TNF-α对人脐静脉内皮细胞表达MCP-1及IL-8的影响

本研究探讨肿瘤坏死因子仪（TNF-α）刺激人脐静脉内皮细胞（HUVEC）后对细胞表达单核细胞趋化蛋白-1（MCP-1）和白介素-8（IL-8）的影响及其可能的分子机制。用RT—PCR的方法检测MCP-1和IL-8mRNA的表达,免疫荧光染色法检测HUVEC核内转录因子KB（NF-κBp65）的激活。结果表明：TNF-α刺激HUVEC后,细胞内MCP-1和IL-8mRNA的表达增强,且有时相性变化;IL-8mRNA表达8小时达到高峰,MCP-1mRNA表达12小时达到高峰。细胞核内NF-κBp65蛋白表达增强,在感染后0．5小时开始增加,1小时达峰值。然后,胞浆染色逐渐减弱,而核染色增强,表明转录因子NF-κBp65明显的核移位。结论：TNF-α刺激HUVEC后能增加细胞内MCP-1、IL-8mRNA和NF-κBp65蛋白质的表达,提示TNF-α可能通过NF-κB途径诱导血管内皮细胞MCP-1和IL-8等炎症介子的过度表达。     

核转录因子-κB在慢性间歇低氧大鼠各脏器的表达及其介导的炎症因子变化

目的：观察核转录因子-κB(NF-κB)在正常大鼠及慢性间歇低氧大鼠各脏器的表达差异及其信号通路介导的慢性间歇低氧大鼠机体多种炎症因子变化。方法：将30只雄性大鼠,随机分为对照组及间歇低氧组各15只。对照组常规饲料喂养,关灯睡眠;间歇低氧组每日间歇缺氧7 h。8周后处死2组大鼠,使用酶联免疫吸附法(ELISA)检测血清单核细胞趋化蛋白-1(MCP-1)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、IL-8、C-反应蛋白(CRP)。使用蛋白质印迹法(Western Blotting)分别检测2组大鼠NF-κB的蛋白表达。结果：间歇低氧组NF-κB表达较对照组增加,差异有统计学意义(P<0.01),MCP-1、TNF-α、IL-6、IL-8、CRP水平均较对照组明显增高,差异有统计学意义(P<0.01)。结论：炎症因子在慢性间歇低氧大鼠机体高表达,源于慢性间歇低氧引起的机体免疫紊乱,NF-κB介导的信号通路激活,介导了炎症反应。     

川崎病并发冠脉损伤患儿血清活化HUVECs NF-κB促使MMP-9 mRNA水平上调的实验研究

目的 探讨脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)在川崎病(kawasaki disease,KD)并发冠状动脉损伤(coronary artery lesions,CALs)患儿血清作用下,核因子-κB(nuclear factor-kappa B,NF-κB)对基质金属蛋白酶9(matrix metalloproteases 9,MMP9)mRNA转录的影响。方法将HUVECs分为4组,分别为正常血清组(control组)、一般发热血清组(F组)、无冠脉损伤组(non-CALs组)和冠脉损伤组(CALs组)。免疫共沉淀(co-immunoprecipitation,ChIP)检测HUVECs中NF-κB p65与MMP-9启动子结合情况,RT-PCR检测HUVECs MMP-9mRNA水平。结果 与对照组相比,经KD患儿,特别是并发CALs患儿血清作用后,HUVECs NF-κB p65可直接结合在MMP-9启动子上,同时MMP-9 mRNA呈高表达。结论 HUVECs在KD并发CALs患儿血清作用下,NF-κB p65可直接结合在MMP-9启动子上促进其转录。     

乳香酯对ox-LDL诱导的巨噬细胞表达单核细胞趋化因子的影响

目的探讨乳香酯对氧化型低密度脂蛋白(ox-LDL)所致的巨噬细胞单核细胞趋化因子(MCP-1)释放的影响及相关分子机制。方法以ox-LDL、ox-LDL加不同浓度的乳香酯处理人单核细胞THP-1,采用酶联免疫吸附测定细胞上清液中MCP-1的浓度,免疫印迹法检测细胞质中p65/NF-κB和IκBα的表达水平以及细胞核内p65/NF-κB的表达水平。结果乳香酯能够显著减少ox-LDL所致的巨噬细胞MCP-1的表达,且p65/NF-κB的核移位减少,IκBα的降解水平明显降低。结论乳香酯减少ox-LDL所致的巨噬细胞MCP-1的表达与其调节磷酸化IκBα的表达和NF-κB的核-浆转位有关。     

RNAi抑制IKK/NF-κB信号通路对子宫内膜异位症腺上皮细胞中MMP-9的表达及细胞侵袭性的影响

目的探讨小干扰RNA(siRNA)沉默IκB激酶(IKK)/核因子κB(NF-κB)信号通路对子宫内膜异位症在位内膜腺上皮细胞中基质金属蛋白酶-9(MMP-9)的表达及细胞侵袭性的影响。方法根据基因数据库IKKα的c DNA序列,设计构建合成IKKαsiRNA转染10例EMs患者在位内膜原代腺上皮细胞,设立空白对照组(不加任何siRNA的等比例转染试剂)、阴性对照组(与IKKα同源性极低的无意义链siRNA)及实验组(IKKαsiRNA)。采用Realtime PCR、Western blot、Transwell等方法分别检测IKKαsiRNA转染前后NF-κB、MMP-9 mRNA和蛋白表达及腺上皮细胞侵袭性的变化。结果与空白对照组和阴性对照组比较,实验组EMs腺上皮细胞中NF-кB、MMP-9蛋白及mRNA表达降低,差异有统计学意义(P0.05);IKKαsiRNA转染后EMs腺上皮细胞的细胞侵袭性明显降低,差异有统计学意义(P0.05)。结论 RNA干扰可抑制IKK/NF-κB信号通路进而显著降低细胞的侵袭,这种抑制作用可能是通过下调MMP-9的表达进而改变其侵袭作用诱导EMs的发生。     

脂多糖对血管平滑肌细胞表达MMP-9的影响及可能机制

目的探讨脂多糖(lipopolysaccharides,LPS)对培养的大鼠血管平滑肌细胞(vascular smooth musch cell,VSMC)表达基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)的影响及可能的分子机制。方法贴块法进行血管平滑肌细胞培养,细胞免疫化学检测MMP-9的蛋白表达及核因子NF-κB激活情况,原位分子杂交分析MMP-9mRNA表达。结果LPS是MMP-9的强诱导物,可在蛋白及mRNA水平诱导MMP-9的表达,LPS对其诱导作用强度与LPS的浓度呈正相关。与正常组比较LPS刺激15min即有NF-κB p65核转移,30min达高峰,1h后减弱。结论LPS诱导平滑肌细胞MMP-9表达,其作用呈浓度时间依赖性,MMP-9表达升高可能与LPS激活NF-κB有关。MMP-9表达升高和NF-κB激活提示LPS在动脉粥样硬化发病过程中可能起一定作用。     

C反应蛋白激活单核细胞核因子-кB及辛伐他汀干预

目的:观察C反应蛋白(CRP)刺激人外周血单核细胞核因子-κB(NF-κB)活化及白细胞介素-6(IL-6)表达,予辛伐他汀干预,探讨CRP/NF-κB在动脉粥样硬化(AS)致病机制中的作用和辛伐他汀的抗AS效应。方法:密度梯度离心法分离人外周血单核细胞,免疫细胞化学观察CRP致单核细胞NF-κBp65活化的时间效应,ELISA法观察IL-6产生的时间-浓度效应,其峰值分别与辛伐他汀抑制剂组比较。结果:CRP刺激单核细胞NF-κB活化呈时间依赖性,高峰在2h,单核细胞表达IL-6呈时间与浓度依赖性,在24h达高峰。辛伐他汀(1×10-8mol/L～1×10-6mol/L)呈剂量依赖性抑制NF-κB活化与IL-6表达。结论:CRP激活正常人单核细胞NF-B信号途径,并诱导单核细胞产生IL-6,辛伐他汀抑制单核细胞NF-κB活性与IL-6表达,提示CRP/NF-κB信号途径在促进AS发病机制中起重要作用,辛伐他汀抑制NF-κB有可能减轻或抑制AS进展。     

C反应蛋白激活单核细胞核因子-κB 及辛伐他汀干预

目的观察C反应蛋白(CRP)刺激人外周血单核细胞核因子-κB(NF-κB)活化及白细胞介素-6(IL-6)表达,予辛伐他汀干预,探讨CRP/NF-κB在动脉粥样硬化(AS)致病机制中的作用和辛伐他汀的抗AS效应.方法密度梯度离心法分离人外周血单核细胞,免疫细胞化学观察CRP致单核细胞NF-κB p65活化的时间效应,ELISA法观察IL-6产生的时间-浓度效应,其峰值分别与辛伐他汀抑制剂组比较.结果CRP刺激单核细胞NF-κB活化呈时间依赖性,高峰在2 h,单核细胞表达IL-6呈时间与浓度依赖性,在24 h达高峰.辛伐他汀(1×10-8mol/L～1×10-6mol/L)呈剂量依赖性抑制NF-κB活化与IL-6表达.结论CRP激活正常人单核细胞NF-B信号途径,并诱导单核细胞产生IL-6,辛伐他汀抑制单核细胞NF-κB活性与IL-6表达,提示CRP/NF-κB信号途径在促进AS发病机制中起重要作用,辛伐他汀抑制NF-κB有可能减轻或抑制AS进展.     

罗格列酮对冠心病患者巨噬细胞表达核因子-κB和金属蛋白酶-9的影响

目的 探讨罗格列酮(Ros)干预在调节冠心病患者外周血单核细胞源性巨噬细胞(MDMs)表达核因子-κB(NF-κB)、金属蛋白酶-9(MMP-19)中的作用及可能机制.方法 本研究为临床病例对照研究.于2007年3月至4月间,从湘雅二医院心内科行冠脉造影患者中,选取急性冠脉综合征患者48例(ACS组)、稳定型心绞痛(SA)患者20例(对照组)为研究对象,排除脑血管意外、急性感染和创伤、肝肾功能不全、肿瘤等患者.提取外周血单个核细胞,用巨噬细胞集落刺激因子刺激,转化为MDMs;随机分亚组后,分别用0μmol/L1μmol/L,10 μmol/L,20 μmol浓度Ros干预48h;RT-PCR检测各亚组MDMs表达过氧化物酶增殖体激活受体-γ([PPAR-γ)和MMP-9 mRNA,免疫组化法检测NF-κB P65表达强度.用ANOVA检验比较组间及亚组内MDMs在表达PPAR-γ,MMP-9,NF-κB P65的差异.结果 干预前,ACS组MDMs表达PPAR-γmRNA水平低于对照组,表达NF-κB P65及MMP-9mRNA水平高于对照组;Ros干预后,ACS组及对照组PPAR-γmRNA表达明显上调,ACS组PPAR-γ的表达量与Ros浓度呈正变关系;MMP-9 mRNA表达下调,在ACs组其下调程度与Ros浓度呈反变关系;两组NF-κB P65表达量均呈现非剂量依赖性降低.结论 ACS患者外周血MDMs的PPAR-γRNA表达被抑制、NF-γB活性及MMP-9 mRNA表达增强.Ros干预可通过增加PPAR-γ的表达,从而抑制NF-κB活性和MMP-9的表达.     

靶向核转录因子-κB P65小干扰RNA对脓毒症所致小鼠急性肝损伤的影响

目的 探讨靶向核转录因子(NF)-κB P65 小干扰RNA(siRNA)对脓毒症所致小鼠急性肝损伤的保护作用.方法 将70只雄性昆明小鼠随机分为四组:即健康对照组、脓毒症组、特异干扰组和乱序对照组,后三组每组均设置术后6、12 h两个时间点,每组每个时间点10只小鼠;术前1 h特异干扰组尾静脉注射NF-κB P65 siRNA逆转录病毒,乱序对照组注射Scrambled siRNA逆转录病毒,健康对照组及脓毒症组分别注射等体积生理盐水;除健康对照组小鼠均行盲肠结扎穿孔(CLP)法构建脓毒症急性肝损伤模型;于术后6、12 h留取肝组织标本,检测组织病理学改变,NF-κB P65蛋白表达水平,TNF-α mRNA及蛋白的表达水平.结果.与脓毒症组和乱序对照组比较,特异干扰组术后6、12 h肝内NF-κB P65蛋白的表达均降低,肝脏病理损害均减轻;特异干扰组术后6 h肝内TNF-α mRNA及蛋白水平显著降低(P<0.05).结论.靶向NF-κB P65 siRNA抑制NF-κB的表达后,能够抑制脓毒症所致过度炎症反应,减轻急性肝损伤.     

核因子κBp65特异性小干涉核糖核酸抑制肿瘤坏死因子α及白细胞介素1β诱导的软骨细胞中基质金属蛋白酶9的表达

背景：小干涉核糖核酸是核糖核酸干涉的起始诱导物，在细胞内引起强烈的核糖核酸干涉，降解目的基因的信使核糖核酸，以控制目的基因表达。目的：利用核因子κBp65特异性小干涉核糖核酸抑制软骨细胞中肿瘤坏死因子α和白细胞介素1β诱导的核因子κB的活性及其基质金属蛋白酶9的表达，观察在软骨细胞中核因子κBp65与细胞因子的关系。设计、时间和地点：单一样本观察．细胞学体外实验，于2006—09／2007—09在北京大学医学部中心实验室完成。材料：SD大鼠关节软骨细胞。方法：利用质脂体将筛选优化好的核因子κBp65特异性小于涉核糖核酸转染软骨细胞，特异性抑制核因子κBp65的表达，继而抑制肿瘤坏死因子α和白细胞介素1β诱导的核因子κB的活性及基质金属蛋白酶9的表达。主要观察指标：利用电泳迁移率试验检测核因子κB的活性，反转录聚合酶链反应和蛋白质免疫印记法分析从信使核糖核酸和蛋白质两水平检测基质金属蛋白酶9的表达。结果：核因子κBp65特异性小干涉核糖核酸抑制核因子κBp65的表达，降低肿瘤坏死因子α和白细胞介素1β诱导的核因子κB的转录活性，抑制肿瘤坏死因子α和白细胞介素1β诱导的基质金属蛋白酶9的表达。 结论：在软骨细胞中核因子κBp65与肿瘤坏死因子α和白细胞介素1β关系密切。     

核因子κB途径介导C反应蛋白对肺动脉平滑肌细胞的促炎作用

目的 观察了C反应蛋白(CRP)对培养的肺动脉平滑肌细胞(hPASMCs)炎性因子白介素-6(IL-6)的影响,探讨CRP对肺血管疾病的可能作用.方法 体外培养hPASMCs,以不同质量浓度的CRP(5～200μg/mL)刺激不同时间(0,3,6,9,12,18,24 h).核因子κB(NF-κB)的活性以非变性凝胶电泳迁移率(EMSA)方法进行分析.IL-6 mRNA和蛋白水平以Real-time PCR和ELISA方法进行检测.结果 CRP以浓度依赖的方式促进hPASMCs IL-6的合成.与对照组相比,CRP 200 μg/mL使IL-6的合成增加2.8倍.CRP显著诱导NF-κB在bPASMCs的激活.CRP对hPASMCs的促炎作用受到细胞表面FCγⅡa受体特异性抗体的抑制.结论 CRP促进体外培养的hPASMCs对IL-6的表达,这一作用是通过细胞表而FcγⅡa受体亚型和NF-κB的核内转位激活而介导的.提示CRP在肺动脉高压的发病中有重要作用.

Abstract：

Objective To examine the impact of C-reactive protein (CRP) on the expression of interleukin-6 (IL-6), inflammatory cytokine, in cultured human pulmonary artery smooth muscle cells (hPASMCs) in order to find out the cause of pulmonary artery hypertension (PAH). Method The hPASMCs were cultured and stimulated by different concerntrations of CRP (5 - 200 μg/ml) for different lengths of time. The activity of nuclear factor-κB (NF-κB) was evaluated by electrophoretic gel mobility shift assay (EMSA). The expression of IL-6 mRNA and the level of IL-6 protein were measured by using real-time PCR and ELISA, respectively. Results CRP increased IL-6 production in hPASMCs in a dose-dependent manner. The increase in IL-6 at concerntration of 200 μg/mL in the CRP group was as high as 2.8times that in the control group. CRP also significantly induced the activation of NF-κB in hPASMCs. The effect of CRP on the inflammatory cytokine, IL-6, was inhibited by the specific FcγⅡa receptor antibody.Conclusions In vitro, CRP increases the production of IL-6 in hPASMCs mediated by FcγⅡa receptor and NF-κB translocation. These data offer important insights into the role of CRP in the pathogenesis of PAH.     

Guanine-nucleotide exchange factor H1 mediates lipopolysaccharide-induced interleukin 6 and tumor necrosis factor α expression in endothelial cells via activation of nuclear factor κB

The development of sepsis is multifactorial. Tissue damage and organ dysfunction may be caused not only by the microorganisms but also by the inflammatory mediators released in response to the infection. Interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) levels in serum are well known to be upregulated in humans with sepsis and can be used to predict outcome. Using human umbilical vein endothelial cells, we analyzed the role of guanine-nucleotide exchange factor H1 (GEF-H1) on lipopolysaccharide (LPS)-dependent IL-6/TNF-α expression in endothelial cells. Lipopolysaccharide upregulated IL-6 secretion in a dose- and time-dependent manner. Specific inactivation of RhoA/Cdc42/Rac1 by Clostridium difficile toxin B-10463 (TcdB-10463) reduced LPS-induced nuclear factor κB (NF-κB) p65 phosphorylation, IL-6/TNF-α messenger RNA (mRNA), and IL-6/TNF-α protein productions. Guanine-nucleotide exchange factor H1 protein expression remained on a high level among 1 to 9 h in response to LPS challenge of endothelial cells. Inhibition of GEF-H1 by specific small interfering RNA or inactivation of Rho-associated kinase with Y-27632 not only significantly reduced LPS-induced p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) activities but also blocked LPS-induced NF-κB translocation and activation, thereby inhibiting IL-6/TNF-α mRNA and protein productions. Furthermore, SB203580 (p38 inhibitor) but not PD98059 (ERK1/2 inhibitor) blocked LPS-induced NF-κB activation; however, both inhibitors significantly suppressed IL-6/TNF-α mRNA and protein expression. In summary, our data suggest that LPS rapidly upregulates GEF-H1 expression. Activated Rho-associated kinase by GEF-H1 subsequently activates p38 and ERK1/2, thereby increasing IL-6/TNF-α expression in endothelial cells. P38 and ERK1/2 regulate LPS-induced IL-6/TNF-α expression through an NF-κB-dependent manner and an NF-κB-independent manner, respectively.     

整合素连接激酶通过核因子κB途径介导基质金属蛋白酶9上调促进肺癌细胞迁移和侵袭

目的 探讨整合素连接激酶（ILK）促进肺癌细胞侵袭的相关分子机制.方法 通过细胞转染、siRNA干扰、细胞划痕实验、实时定量PCR、Western Blot方法探讨ILK和基质金属蛋白酶9（MMP-9）在肺癌A549细胞中的表达及相互关系.结果 在肺癌A549细胞系中,过度表达的ILK诱导MMP-9的表达（P＜0.01）;加入MMP-9抑制剂多西环素显著影响了转染组细胞划痕愈合能力（P＜0.01）,加入抗-MMP-9中和抗体则严重阻碍了细胞迁移能力（P＜0.01）,体外基底膜侵袭实验亦得到了相同的结果（P＜0.01）.ILK的过度表达促进磷酸化和核因子-κB（NF-κB）亚单位p65的核易位（P＜0.01）,并且NF-κB抑制剂BAY11-7028和NF-κBp65siRNA能抑制ILK高表达细胞系中MMP-9的上调（P＜0.01）.结论 本研究结果表明,ILK的过度表达可促进肺癌细胞迁移和侵袭,并且是通过NF-κB途径介导MMP-9上调来实现这一过程.     

Ox-LDL-induced LOX-1 expression in vascular smooth muscle cells: role of reactive oxygen species

Oxidized low density lipoprotein (ox-LDL) and lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) have been implicated in the development of atherosclerosis. This study was designed to investigate the expression regulation of LOX-1 by ox-LDL and the potential underlying mechanisms in cultured rat vascular smooth muscle cells (VSMCs). VSMCs were treated with ox-LDL, and the expressions of LOX-1 mRNA and proteins were determined by RT-PCR and western blotting, respectively. The intracellular reactive oxygen species (ROS) production was monitored by flow cytometry with fluorescence probe, DCFH(2) -DA. The effect of several inhibitors including aspirin, NDGA, allopurinol, apocynin, and rotenone on ox-LDL-induced ROS formation and LOX-1 expression was also investigated. The roles of NF-κB p65 and JNK were explored. Ox-LDL significantly induced LOX-1 expression at both mRNA and protein levels in a dose-dependent and time-dependent manner. Aspirin, NDGA, and preconditioned apocynin suppressed ox-LDL-induced intracellular ROS production and LOX-1 expression, while allopurinol and rotenone failed to do so. Vitamin C and N-acetyl-l-cysteine demonstrated similar effect. Furthermore, both NF-κB p65 expression and phosphorylated JNK (p-JNK) to JNK expression ratio were elevated after ox-LDL treatment. In addition, the NF-κB inhibitor PDTC and JNK inhibitor SP600125 pretreatment partly abolished ox-LDL-induced LOX-1 expression. These findings suggested that ROS mediated ox-LDL-induced LOX-1 expression in VSMCs through NF-κB and JNK signaling pathways.     

Activation of RAW264.7 macrophage by Exopolysaccharide from Aphanothece halaphytica (EPSAH) and the underlying mechanisms

Exopolysaccharide from Aphanothece halophytica (EPSAH), a potent antitumor agent and immunological adjuvant, was investigated for the activation effect on RAW264.7 macrophages and the underlying mechanisms. EPSAH could significantly enhance macrophage phagocytosis and the secretion of nitric oxide, increase the mRNA expression levels of the pro-inflammatory cytokines (IL-1β, IL-6, IL-12, and TNF-α), anti-inflammatory cytokine IL-10, and chemokines (MCP-1 and MIP-1α). When RAW264.7 cells were treated with EPSAH, the mRNA expression of TLR4 and its downstream molecules TRAF6 and MyD88 were upregulated. When TLR4 was blocked using a TLR4-specific neutralizing antibody, nitric oxide secretion from the macrophages was significantly inhibited. EPSAH was further shown to induce phosphorylation of the mitogen-activated protein kinases (MAPKs) ERK, JNK, and p38, and promote cytoplasmic IκB phosphorylation and increase nuclear NF-κB p65 levels remarkably in RAW264.7 cells. These data demonstrate the capacity of EPSAH to induce macrophage activation possibly via TLR4/MyD88 pathway, which leads to the activation of its main signaling downstream molecules MAPKs and NF-κB.