CCK-8对内毒素休克大鼠肺泡巨噬细胞吞噬及p38MAPK表达的影响

目的研究八肽胆囊收缩素(CCK-8)对脂多糖(LPS)引起的内毒素休克(ES)大鼠肺泡巨噬细胞吞噬功能、支气管肺泡灌洗液(BALF)中MDA含量变化以及大鼠肺泡巨噬细胞p38MAPK表达的改变.方法使用生理多道记录仪,观察尾静脉注入LPS(8mg/kgiv)复制的大鼠ES模型、LPS注入前10min尾静脉注入CCK-8(40μg/kgiv)、单独注入CCK-8(40μg/kgiv)或生理盐水(对照)的四组大鼠血压的改变,于LPS注入2h进行支气管肺泡灌洗,获BALF,从中分离肺泡巨噬细胞,检测吞噬白色念珠菌能力的改变,以吞噬率和吞噬指数表示;分离正常大鼠肺泡巨噬细胞,体外刺激30min后,免疫组化观察p38MAPK表达的变化.结果[HTSS〗CCK-8可逆转LPS引起的大鼠平均动脉血压的下降.LPS注入2h肺泡巨噬细胞吞噬能力显著增强,吞噬指数由对照组的2.43±0.41增加到3.80±0.60(P＜0.05),CCK-8抑制LPS诱导的肺泡巨噬细胞吞噬能力增强,但与对照组相比,CCK-8可增强吞噬能力.LPS组BALF中MDA含量显著增加(P＜0.05),CCK-8±LPS组MDA含量明显低于LPS组.免疫组化显示LPS可激活p38MAPK,CCK-8可增强p38MAPK的表达.讨论和结论LPS致BALF中MDA含量增高,提示LPS刺激下肺泡巨噬细胞激活,吞噬功能增强,引起一系列过强的炎症反应.CCK-8可明显减少BALF中MDA含量,抑制LPS引起的巨噬细胞吞噬功能的增强,但本身可增强巨噬细胞吞噬功能,其机制可能与p38MAPK途径有关.p38MAPK为多种信号传导途径的交汇点.CCK-8可能通过激活p38MAPK途径调节ES大鼠的免疫功能,二者可能存在微妙的平衡.     

没食子儿茶素没食子酸酯对脂多糖激活小鼠巨噬细胞系p38MAPK信号通路的作用研究

目的:探讨没食子儿茶素没食子酸酯对巨噬细胞中由脂多糖(LPS)激活的p38MAPK的作用特性及对肿瘤坏死因子(TNF-α)表达的影响。方法:在体外培养的小鼠巨噬细胞系中用Westernblotting检测p38MAPK磷酸化水平,应用酶联免疫吸附法检测巨噬细胞表达TNF-α的水平,利用电镜观察EGCG对LPS结构的影响。结果:LPS刺激巨噬细胞引起p38MAPK磷酸化程度和TNF-α表达明显增高,EGCG对LPS激活的p38MAPK磷酸化和TNFα的表达有明显的抑制作用,EGCG对LPS的结构无显著影响。结论:EGCG对LPS无直接的拮抗作用,而是通过干预体内信号通路发挥其抑制作用,p38MAPK可能是EGCG抑制LPS诱导巨噬细胞表达TNF-α的重要通路之一。     

金雀异黄素对脂多糖诱导的巨噬细胞MAPK和TLR信号转导通路的影响

目的研究金雀异黄素对脂多糖(LPS)诱导的巨噬细胞丝裂原活化蛋白激酶(MAPK)信号转导通路和Toll样受体(TLR)通路的影响。方法用100 ng/mL脂多糖和金雀异黄素分别处理RAW264.7巨噬细胞不同时间后,采用Western blot法检测对MAPK信号通路蛋白磷酸化的影响;采用RT2 ProfilerTMPCR芯片检测金雀异黄素对LPS诱导的TLR信号转导通路基因表达的影响。结果 LPS能够显著诱导蛋白p38和p42/44磷酸化,激活MAPK信号通路,金雀异黄素能够加强其作用,同时,LPS能够显著诱导TLR信号转导通路的细胞因子基因表达,包括IFN-β、IL-10、IL-1α、IL-1β、IL-6、TNF-α、集落刺激因子2(CSF-2)、CSF-3、趋化因子CCL2和CXCL10、环氧合酶2(COX-2)、NF-κB1和IκB-α等,金雀异黄素能够显著降低这些上调基因的表达。结论金雀异黄素能够显著增强LPS激活的MAPK信号通路并抑制TLR信号通路的激活。     

异丙酚通过NLRP3/IL-1β信号通路影响脂多糖诱导的肺泡巨噬细胞M1/M2表型分化

目的研究异丙酚对脂多糖诱导的肺泡巨噬细胞的免疫应答影响及相关机制进行初步探讨。方法分离大鼠原代肺泡巨噬细胞(AMs),并将细胞分为4组:对照组(control组:常规培养AMs),脂多糖组(LPS组:加入终质量浓度为1 000 ng/L脂多糖),异丙酚组(P组:加入终浓度为25μmol/L异丙酚),脂多糖+异丙酚组(LPS+P组:加终质量浓度为1 000 ng/L的LPS和25μmol/L异丙酚)。4组细胞常规培养24 h后,应用ELISA检测4组细胞培养上清液中IL-1β、IL-10、TNF-α的含量;细胞流式实验检测4组细胞表面F4/80、CD16/32、CD206分子表型所占比例;免疫磁珠分选F4/80+细胞,并利用RT-PCR检测细胞中iNOS、CD206的mRNA表达变化;最后应用RT-PCR及Western blot实验检测4组细胞中NLRP3/IL-1β信号通路中NLRP3、IL-1β及Caspase-1的mRNA及蛋白表达变化。结果 ELISA实验显示,异丙酚能够明显抑制LPS所诱导的AMs对促炎因子IL-1β、TNF-α的分泌,且促进细胞对抑炎因子IL-10的分泌;细胞流式及RT-PCR实验结果显示,异丙酚能够明显抑制LPS所诱导的AMs向M1型巨噬细胞分化,并促进细胞向M2型巨噬细胞分化;RT-PCR及Western blot实验结果显示,异丙酚能够明显抑制LPS所诱导的AMs中NLRP3/IL-1β信号通路中NLRP3、IL-1β及Caspase-1的m RNA及蛋白表达。结论异丙酚能够通过下调NLRP3/IL-1β信号通路抑制LPS所诱导的AMs向M1表型的分化,并促进细胞向M2表型的分化。     

CpG寡聚脱氧核苷酸(CpG ODN)协同促进脂多糖诱导的小鼠巨噬细胞增殖与迁移

目的探讨CpG寡聚脱氧核苷酸(CpG ODN)对脂多糖(LPS)诱导的巨噬细胞增殖与迁移能力的影响及机制。方法使用1 mg/L LPS处理小鼠RAW264.7巨噬细胞建立体外炎症细胞模型,采用CCK-8法检测CpG ODN(500 nmol/L)对LPS诱导的巨噬细胞增殖的影响,采用Transwell~(TM)实验检测CpG ODN对细胞迁移能力的影响;采用Western blot法检测p38丝裂原激活蛋白激酶(MAPK)、 c-Jun氨基末端激酶(JNK)、胞外信号调节激酶(ERK)、核因子κBp65(NF-κB p65)的蛋白磷酸化水平,同时使用以上通路的抑制剂SB203580、 SP600125、 PD98059、 BAY11-7082,探讨CpG ODN发挥效应的机制;采用实时定量PCR检测CpG ODN对LPS诱导产生的单核细胞趋化蛋白1(MCP-1)、环加氧酶2(COX2)mRNA水平的影响。结果 CpG ODN协同促进LPS诱导的巨噬细胞增殖与迁移,并促进COX2、 MCP-1的转录,增强JNK、 ERK信号通路蛋白的磷酸化水平,并且JNK与ERK信号通路激酶抑制剂可有效降低CpG ODN的协同效应。结论 CpG ODN可通过JNK与ERK途径协同促进LPS诱导的巨噬细胞增殖与迁移并促进COX2、 MCP-1的转录。     

血管活性肠肽对肺泡巨噬细胞活化和p38MAPK信号通路影响的实验研究

目的 研究血管活性肠肽（vasoactive intestinal peptide,VIP）对肺泡巨噬细胞活化的影响。方法 原代小鼠肺泡巨噬细胞分为Normal组（正常对照）、LPS组（脂多糖刺激巨噬细胞活化）、LPS+VIP组(脂多糖刺激巨噬细胞活化后,10^-6mol/L VIP干预)。3 C57BL/6小鼠随机分为Normal组（正常对照）,ALI组（急性肺损伤）,ALI+VIP组(急性肺损伤后,10^-8mol/L VIP气管滴入)。免疫荧光法检测肺泡巨噬细胞标记物CD86和白细胞介素-6（interleukin-6,IL-6）的表达;ELISA法检测细胞培养上清和血清中肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、IL-6的含量;RT-PCR法检测巨噬细胞和肺组织内TNF-α、IL-6、p38丝裂原活化蛋白激酶（mitogen activated protein kinase, MAPK）mRNA的表达水平;Western blot法检测各组巨噬细胞和肺组织内TNF-α、IL-6、p38MAPK、p-p...     

血管活性肠肽对肺泡巨噬细胞活化和p38MAPK信号通路影响的实验研究

目的 研究血管活性肠肽（vasoactive intestinal peptide,VIP）对肺泡巨噬细胞活化的影响。方法 原代小鼠肺泡巨噬细胞分为Normal组（正常对照）、LPS组（脂多糖刺激巨噬细胞活化）、LPS+VIP组(脂多糖刺激巨噬细胞活化后,10^-6mol/L VIP干预)。3 C57BL/6小鼠随机分为Normal组（正常对照）,ALI组（急性肺损伤）,ALI+VIP组(急性肺损伤后,10^-8mol/L VIP气管滴入)。免疫荧光法检测肺泡巨噬细胞标记物CD86和白细胞介素-6（interleukin-6,IL-6）的表达;ELISA法检测细胞培养上清和血清中肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）、IL-6的含量;RT-PCR法检测巨噬细胞和肺组织内TNF-α、IL-6、p38丝裂原活化蛋白激酶（mitogen activated protein kinase, MAPK）mRNA的表达水平;Western blot法检测各组巨噬细胞和肺组织内TNF-α、IL-6、p38MAPK、p-p...     

三七皂苷Rg1抑制脂多糖诱导小胶质细胞激活的机制研究

目的:探讨中药有效成分三七皂苷Rg1(Ginsenoside Rg1,Rg1)对抑制脂多糖(lipopolysaccharide,LPS)诱导的小胶质细胞株BV-2细胞激活的机制。方法:用LPS刺激BV-2细胞构建激活模型,采用四甲基偶氮唑蓝比色法(MTT)检测Rg1对BV-2细胞的活力影响,蛋白质免疫印迹(Western Blot)方法检测不同浓度Rg1(10、20和40μmol/L)对磷酸化的核因子-κB抑制蛋白-α(inhibitorκB-α,IκB-α)和反应结合蛋白(cAMP-responseelement binding protein,CREB)以及促分裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)家族的细胞外信号调节激酶(extracellular signal-regulated kinase 1/2,ERK1/2)、c-Jun氨基端激酶(c-Jun N-terminal kinase,JNK)和p38促分裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)等细胞信号通路蛋白的表达及其变化规律。结果:不同浓度的Rg1明显抑制了LPS诱导的磷酸化IκB-α和CREB蛋白表达以及MAPKs通路(ERK1/2,JNK,p38 MAPK)磷酸化蛋白表达,并且对p38 MAPK表达的影响呈剂量依赖性。结论:Rg1可能通过抑制MAPKs的磷酸化来调控LPS诱导的小胶质细胞株BV-2细胞激活,发挥其神经抗炎的作用。     

p38蛋白激酶对大鼠肺泡巨噬细胞活化机制的调控

目的:探讨p38蛋白激酶对LPS诱导大鼠肺泡巨噬细胞激活机制的作用。方法:提取细胞核蛋白,采用Western印迹分析p38蛋白激酶水平。用放射免疫法检测细胞上清TNF-α、IL-8的含量。结果:LPS显著增加肺泡巨噬细胞TNF-α、IL-8的合成,呈剂量依赖性诱导p38的活化。特异性p38蛋白激酶抑制剂SB203580能显著降低LPS诱导的肺泡巨噬细胞核蛋白p38的含量及细胞上清TNF-α、IL-8水平。结论:LPS刺激肺泡巨噬细胞释放炎性细胞因子TNF-α、IL-8受p38蛋白激酶调控。     

LPS触发的TLR4对NF-κB和IRF3信号通路的调控差异

目的探讨脂多糖(LPS)触发的Toll样受体4(TLR4)对NF-κB和干扰素调节因子3(IRF3)信号通路的调控差异。方法 LPS刺激小鼠原代腹腔巨噬细胞,用免疫荧光法检测TLR4及两种转录因子p65及IRF3的定位,用Western blot方法检测转录因子p65和IFR3的磷酸化水平。结果巨噬细胞经LPS刺激30 min内,细胞膜上的TLR4荧光强度增加(P0.01),胞质中TLR4荧光强度显著增加(P0.01),且与早期内体抗原1(early endosome antigen 1,EEA1)共定位;当LPS刺激90和180 min时,胞膜和胞质内的TLR4信号均显著下降(P0.01),与EEA1共定位的信号也明显减少(P0.01)。静息状态下,TLR4的下游信号通路分子p65和IRF3的荧光信号均出现在胞质中;LPS刺激后,两者的荧光信号在细胞核中逐渐增加(P0.05),但p65荧光信号比IRF3增强得更早,且更持久。p65磷酸化的修饰也明显早于IRF3,且持续时间更长。结论 TLR4活化后的定位变化导致其下游IRF3信号通路的传递时间明显延后,且比NF-κB信号通路短暂。     

Serum factors, cell membrane CD14, and beta2 integrins are not required for activation of bovine macrophages by lipopolysaccharide.

The role of serum factors such as lipopolysaccharide (LPS)-binding protein (LBP) and of macrophage-expressed CD14 and beta2 integrins in the activation of bovine macrophages by LPS was investigated. Macrophage activation was determined by measuring tumor necrosis factor production, NO generation, and upregulation of procoagulant activity by LPS (Escherichia coli O55:B5) at concentrations of 100 pg/ml to 100 ng/ml. The 50% effective dose for LPS was 1 order of magnitude higher than that for activating human macrophages. Macrophages were activated by LPS in the presence of serum or in the presence of albumin demonstrated to be free of LBP. The capacity to react to LPS in the absence of LBP was not due to the acquisition of LBP during a previous culture in serum. It was then established which CD14-specific antibodies block LPS binding to monocytes. Among the CD14-specific antibodies recognizing bovine mononuclear phagocytes (60bca, 3C10, My4, CAM36, VPM65, CMRF31, and TUK4), the first four blocked the binding of LPS-fluorescein isothiocyanate to bovine monocytes at low concentrations. Anti-CD14 antibodies did not block LPS-mediated activation of bovine bone marrow-derived macrophages, monocyte-derived macrophages, and alveolar macrophages. This was observed in experiments in which anti-CD14 concentrations exceeded the 50% inhibitory dose by >30-fold (3C10 and My4) or >300-fold (60bca), as defined in the binding assay described above. Monocyte-derived macrophages from an animal deficient in beta2 integrins and control macrophages were activated by similar concentrations of LPS, suggesting that beta2 integrins are not important bovine LPS receptors. Thus, in bovine macrophages, LPS recognition pathways which are independent of exogenous LBP, of membrane-expressed CD14, and of beta2 integrins may exist.     

Surfactant protein a inhibits lipopolysaccharide-induced immune cell activation by preventing the interaction of lipopolysaccharide with lipopolysaccharide-binding protein

Pulmonary surfactant protein (SP)-A, an innate immune molecule, modifies lipopolysaccharide (LPS)-induced cell responses. Because SP-A avidly binds to the deep rough (Re) mutant of LPS, we first investigated the functional consequences of this interaction and found that preincubation of Re-LPS with SP-A significantly and in a dose-dependent manner decreased the sensitivity of rat alveolar macrophages and human mononuclear cells to Re-LPS-induced activation at limited amounts of LPS-binding protein (LBP). At high LBP concentrations, the SP-A-mediated cellular inhibition of Re-LPS-induced activation was abrogated. Because LBP-catalyzed binding of LPS to CD14 is essential for low-dose LPS-induced signaling, we then hypothesized that SP-A inhibits Re-LPS-induced immune cell activation via inhibiting the binding of Re-LPS to LBP. Binding competition experiments employing a surface plasmon resonance technique showed that Re-LPS preincubated with SP-A bound to LBP to a significantly lesser extent than Re-LPS alone. For enhanced cellular association of [(3)H]LPS/SP-A complexes to occur, the expression of membrane-bound CD14 by human embryonic kidney cells 293 was not essential. Therefore, the ability of SP-A to inhibit immune cell activation by Re-LPS may be due to its ability to block the binding of Re-LPS to LBP and prevent the initiation of the LBP/CD14 pathway for inflammatory reactions in the lung.     

Roles for tumor necrosis factor alpha and nitric oxide in resistance of rat alveolar macrophages to Legionella pneumophila.

Legionella pneumophila is an intracellular parasite of alveolar macrophages, and recovery from legionellosis is associated with activation of alveolar macrophages to resist intracellular bacterial replication. Gamma interferon (IFN-gamma) is known to activate alveolar macrophages to suppress L. pneumophila, but the role of macrophage-derived cytokines in modulating alveolar macrophage resistance is unknown. To test the hypothesis that macrophage-derived mediators contribute to the resistance of alveolar macrophages to L. pneumophila, we incubated adherent rat alveolar macrophages with Escherichia coli lipopolysaccharide (LPS), recombinant tumor necrosis factor alpha (TNF-alpha), recombinant IFN-gamma, neutralizing anti-TNF-alpha, and/or N(G)-monomethyl-L-arginine (L-NMMA) for 6 h before challenge with L. pneumophila. Monolayers were sonically disrupted and quantitatively cultured on successive days. We also measured bioactive TNF-alpha release by infected macrophages in the presence or absence of IFN-gamma. We found that pretreatment of alveolar macrophages with LPS or, to a lesser degree, TNF-alpha, significantly inhibited intracellular replication of L. pneumophila. Both LPS and TNF-alpha acted synergistically with IFN-gamma at less than the maximally activating concentration to suppress L. pneumophila growth. The independent and coactivating effects of LPS were blocked by anti-TNF-alpha. Killing of L. pneumophila by IFN-gamma at the maximally activating concentration was inhibited by anti-TNF-alpha. The synergistic effects of TNF-alpha. or LPS in combination with IFN-gamma were inhibited by L-NMMA. Infected alveolar macrophages secreted TNF-alpha in proportion to the bacterial inoculum, and secretion of TNF-alpha was potentiated by cocultivation with IFN-gamma. These data indicate that secretion of TNF-alpha is an important autocrine defense mechanism of alveolar macrophages, serving to potentiate the activating effects of IFN-gamma through costimulation of nitric oxide synthesis.     

Prophylactic effect of JTE-607 on LPS-induced acute lung injury in rats with CINC-1 inhibition

OBJECTIVE AND DESIGN: JTE-607, a multiple cytokine inhibitor, was evaluated in lipopolysaccharide (LPS)-induced acute lung injury in rats in vivo and in vitro. MATERIALS AND METHODS: LPS instillation into airways of rats was performed. JTE-607 at 3-30 mg/kg and dexamethasone at 3 mg/kg were administered intravenously at 10 min and 0 min for JTE-607, and 60 min for dexamethasone prior to the LPS instillation (n = 8). Cytokine-induced neutrophil chemoattractant (CINC)-1 level and myeloperoxidase (MPO) activity in lung were measured at 4 h after LPS instillation, and at 24 h for lung wet weight measurement and histological study. LPS-induced CINC-1 production by rat alveolar macrophages were also measured in vitro. RESULTS: JTE-607 and dexamethasone showed a significant reduction of increased CINC-1 level and MPO activity in lung after LPS treatment in vivo. Increased wet weight was also significantly inhibited. Histological studies revealed that JTE-607 and dexamethasone significantly inhibited LPS-induced accumulation of peribronchial neutrophils and eosinophils, and perivascular edema. JTE-607 and dexamethasone suppressed CfNC-1 synthesis by rat alveolar macrophages in vitro with IC50 values of 12.4 microM and 2.3 nM, respectively. CONCLUSIONS: These results indicate that JTE-607 has an inhibitory effect on LPS-induced rat lung inflammation in parallel with CINC-1 reduction. The effect of JTE-607 was suggested to be through direct inhibition of CINC-1 production from rat alveolar macrophages. JTE-607 may thus be efficacious in cytokine-mediated lung inflammation such as acute respiratory distress syndrome.     

盐酸戊乙奎醚抑制脂多糖致急性肺损伤大鼠肺组织p38丝裂原活化蛋白激酶的活化

目的:观察盐酸戊乙奎醚(PHC)对脂多糖(LPS)致急性肺损伤(ALI)大鼠肺组织p38丝裂原活化蛋白激酶(p38MAPK)、c-jun氨基末端激酶(JNK)活化的影响。方法:SD大鼠随机分为对照组、LPS模型组（5 mg/kg LPS,iv）和LPS+PHC高、中、低(3.0、1.0和0.3 mg/kg)3个剂量组,每组6只,进行PHC对肺组织p38MAPK、JNK表达的量效性分析;另取大鼠在注入NS后即刻0（对照组）和注射LPS后2 h、4 h、6 h和12 h共5个时点,每时点6只,进行肺组织p38MAPK、JNK表达的时效性分析。蛋白免疫印迹法检测肺组织p38MAPK、JNK的表达。结果:LPS模型组大鼠肺组织磷酸化p38MAPK、JNK的表达显著高于对照组（P＜0.05);PHC高剂量组显著抑制LPS诱导的大鼠肺组织磷酸化p38MAPK表达（P＜0.05);PHC在造模后6 h时最能有效抑制磷酸化p38MAPK上调。与LPS模型组相比,PHC高、中、低剂量组磷酸化JNK的表达均无显著差异(均P＞0.05);造模后不同时点,PHC对磷酸化JNK的表达均无抑制作用。结论:PHC抑制LPS诱导的ALI大鼠肺组织p38MAPK活化,但不能抑制JNK活化,PHC对LPS诱导大鼠ALI的拮抗作用可能与其抑制p38MAPK的活化有关。     

Propofol Protects Rats and Human Alveolar Epithelial Cells Against Lipopolysaccharide-Induced Acute Lung Injury via Inhibiting HMGB1 Expression

High-mobility group box 1 (HMGB1) plays a key role in the development of acute lung injury (ALI). Propofol, a general anesthetic with anti-inflammatory properties, has been suggested to be able to modulate lipopolysaccharide (LPS)-induced ALI. In this study, we investigated the effects of propofol on the expression of HMGB1 in a rat model of LPS-induced ALI. Rats underwent intraperitoneal injection of LPS to mimic sepsis-induced ALI. Propofol bolus (1, 5, or 10 mg/kg) was infused continuously 30 min after LPS administration, followed by infusion at 5 mg/(kg?·?h) through the left femoral vein cannula. LPS increased wet to dry weight ratio and myeloperoxidase activity in lung tissues and caused the elevation of total protein and cells, neutrophils, macrophages, and neutrophils in bronchoalveolar lavage fluid (BALF). Moreover, HMGB1 and other cytokine levels were increased in BALF and lung tissues and pathological changes of lung tissues were excessively aggravated in rats after LPS administration. Propofol inhibited all the above effects. It also inhibited LPS-induced toll-like receptor (TLR)2/4 protein upexpression and NF-κB activation in lung tissues and human alveolar epithelial cells. Propofol protects rats and human alveolar epithelial cells against HMGB1 expression in a rat model of LPS-induced ALI. These effects may partially result from reductions in TLR2/4 and NF-κB activation.