的保护作用及其机制。方法试验一:健康♂SD大鼠随机分为对照组、模型组和AG治疗组。模型组、AG治疗组静脉注射LPS(5mg·kg-1)复制内毒素性肺损伤模型。LPS3h和6h后腹腔注射AG(100mg·kg-1,AG治疗组)和生理盐水(对照组及LPS组)。原位杂交法(ISH)和Western blot法分别测定肺组织肺表面活性物质蛋白A(SP-A)mRNA及蛋白水平;留取肺泡灌洗液(BALF),测定BALF中的总磷脂(TPL)和总蛋白(TP)。试验二:体外分离培养大鼠肺泡巨噬细胞,以LPS(终浓度10mg·L-1)损伤巨噬细胞,观察AG对肺泡巨噬细胞的影响。结果与对照组比较,大鼠肺损伤后SP-A mRNA及蛋白表达在LPS6h、9h后明显下降;BALF中TPL明显减少,TP增多。肺损伤3h用AG治疗3h后,SP-A mRNA阳性细胞表达及蛋白明显增强,BALF中TPL较LPS组相同时间点明显上升,TP降低,病理显示肺损伤减轻。体外实验显示,与正常对照组相比,LPS处理巨噬细胞后,细胞培养上清中NO、TNF-α、IL-6和LDH浓度明显增高,AG明显减少LPS所致的LDH和NO的释放,降低TNF-α和IL-6浓度。结论肺损伤后早期给予AG可通过增强肺表面活性物质表达减轻内毒素性肺损伤。AG可抑制LPS对巨噬细胞的作用。     

糖皮质激素对内毒素耐受的影响

目的应用地塞米松(dexamethasone,Dex)干预小鼠内毒素耐受的形成,研究糖皮质激素对内毒素耐受的影响。方法以昆明鼠为实验对象进行分组,ⅠA组为健康对照组,ⅠB组为单次直接致死剂量脂多糖(lipopolysaccharide,LPS)(2LD50=8 mg/kg)对照组,Ⅱ组为标准内毒素耐受组,ⅢA组为初始LPS刺激前高剂量Dex(10 mg/kg)干预组,ⅢB组为终末致死量LPS前高剂量Dex干预组,ⅣA组为初始LPS刺激前低剂量Dex(1mg/kg)干预组,ⅣB组为终末致死量LPS前低剂量Dex干预组。完成实验处置后3 h取血标本,应用ELISA方法检测血清TNF-α和IL-10水平。结果①ⅠA组TNF-α、IL-10水平极低,ⅠB组TNF-α、IL-10水平较ⅠA组显著增高(P<0.05,P<0.01),Ⅱ组TNF-α、IL-10水平较ⅠB组显著下降(P<0.05,P<0.01),但仍高于ⅠA组。②各Dex干预组TNF-α水平与Ⅱ组比较:ⅢA、ⅣA组TNF-α水平与Ⅱ组TNF-α水平差异无统计学意义(P>0.05),ⅢB、ⅣB组TNF-α水平低于Ⅱ组,差异有统计学意义(P<0.05)。③各Dex干预组IL-10水平与Ⅱ组比较:ⅢA、ⅣA组IL-10水平高于Ⅱ组,差异有统计学意义(P<0.05),ⅢB、ⅣB组IL-10水平与Ⅱ组比较差异无统计学意义(P>0.05)。结论①LPS刺激可导致小鼠体内TNF-α水平、IL-10水平升高。内毒素耐受时,小鼠体内TNF-α、IL-10水平升高的程度明显降低。②终末LPS前一定时间内予以Dex干预可以促进内毒素耐受的形成,Dex剂量过高可能不会带来更多受益。③IL-10对内毒素耐受的形成可能不具有决定作用。     

哮喘大鼠血管内皮生长因子表达与γ-干扰素、白介素-4变化的相关性及地塞米松的干预

目的:观察血管内皮生长因子(vascular endothelial growthfactor,VEGF)、γ-干扰素(IFN-γ)、白介素-4(IL-4)在大鼠哮喘急性模型中的表达情况,探讨Th细胞亚群的失衡与其表达的相关性.方法:36只清洁级雄性sD大鼠随机分为生理盐水对照组(C组)、哮喘组(A组)和地塞米松干预组(D组),以卵清白蛋白(ovalbumin,OVA)致敏激发法制备大鼠哮喘急性模型.末次激发24 h后腹腔注射麻醉,心脏取血,右肺行支气管肺泡灌洗留取灌洗液(BALD.应用酶联免疫吸附(ELISA)法测定血清和BALF中IL-4、IFN-γ和VEGF的浓度.结果:A组血清和BALF中IL-4、VEGF的水平均分别显著高于C组,D组血清和BALF中IL-4、VEGF的水平均分别显著低于A组(P<0.01),C组和D组相比差异均无统计学意义(P>0.05).A组血清和BALF中IFN.,的水平均显著低于C组(P<0.01),D组血清、BALF中的IFN-γ水平均显著高于A组(P<0.01)而低于C组(P<0.01).大鼠BALF中IL-4浓度与VEGF浓度呈显著正相关(r=0.797,P<0.01),IFN-γ浓度与VEGF浓度呈显著负相关(r=-0.568,P相似文献   

孕酮对幼年大鼠感染性脑水肿水通道蛋白4表达的影响

目的 观察内毒素(LPS)致幼年大鼠感染性脑水肿后,腹腔注射孕酮(Prog)对水通道蛋白4(AQP4)表达的影响.方法 将90只幼年大鼠随机分为对照组(C,n=10),内毒素组(LPS,n=40)和内毒素+孕酮组(LPS+Prog,n=40).建立LPS致大鼠感染性脑水肿模型后1 h,LPS+Prog组和LPS组分别腹腔注射孕酮和生理盐水,以后每6小时注射一次.手术后6、12、24和48 h处死动物,分别行脑组织含水量的测定,用免疫组化和RT-PCR检测脑组织内AQP4表达水平.结果 LPS组脑组织含水量明显高于C组和LPS+Prog组(P<0.01).在LPS注射6 h时,幼鼠脑组织 AQP4表达明显增加,12 h达高峰.LPS+Prog组在LPS注射后12 h时,脑组织中AQP4在蛋白水平和mRNA水平的表达均低于LPS组(P<0.01).结论 孕酮可能在转录和翻译水平下调AQP4的表达,从而抑制LPS诱导的幼年大鼠感染性脑水肿的发生发展.     

奥拉帕尼通过PARP-1通路调控脂多糖诱导的A549细胞炎症反应

目的探讨奥拉帕尼对脂多糖(LPS)诱导的肺泡上皮细胞炎症损伤的保护作用。方法体外培养的肺泡上皮细胞(A549)同时加入LPS 10mg·L~(-1)+奥拉帕尼10和25μmol·L~(-1)孵育24 h。酶联免疫吸附试验(ELISA)测定白细胞介素6(IL-6),IL-8和IL~(-1)0释放;实时PCR技术检测肿瘤坏死因子α(TNF-α)、IL~(-1)β、IL-6、IL-8和细胞间黏附分子1(ICAM-1)mRNA的表达水平;流式细胞术检测细胞活性氧(ROS)水平;Western印迹法检测细胞聚腺苷二磷酸核糖聚合酶1(PARP-1)蛋白表达水平和NF-κB通路相关蛋白磷酸化水平。结果与LPS 10 mg·L~(-1)损伤组相比,奥拉帕尼10和25μmol·L~(-1)显著降低LPS诱导的A549细胞IL-6,IL-8的释放(P<0.01)和ROS水平(P<0.01),升高IL~(-1)0水平(P<0.01),抑制了LPS诱导的TNF-α,IL~(-1)β,IL-6,IL-8和ICAM-1 mRNA表达水平(P<0.01),抑制LPS诱导的PARP-1蛋白表达和NF-κB通路磷酸化激活(P<0.01)。结论奥拉帕尼可有效缓解LPS诱导的肺泡上皮细胞炎症损伤和氧化应激,其作用机制可能为奥拉帕尼通过下调PARP-1的表达,继而影响NF-κB通路的活化,最终抑制了相关炎症因子的表达和释放。     

IL-17和RORγt在类风湿关节炎CD4+T细胞中的表达及临床意义

目的探讨类风湿关节炎(RA)患者外周血CD4+ T细胞中白细胞介素(IL)-17、孤核受体(RORγt)表达及意义。方法 35例RA患者分为病情活动组(A组,20例)和RA病情稳定组(B组,15例);另设健康人作对照(C组,15例)。取外周血单个核细胞(PBMC),免疫磁珠阴选CD4+ T细胞,通过流式细胞术(FCM)检测CD4+ T细胞内IL-17蛋白水平;非特异性刺激剂(A-CD3、A-CD28)刺激CD4+ T细胞后,RT-PCR检测IL-17、RORγt mRNA水平。结果与B组和C组比较,A组IL-17蛋白水平和IL-17、RORγt mRNA表达均显著增高(P<0.01),且B组各指标也高于C组(P<0.05)。A-CD3/A-CD28刺激CD4+ T细胞12h后,IL-17、RORγt mRNA表达水平较刺激24h明显增加(P<0.01),且两组各指标亦均高于未刺激组(P<0.05)。结论 RA患者外周血CD4+ T细胞中IL-17、RORγt mRNA高表达,这可能与RA病情活动相关。     

蟾蜍灵对脂多糖诱导的大鼠肾小球系膜细胞IL-6和TGF-β1表达的影响

目的 探讨蟾蜍灵对脂多糖(LPS)诱导的大鼠肾小球系膜细胞(GMC)增殖及白细胞介素6(IL-6)和转化生长因子β1(TGF-β1)表达的影响.方法 体外培养大鼠肾小球系膜细胞,分为蟾蜍灵干预(A)组、LPS刺激(B)组和对照(C)组,应用台盼兰拒染法检测蟾蜍灵对GMC的细胞毒性作用,MTT观察LPS诱导的GMC增殖的变化,RT-PCR检测TGF-β1和IL-6 mRNA的相对表达.结果 与B组相比,A组蟾蜍灵10-8mol/L和10-7mol/L明显抑制GMC增殖,A492nm值分别为[(0.818±0.058)和(0.574±0.182),P<0.01].B组IL-6和TGF-β1 mRNA相对表达量较C组和A组明显减少(P<0.01).结论 蟾蜍灵明显抑制LPS诱导的大鼠系膜细胞增殖及其IL-6和TGF-β1的表达.     

脂多糖对人肠微血管内皮细胞水通道蛋白1表达及功能的影响

目的 研究脂多糖(LPS)对人肠微血管内皮细胞(HIMEC)水通道蛋白(AQP)1表达及功能的影响.方法 40份HIMEC均分为A、B、C、D四组.A、B、C组分别经浓度为0.1、1、10 mg/L的LPS作用;D组为对照组,只加培养基.各组细胞培养12 h.Western blot法检测HIMEC AQP-1的表达;RT-PCR检测HIMEC AQP-1 mRNA的表达;放射法测定HIMEC的摄水功能.结果 随着作用于HIMEC的LPS浓度增加,AQP-1蛋白及其mRNA表达和HIMEC的摄水功能均逐步减少(P＜0.05).结论 HIMEC AQP-1表达与LPS浓度呈负性相关.     

谷氨酰胺对内毒素诱导大鼠肺组织氧化应激反应的影响

目的探讨谷氨酰胺对内毒素(LPS)诱导大鼠肺组织氧化应激反应的影响。方法雄性SD大鼠50只,随机分为5组(n=10):对照组(A组);LPS组(B组),股静脉注射LPS 5 mg/kg;C、D、E组为谷氨酰胺+LPS组,分别于LPS注入前1 h时,LPS注入同时,LPS注入后1 h时,均静脉注射谷氨酰胺0.75 g/kg。于注射LPS后4 h时处死大鼠,测定肺组织超氧化物歧化酶(SOD)活性、丙二醛(MDA)、一氧化氮(NO)、总一氧化氮合酶(tNOS)水平,诱导型一氧化氮合酶(iNOS)活性及iNOS mRNA表达。结果与A组比较,B组肺组织SOD活性下降,MDA、NO、tNOS水平、iNOS活性及iNOS mRNA表达均升高(P<0.05及P<0.01);与B组比较,C、D组肺组织SOD活性升高,MDA、NO、tNOS水平、iNOS活性及iNOS mRNA表达降低(P<0.05及P<0.01),E组上述各项指标比较差异无统计学意义(P>0.05)。结论谷氨酰胺早期给药可减轻LPS诱导肺组织氧化应激反应。     

脂联素干预TNF-α作用后的巨噬细胞脂联素受体mRNA表达水平及其吞噬脂质变化

目的探讨脂联素干预TNF-α作用后的巨噬细胞表面脂联素受体mRNA表达水平及其吞噬脂质的变化。方法单核巨噬细胞系用佛波酯诱导,使之分化为巨噬细胞;进而采用不同浓度的TNF-α(0、0.1、1、10、100ng/ml)作用24h(以TNF-α0ng/ml为空白对照组),采用RT-PCR技术观察巨噬细胞表面脂联素受体R1、R2mRNA表达水平。选择0、10ng/ml浓度TNF-α作用后的巨噬细胞,用0.2mg/ml低密度脂蛋白混悬生长液作用24h,油红O染色和比色定量检测巨噬细胞吞噬脂质变化;同时TNF-α作用后的巨噬细胞,进一步用人重组脂联素20μg/ml干预24h,再次观察巨噬细胞表达脂联素受体mRNA水平及吞噬脂质变化。结果巨噬细胞用不同浓度的TNF-α作用后,随着TNF-α浓度梯度增加,脂联素受体R1、R2mRNA表达水平逐渐下降(P<0.05)。TNF-α10ng/ml作用后的巨噬细胞吞噬脂质的量明显高于TNF-α空白对照组(P<0.05);同时10ng/mlTNF-α作用后的巨噬细胞,进一步用脂联素干预,巨噬细胞表达脂联素受体R1、R2mRNA水平均较干预前上调(P<0.05),吞噬脂质的量较干预前减少(P<0.05)。结论脂联素及其受体在调节巨噬细胞吞噬脂质过程中产生重要作用,该作用可能成为预防和治疗动脉粥样硬化发生发展的新靶标。     

Toll-like receptor priming sensitizes macrophages to proinflammatory cytokine gene induction by deoxynivalenol and other toxicants.

Activation of the innate immune system might predispose a host to toxicant-induced inflammation. In vitro macrophage models were employed to investigate the effects of preexposure to Toll-like receptor (TLR) agonists on induction of proinflammatory cytokine gene expression by the trichothecene mycotoxin deoxynivalenol (DON) and other toxicants. Priming of the murine RAW 264.7 macrophage line or peritoneal murine macrophages with the TLR4 agonist lipopolysaccharide (LPS) at 100 ng/ml for 4, 8, and 16 h significantly increased DON-induced IL-1beta, IL-6, and TNF-alpha mRNA expression as compared to LPS or DON alone. The minimum LPS concentration for sensitization of both cell types was 1 ng/ml. LPS priming also potentiated IL-1beta mRNA induction by DON in human whole-blood cultures, suggesting the relevance of the murine findings. As observed for LPS, preexposure to TLR agonists including zymosan (TLR2), poly (I:C) (TLR3), flagellin (TLR5), R848 (TLR7/8), and ODN1826 (TLR9) sensitized RAW 267.4 cells to DON-induced proinflammatory gene expression. Amplified proinflammatory mRNA expression was similarly demonstrated in LPS-sensitized RAW 264.7 cells exposed to the microbial toxins satratoxin G, Shiga toxin, and zearalenone as well as the anthropogenic toxicants nickel chloride, triphenyltin, 2,4-dinitrochlorobenzene, and 2,3,7,8-tetrachlorodibenzodioxin. The results suggest that prior TLR activation might render macrophages highly sensitive to subsequent induction of proinflammatory gene expression by xenobiotics with diverse mechanisms of action.     

The influence of ezetimibe on classical and alternative activation pathways of monocytes/macrophages isolated from patients with hypercholesterolemia

Macrophages are crucial for the development of atherosclerotic plaques. Classically activated macrophages contribute to plaque growth and destabilization, while alternatively activated macrophages increase plaque stability. Here, we assessed the influence of ezetimibe on the activation of monocyte-derived macrophages isolated from patients with hypercholesterolemia (total cholesterol 263.4?±?12.5 mg/dl, low-density lipoprotein cholesterol 179.7?±?11.3 mg/dl, triglycerides 123.9?±?11.4 mg/dl). Cells were stimulated with 1 μg/ml lipopolysaccharide (LPS) or 1 μg/ml LPS plus 22 ng/ml ezetimibe. Control cells were left unstimulated. The expression of classical activation markers (interleukin-1β (IL-1β), nitric oxide (NO), and inducible nitric oxide synthase (iNOS)) and alternative activation markers (mannose receptor (MR) and arginase-1 (Arg1)) was determined after 48 h. The employed analytical methods included enzyme-linked immunosorbent assay, Griess reaction, real-time polymerase chain reaction, and Western blotting. LPS increased the secretion of IL-1β and NO and the expression of iNOS mRNA, iNOS protein, and Arg1 protein. It did not affect the expression of MR or Arg1 mRNA. In comparison to LPS stimulation, co-stimulation with ezetimibe decreased the secretion of IL-1β and the expression of iNOS mRNA and protein, while it increased MR mRNA and protein expression. Co-stimulation with ezetimibe did not change the secretion of NO or the expression of Arg1. The results suggest that ezetimibe in inflammatory in vitro conditions contributes to the suppression of classical and promotion of the alternative macrophage activation.     

Suppressive activity of fexofenadine hydrochloride on the production of eosinophil chemoattractants from human nasal fibroblasts in vitro

The influence of fexofenadine hydrochloride (FEX; CAS 138452-21-8) on the production of eosinophil chemoattractants, RANTES and eotaxin, from nasal polyp fibroblasts (NPFs) was examined in vitro. Seventh to tenth generation NPFs were cultured with or without 1 microg/ml lipopolysaccharide (LPS) in the presence of various concentrations of FEX. After 24 h, the culture supernatants were obtained and assayed for eosinophil chemoattractants by enzyme-linked immunosorbent assay (ELISA). FEX at a dose of more than 250 ng/ml (but not 100 ng/ml) inhibited RANTES and eotaxin production in response to LPS stimulation, when the agent was added at starting of cell cultures. FEX also showed suppressive effect on RANTES and eotaxin production from NPFs after stimulation with tumor necrosis factor alpha (TNF-alpha). However, the addition of FEX at 12 h after culture could not inhibit factor production. The influence of FEX on messenger RNA (mRNA) expression in NPFs was then examined. The addition of FEX at 100 ng/ml scarcely affected the expression of RANTES and eotaxin mRNA. On the other hand, 250 ng/ ml of FEX significantly inhibited these mRNA expressions that were enhanced by LPS stimulation.     

Differential upregulation of TNF-alpha, IL-6, and IL-8 production by deoxynivalenol (vomitoxin) and other 8-ketotrichothecenes in a human macrophage model.

The effects of deoxynivalenol (DON or vomitoxin) and four closely related 8-ketotrichothecenes on proinflammatory cytokine and chemokine production were evaluated in a clonal human macrophage model. U-937 cells, which represent a human monocytelike histocytic lymphoma, were differentiated into macrophages by preincubation with phorbol 12-myristate 13-acetate (PMA). Differentiated macrophages were incubated with DON in the absence or presence of lipopolysaccharide (LPS), and supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA) for the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), and for the chemokine interleukin-8 (IL-8). In the absence of LPS, DON at 500 or 1,000 ng/ml upregulated TNF-alpha production as early as 3 h and up to 6 h, whereas 100 to 1,000 ng/ml of DON significantly increased production of IL-6 from 3 to 24 h and IL-8 from 6 to 48 h. In cells costimulated with 0.2 microg/ml LPS, DON at 500 or 1000 ng/ml markedly superinduced TNF-alpha and IL-8 production. Although 100 ng/ml of DON also potentiated LPS-induced IL-6 production, 500 or 1,000 ng/ ml of the toxin suppressed the LPS-induced IL-6 response. Four other 8-ketotrichothecenes, fusarenon X, nivalenol, 3-acetyl DON, and 15-acetyl DON, were also capable of upregulating or suppressing TNF-alpha, IL-6, and IL-8 production at concentrations similar to that of DON. In total, the results suggest that DON and other 8-ketotrichothecenes have the potential to both directly induce and superinduce proinflammatory cytokine and chemokine expression in human macrophages, even at toxin concentrations that are cytotoxic.     

丙泊酚对体外内毒素激活人中性粒细胞NF-κB亚单位p65蛋白的影响

目的观察丙泊酚对内毒素(LPS)激活后人体中性粒细胞(PMN)NF-κB p65的表达及对PMN凋亡的影响。方法采集20例健康志愿者外周静脉血,分离PMN后分为五组:C组,加入生理盐水作为对照;LPS组,加入LPS 100ng/L);P1组,LPS+丙泊酚2μg/ml;P2组,LPS+丙泊酚4μg/ml;P3组,LPS+丙泊酚8μg/ml。孵育2.5h后,提取RNA;用RT-PCR法检测p65mRNA,Western blot检测p65蛋白含量,流式细胞术检测PMN凋亡。结果 LPS 100ng/L可以显著激活PMN,使p65表达大量增加。实验剂量的丙泊酚均可不同程度抑制LPS激活后PMN p65的表达(P<0.05);2-8μg/ml浓度的丙泊酚可剂量依赖性增加LPS导致的PMN凋亡(P<0.05或P<0.01)。结论丙泊酚2-8μg/ml可抑制PMN p65的激活,促进LPS激活后的PMN凋亡。     

AFLATOXIN B1-INDUCED SUPPRESSION OF NITRIC OXIDE PRODUCTION IN MURINE PERITONEAL MACROPHAGES

Aflatoxin B1 (AFB1), a potent hepatocarcinogen, is known to impair specific and nonspecific immune responses. AFB1 mainly decreases lymphocyte functions and may also affect macrophages assisting lymphocyte functions. Macrophages play an important role in a host defense against tumors and bacteria. Furthermore, some macrophage products, including nitric oxide (NO), may be involved in cytotoxicity. The effect of aflatoxin B1 (AFB1) was investigated on NO production from murine peritoneal macrophages. Macrophages were pretreated with AFB1 for 24 h and then stimulated with lipopolysaccharide (LPS) for 24 h. AFB1 at 10 or 50 mu M reduced the production of NO. Compared to vehicle control, there was a greater reduction of NO production with increased AFB1 pretreatment and LPS stimulation. AFB1 at 10 or 50 mu M decreased inducible nitric oxide synthase (iNOS) activity about 24% and 28%, respectively, after stimulation with 1 mug/ml LPS and about 12% and 24%, respectively, after stimulation with 10 mug/ml LPS. AFB1 pretreatment also decreased the synthesis of iNOS protein and the mRNA of macrophages. Taken together, these results suggest that AFB1 pretreatment reduces NO production from murine peritoneal macrophages stimulated by LPS, which is mediated by the reduction of iNOS activity, mRNA, and protein.     

Nicotine enhancement of lipopolysaccharide/interferon-gamma-induced cytotoxicity with elevating nitric oxide production

Nicotine has been shown to induce relaxation via nitric oxide (NO) production with activation of endothelium nitric oxide synthase (eNOS), however the effect of nicotine on lipopolysaccharide/interferon-gamma (LPS/IFN-gamma)-induced NO production and inducible NOS (iNOS) gene expression is still undefined. Here, nicotine alone did not affect the NO and PGE2 production in RAW264.7 and primary peritoneal macrophages. Interestingly, nicotine showed the dose-dependent stimulatory effect on LPS (20 ng/ml)/IFN-gamma (10 ng/ml)-induced NO but not PGE2 production in both cells. Although nicotine stimulates NO production in the presence of LPS/IFN-gamma, LPS at the dose of 20 ng/ml, nicotine showed no obvious inductive effect on the expression of iNOS protein by Western blotting in both cells. However, nicotine significantly stimulates LPS (2.5, 5 ng/ml)/IFN-gamma (10 ng/ml)-induced iNOS expression and NO production in RAW264.7 cells. Cytotoxicity assay showed that nicotine enhanced LPS (20 ng/ml) and IFN-gamma (10 ng/ml)-induced cytotoxicity, which was inhibited by an NOS inhibitor N-nitro-L-arginine (NLA) in RAW264.7 cells. Direct and indirect NOS activity assays indicated that nicotine did not affect NOS activity. And, iNOS protein stability was not changed by nicotine after LPS/IFN-gamma treatment. These data indicates that nicotine may potentiate LPS/IFN-gamma-induced cytotoxic effects by enhancing NO production; enhancing iNOS gene expression induced by LPS/IFN-gamma is involved. A cross-talk between inflammation and smoking was proposed in the present study.     

Induction of secretory and tumoricidal activities in peritoneal macrophages by ginsan

The immunomodulatory effect of ginsan based on the production of cytokines and the activation of macrophage was studied. Murine peritoneal macrophages (PM) on in vitro treatment with ginsan isolated from Panax ginseng induced mRNA of cytokines such as tumor necrosis factor (TNF)-alpha, interleukin-1 (IL-1)beta, interleukin-6 (IL-6) and interleukin-12 (IL-12); TNF-alpha mRNA induction was maximum within 3 h, IL-6 mRNA was gradually induced up to 24 h, and IL-1beta and IL-12 mRNA were highly induced at 24 h. IL-1beta and IL-6 protein levels also increased within 24 h in a dose-dependent manner and reached a maximum with 100 microg/ml ginsan. IL-12 was induced after 3 days and a high level of induction was detected after 4 days post treatment. Ginsan enhanced the lytic death of L929 cells through TNF-alpha activation. The mRNA expression of nitric oxide synthase (iNOS) was highly induced after 24 h treatment of ginsan, and then NO production was maximum after 48-h treatment with a low dose of 1 microg/ml. The level of iNOS mRNA induction by ginsan was slightly less than that of macrophages activating agents such as LPS plus IFN-gamma. The tumoricidal activity of macrophage cultured with ginsan on Yac-1 cells was enhanced in a dose-dependent manner; growth inhibition increased 1.6-fold with 100 microg/ml ginsan. These results suggest that ginsan exerts as an effective immunomodulator and enhances antitumor activity of macrophages.