烧伤血清对内皮细胞IκBα降解和NF-κB活化的影响

目的 了解烧伤血清对内皮细胞抑制性κB(IκBα)降解、核因子-κB(NF-κB)活化的影响,进一步探讨烧伤血清诱导内皮细胞活化分泌细胞因子的机制。方法 采用体外培养的内皮细胞,分别用正常人血清(对照组)、烧伤患者血清、烧伤患者血清+吡咯烷二硫代氨基甲酸盐(PDTC)刺激内皮细胞。采用Western印迹法检测血清刺激30、60、90、120min后内皮细胞IκBα蛋白降解情况,电泳迁移率分析检测血清刺激30、60、120、240min后NF-κB活性的变化。结果 烧伤血清刺激内皮细胞后30min,IκBα发生明显降解,刺激后60min达高峰,2h后表达逐渐升高;烧伤血清刺激内皮细胞后30min,NF-κB活性迅速升高,30~60min达高峰,2h后逐渐回复基础状态。PDTC能有效抑制烧伤血清作用条件下内皮细胞IκBα降解、NF-κB活化。结论 烧伤血清可诱导内皮细胞IκBα降解,活化NF-κB,从而在烧伤后内皮细胞分泌细胞因子过程中起重要作用。     

NF-κB/IκB信号通路介导血管紧张素Ⅱ诱导的系膜细胞促炎性细胞因子表达

目的：探讨核因子—κB(NF—κB)／IκB信号通路在血管紧张素Ⅱ(AngⅡ)诱导的肾小球系膜细胞促炎性细胞因子表达中的作用。方法：应用核酸酶保护法检测系膜细胞肿瘤坏死因子α(TNF—α)、IL-1α和IL-1β mRNA表达；应用凝胶电泳迁移率和Western blot检测肾小球系膜细胞中NF—κB活化、p65亚基核转位以及IκBα和IκBβ的降解。结果：正常培养状态下，系膜细胞可组成型表达TNF—α和IL—1β，而不表达IL-1αmRNA，AngⅡ刺激后促炎性细胞因子表达显著上调，NF—κB特异性抑制剂2-硫代氨基甲酸吡咯烷显著抑制AngⅡ诱导的促炎性细胞因子基因表达；AngⅡ诱导系膜细胞NF—κB活化，p65核转位及胞质内IκBα和IκBα的降解。结论：AngⅡ诱导肾小球系膜细胞中促炎性细胞因子表达可能通过NF—κB／IκB信号转导通路来实现。     

多黏菌素B抗炎作用机制的研究

目的观察多黏菌素B(PMB)和内毒素(LPS)共同处理大鼠肺泡巨噬细胞(PAM)后对核因子-κB(NF-κB)信号通路的变化。方法分离、培养大鼠PAM,分为正常对照组、LPS刺激组及PMB干预组。采用原位杂交(ISN)技术、凝胶电泳迁移率改变(EMSA)及ELISA法,检测刺激后0、15、30、60、120和240min各时相点PAM中IKK-βmRNA及IκB-α的表达、NF-κB的活性和TNF-α的含量。结果LPS刺激组IKK-βmRNA的水平在刺激后15min出现升高,30min达高峰,60min后逐渐下降;IκB-α的水平的变化趋势在各时相点与IKK-βmRNA刚好相反;NF-κB活性的峰值相出现在60min,15min出现升高,120min后逐渐下降;TNF-α的含量峰值相出现在60min,30min出现升高,120~240min后恢复至刺激前水平。PMB干预组NF-κB的活性与TNF-α的含量虽较刺激前和正常对照组升高,但均显著低于LPS刺激组(P<0.01)。IκB-α水平的最低值显著高于LPS刺激组(P<0.01);而IKK-βmRNA的峰值则显著低于LPS刺激组(P<0.01)。结论LPS能诱导PAM中的IKK-β激活、IκB-α降解和NF-κB活化,并促进TNF-α释放。而PMB则能抑制LPS诱导的IKK-β激活、IκB-α降解、NF-κB活化和TNF-α释放。     

p38蛋白激酶对脂多糖诱导肺泡巨噬细胞NF-κB活化的调控

目的探讨p38蛋白激酶对脂多糖（LPS）诱导肺泡巨噬细胞NF-κB活化的调控机制。方法分离培养肺泡巨噬细胞。设正常对照组、LPS刺激组、SB203580（p38蛋白激酶抑制剂）＋LPS组、PDTC（NF—κB抑制剂）＋LPS组和SB203580＋PDTC＋LPS组。分别采用免疫细胞化学（SP法）和Western blot检测NF—κB、p38蛋白激酶和NF—κB抑制蛋白（I-κB）的表达。结果与正常对照组相比，LPS刺激肺泡巨噬细胞后胞核NF-κB和p38蛋白激酶染色显著增强，而I—κB的表达显著降低（均P〈0．01）。SB203580、PDTC均可显著降低LPS刺激的肺泡巨噬细胞NF-κB，p38蛋白激酶胞核染色阳性细胞的百分比，并显著增强I—κB的表达（均P〈0．05）。同时应用PDTC和SB203580预孵育肺泡巨噬细胞，与LPS刺激组相比，p38蛋白激酶和NF—κB胞核染色阳性细胞百分比均显著降低（P〈0．05），I-κB的表达显著增强（P〈0．05），但分别与SB203580＋LPS和PDTC＋LPS组相比，差异均无显著性意义（均P〉0．05）。结论LPS刺激肺泡巨噬细胞p38蛋白激酶和NF—κB活化；p38蛋白激酶可能通过调节I-κB降解而调控NF—κB的活化，NF—κB可能是p38信号途径下游的最重要位点。     

肾毒血清肾炎大鼠肾组织中NF-κB活化及其意义

目的：探讨肾毒血清性肾炎大鼠肾组织核因子—κB(NF—κB)活化及其意义。方法：肾毒血清肾炎应用兔抗鼠肾小球基膜肾毒血清制备。应用凝胶电泳迁移率(EMSA)和Western blot检测。肾毒血清。肾炎大鼠。肾组织中NF-κB活化及IκBα和IκBβ的降解；采用核酸酶保护法检测肾组织中IL-8表达，并分析其与NF—κB活化的关系。结果：模型组肾组织中IL-8表达显著高于正常对照组；肾毒血清肾炎大鼠肾组织中NF—κB活化显著增强，p65由胞质转移至咆核．咆质内IκBα和IκBβ降解明显增加；NF-κB活化与IL-8表达呈显著正相关。结论：NF—κB／IκB信号通路介导小球肾炎肾组织中IL-8表达。     

核因子-κB活化对大鼠烧伤早期肺组织表达促炎细胞因子的作用

目的 探讨核因子-κB(NF-κB)在大鼠烧伤早期肺组织表达促炎细胞因子中的作用.方法 采用Wistar大鼠Ⅲ度35%TBSA烧伤模型.实验分正常对照组、烧伤组、烧伤后吡咯烷二硫代氨基甲酸盐干预组(PDTC组).大鼠烧伤后1、3、6、12、24 h凝胶电泳迁移率分析法检测肺组织NF-κB活性;逆转录-聚合酶链式反应检测TNFo、IL-8 mRNA的表达.结果 大鼠烧伤后肺组织NF-κB活性在伤后1 h内即迅速增高,并持续增高到伤后24 h.伤后肺组织TNFα、IL-8mRNA表达逐渐增多,6 h达高峰.PDTC组NF-κB活性降低,肺组织TNFα和IL-8 mRNA表达均较对照组明显减少.结论 严重烧伤可活化肺组织NF-κB,从而介导对细胞因子的合成和释放.提示NF-κB活化在烧伤后脏器组织细胞表达释放细胞因子过程中起重要作用.     

NAC对烧伤大鼠单个核细胞内NF-κB活化及细胞因子mRNA表达的作用

目的：研究烧伤大鼠外周血单个核细胞(PBMC)内NF—κB活化及炎性相关细胞因子mRNA表达的规律，探讨NF—κB在严重烧伤后全身炎症反应中的作用机制。方法：选用SD大鼠制成30％TBSA三度烫伤模型，取全血分离单个核细胞，抽提核蛋白及总RNA，用凝胶电泳迁移率变动分析(EMSA)方法测定NF—κB的活化。用RT—PCR方法检测细胞因子mRNA表达的变化。用N-乙酰-L型半胱氨酸(NAC)阻断NF—κB活化，观察NF—κB对细胞因子表达的影响。结果：烧伤后PBMC内的NF-κB明显活化，在细胞核内积聚明显增加；促炎性细胞因子和Th2型细胞因子mRNA高表达。用NAC后NF—κB的活化水平被抑制，细胞因子表达水平相应地降低。结论：严重烧伤后活性氧(OFRs)至NF—κB的信号转导途径被激活，NF—κB过度活化，促炎性细胞因子失控性高表达。NAC可阻断OFRs至NF-κB信号转导途径，抑制细胞因子的过度表达。烧伤后T辅助细胞出现分化，Th2型细胞占优势，抗炎性细胞因子表达也明显增加。     

NF-κB在缺血再灌注肝脏枯否细胞活化中的作用

目的 探讨缺血再灌注肝脏枯否细胞（KCs）的活化机制。方法 Wistar大鼠随机分为肝缺血30min再灌注组（HIR－30min组）、肝缺血60min再灌注组（HIR－60min组）及对照组。EMSA、ELISA法检测HIR后KCs NK-κB激活及KCs培养上清液TNF－α含量。结果 肝缺血30min或60min再灌注后0h KCs NF-κB活性均于HIR后3h达到高峰，肝缺血时间越长，KCs NF－κB激活越明显；KCs培养上清TNF－α含量于HIR后0h增高，HIR后6h达到高峰，肝缺血时间越长，KCs培养上清TNF－α含量越高。结论 NF－κB是缺血再灌注肝脏KCs活化的关键因子。     

严重烫伤后小鼠腹腔巨噬细胞NF-κB、IκB-α、TNF-α的变化及调控

目的观察严重烫伤后不同时相点及运用特异性NF-κB抑制剂PDTC后小鼠腹腔巨噬细胞内NF-κB活性、IκB-α的表达及TNF-α的变化,从信号转导的角度探讨巨噬细胞功能紊乱的机制.方法以15%体表面积Ⅲ度烫伤小鼠为模型,收集腹腔巨噬细胞,采用放射免疫法检测TNF-α的含量,电泳迁移率改变分析法测NF-κB的活性,改良的ELISA及Western Blotting法测IκB-α的表达,反转录-PCR测TNF-αmRNA的表达.结果烫伤后巨噬细胞分泌TNF-α亢进,于伤后12 h达到高峰.NF-κB伤后明显活化,于2 h达到了高峰,早于TNF-α的增多,IκB-α的表达先降低,后升高.予PDTC后NF-wκB活性及TNF-α mRNA表达量均下降.结论烫伤后NF-κB活性及TNF-α表达明显增强,IκB-α对NF-κB在高水平上维持着一种制约关系.烫伤后小鼠腹腔巨噬细胞内NF-κB途径参与TNF-α表达的调控.     

核转录因子κB在TNF-α诱导人气道上皮细胞hBD-2表达中的作用

目的观察肿瘤坏死因子-α(TNF-α)诱导人气道上皮细胞人β防御素-2(hBD-2)的表达及核转录因子κB(NF-κB)在hBD-2表达中的作用.方法用TNF-α和或NF-κB抑制剂PDTC处理体外培养人气道原代上皮细胞,RT-PCR法检测hBD-2 mRNA的表达,Western blot检测细胞质IκB-α蛋白活性变化,凝胶迁移试验(EMSA)检测不同时相NF-κB活性的变化.结果TNF-α刺激0.5 h后可见人气道上皮细胞hBD-2 mRNA表达,并呈时间依赖性;PDTC可抑制hBD-2mRNA表达;NF-κB在TNF-α刺激1 h后明显活化,抗体超迁移率实验结果显示p65-p50异型二聚体NF-κB参与了NF-κB的活化.结论一定剂量的TNF-α可诱导人气道上皮细胞hBD-2 mRNA表达,NF-κB在TNF-α诱导气道上皮细胞hBD-2mRNA起重要的调控作用.     

Effect of nuclear factor-kappaB activation on expression of proinflammatory cytokines in rat lung tissues in early stage of burn injury]

OBJECTIVE: To investigate the effect of nuclear factor-kappaB (NF-kappaB) activation on the expression of proinflammatory cytokines in the lung tissues of rats with early-stage burn injury. METHODS: Wistar rats were randomly divided into 3 groups, namely the normal control, burn, burn and PDTC treatment groups, and in the latter two groups, the rats were subjected to 35% TBSA full-thickness burns. Activation of pulmonary NF-kappaB at 1, 3, 6, 12, and 24 postburn hour (PBH) was tested by electrophoretic mobility shift assay , and the expressions of pulmonary tumor necrosis factor alpha (TNF alpha) and interleukin-8 (IL-8) mRNAs at 3, 6, 12, and 24 h were detected by RT-PCR. RESULTS: Compared to that of the control group, activity of pulmonary NF-kappaB in burned rats was markedly increased within 1 PBH and kept increasing till 24 h. Expressions of pulmonary TNF alpha and IL-8 mRNAs increased gradually, reaching the peak level at 6 PBH, and PDTC could effectively inhibit pulmonary NF-kappaB activation and expression of the pulmonary cytokines induced by the burn injury. CONCLUSION: Severe burn injury may activate pulmonary NF-kappaB, which ultimately leads to secretion of cytokines in the lung tissues.     

Inhibition of NF-κB by mutant IκBα enhances TNF-α-induced apoptosis in HL-60 cells by controlling bcl-xL expression

Backgound The aim of this study was to explore whether the inhibition of nuclear factor-κB (NF-κB)activation by mutant IκBα (S32,36→A) can enhance TNF-α-induced apoptosis of leukemia cells and to investigate the possible mechanism. Methods The mutant IκBα gene was transfected into HL-60 cells by liposome-mediated techniques. G418 resistant clones stably expressing mutant IκBα were obtained by the limiting dilution method. TNF-α-induced NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The expression of bcl-xL was detected by RT-PCR and Western blot after 4 hours exposure of parental HL-60 and transfected HL-60 cells to a variety of concentrations of TNF-α. The percentage of apoptotic leukemia cells was evaluated by flow cytometry (FCM). Results Mutant IκBα protein was confirmed to exist by Western blot. The results of EMSA showed that NF-κB activation by TNF-α in HL-60 cells was induced in a dose-dependent manner, but was almost completely inhibited by mutant IκBα repressor in transfected cells. The levels of bcl-xL mRNA and protein in HL-60 cells increased after exposure to TNF-α, but changed very little in transfected HL-60 cells. The inhibition of NF-κB activation by mutant IκBα enhanced TNF-α-induced apoptosis. Thecytotoxic effects of TNF-α were amplified in a time- and dose-dependent manner. Conclusions NF-κB activation plays an important role in the resistance to TNF-α-induced apoptosis. The inhibition of NF-κB by mutant IκBα could provide a new approach that may enhance the antileukemia effects of TNF-α or even of other cytotoxic agents.     

Myosin light-chain kinase contributes to short-term endothelial cell cytoskeletal alteration induced by serum from burned rats.

OBJECTIVES: To investigate the time-dependent effects of serum from burned rats on cytoskeletal filamentous actin (F-actin) reorganization by visualizing their distribution in human umbilical vein endothelial cell line ECV-304 and evaluate the role of myosin light-chain kinase (MLCK) in this process. METHODS: The serum-starved ECV-304 cells were incubated with the serum from burned rats for 30 min, 1, 2, 4, and 6 h, respectively, and 30 min before or after the incubation, the cells were treated with 5 micromol/L ML-7 for 30 min. F-actin was stained with rhodamine-phalloidin and observed under fluorescence microscope. RESULTS: Under normal condition, F-actin was distributed mainly in the cortical area of the endothelial cells. After stimulation with the burn serum, stress fiber formation could be clearly seen in the endothelial cells, exhibiting a time-dependent enhancement in a time course ranging from 30 min to 6 h. Such an effect could be significantly inhibited by a 30-min pretreatment of the cells with MLCK-specific inhibitor ML-7. Inhibition of MLCK also reversed actin reorganization in the endothelial cells pretreated with the burn serum. CONCLUSION: Serum from burned rats induces characteristic morphological changes in the endothelial cell actin cytoskeleton mainly due to the MLCK activation, an effect that can be reversed by the inhibition of MLCK.     

Pro-apoptotic role of NF-κB pathway inhibition in lipopolysaccharide stimulated polymorphonuclear neutrophils

Objective To investigate the role of nuclear factor kappa B (NF-κB) pathway inhibition in lipopolysaccharide (LPS)-stimulated apoptosis of polymorphonuclear neutrophils (PMNs).Methods Rats with acute lung injury induced by LPS intratracheal instillation and cultured human venous PMNs were studied. Pyrrolidine dithiocarbamate (PDTC) and gliotoxin were used as NF-κB inhibitors. Additionally, to explore the role of extracellularly regulated protein kinase as an upstream signal in NF-κB pathway on regulating LPS-stimulated PMN apoptosis, PD098059, the specific inhibitor of extracellularly regulated protein kinase, was also applied. The lung injury was determined by protein content and PMN numbers in bronchoalveolar lavage fluid. PMN apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) end labeling and DNA fragmentation. IκBα degradation was analyzed by Western blot. NF-κB DNA binding activity was detected by an electrophoretic mobility shift assay.Results (1) The increase of protein content and PMN numbers in bronchoalveolar lavage fluid induced by LPS (100μg per rat) intratracheal instillation were alleviated by PDTC (50, 100, or 200mg/kg, i. p. ) in a dose-dependent manner. (2) PMNs apoptosis in vivo or in vitro was delayed by LPS, and accelerated by PDTC, gliotoxin or PD098059 pretreatment. (3) IκBα degradation and increased NF-KB DNA binding activity mediated by LPS were inhibited by PDTC, gliotoxin or PD098059 pretreatment.Conclusion Inhibition of either NF-κB itself or the upstream signals in NF-κB pathway such as extracellularly regulated protein kinases has therapeutic effect on LPS-induced acute lung injury, in which the dysregulation of PMN apoptosis plays an important role.     

Adenovirus-mediated overexpression of novel mutated IκBα inhibits nuclear factor κB activation in endothelial cells

Nuclear factor κB （NF-κB） overactivation, requiring phosphorylation and degradation of its inhibitor IκBα, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-IκBα, carrying an IκBα gene from human placenta, we optimized a novel IκBα mutant （IκBα） gene, constructed and characterized its replication-deficient recombinant adenovirus （AdIκBαM）, and tested whether AdIκBαM-mediated overexpression of IκBαM could inhibit the NF-κB activation in endothelial cells.     

肌球蛋白轻链激酶在烧伤大鼠血清引起的内皮细胞骨架改变过程中的作用

目的观察烧伤大鼠血清刺激人脐静脉内皮细胞株ECV-304引起的纤维状肌动蛋白在细胞内分布变化的时间效应以及肌球蛋白轻链激酶在其中的作用。方法用烧伤大鼠血清分别孵育细胞30 min、1 h、2 h、4 h和6 h。或在烧伤血清作用30 min之前或之后使用5 μmol/L的ML-7作用30 min。使用罗丹明标记的鬼笔环肽探针特异性结合并显示细胞内的纤维状肌动蛋白,并在Nikon TE-300荧光显微镜下观察照相。结果正常情况下,纤维状肌动蛋白主要分布在内皮细胞内的外周膜部位。使用烧伤大鼠血清进行30 min~12 h的刺激后,内皮细胞内可观察到明显的应力纤维形成,并且随着刺激时间的延长而增强。可以通过使用肌球蛋白轻链激酶特异性抑制剂ML-7预处理细胞30 min来抑制这种效应。此外,ML-7还能够改善烧伤大鼠血清引起的内皮细胞内肌动蛋白重排。结论本研究表明,大鼠烧伤血清能够通过激活肌球蛋白轻链激酶引起明显的细胞内肌动蛋白排列的改变。在烧伤血清刺激细胞后,抑制肌球蛋白轻链激酶可以改善烧伤大鼠血清引起的内皮细胞骨架重排。     

Effect of homocysteine on NF-kappaB activity in cultured rat vascular smooth muscle cells]

OBJECTIVE: To investigate the effect of homocysteine (Hcy) on nuclear factor-kappaB (NF-kappaB) activity in cultured rat vascular smooth muscle cells (VSMCs). METHODS: Rat VSMCs were stimulated with 0.25 mmol/L Hcy. Cells were collected and nuclear protein was extracted at 30 min, 1 h and 2 h following stimulation. NF-kappaB activity was examined by electrophoretic mobility shift assay (EMSA). RESULT: Baseline NF-kappaB nuclear binding in VSMCs increased more than 1.76-fold at 30 min (P<0.01), 1.91-fold at 1h (P<0.01) and 1.84-fold at 2 h (P<0.01) following stimulation of Hcy. CONCLUSION: Hcy can enhance transient mobilization of N-kappaB in VSMCs, which suggests that some regulating effect of Hcy on VSMCs might be exerted through NF-kappaB activation pathway.     

NF-KB激活对缺血再灌注心肌TNF-α和IL-1β表达的影响

目的 探讨心肌缺血再灌注损伤时NF-κB激活对TNF-α和IL-1β表达的影响.方法 65只SD大鼠随机分为3组:假手术组(n=5);对照组(心肌缺血30 min后再分别灌注0、15、30、60、120、240 min,每个时间点n=5);PDTC干预组,术前给予PDTC 15 mg/kg,其它与对照组相同.RT-PCR检测TNF-a和IL-1β mRNA表达,电泳迁移率实验(EMSA)检测NF_KB活性,硫代巴比妥酸法测定心肌MDA含量.结果 TNF-α和IL-1β表达在再灌注开始之前即有升高,分别在再灌注30和60 min达到高峰,再灌注120 min仍维持较高水平;NF-KB在再灌注15 min开始激活,至再灌注60 min达到高峰.PDTC干预组的NF-KB激活被阻断,与对照组各时间点相比,TNF-a和IL-1β mRNA表达均不同程度下降,MDA含量减少.结论 NF-KB激活对缺血再灌注心肌细胞因子表达具有重要作用,抑制NF-KB信号通路可能对于再灌注损伤具有潜在的治疗价值.