稀土镧化合物调控核因子κB信号通路的分子机制
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摘要
研究背景核因子-κB( nuclear factor-kappa B , NF-κB)是一种广泛存在于哺乳动物多种细胞具有多向性调节作用的蛋白质分子,是细胞中一个对环境改变发生应激反应的转录因子,它调节大量与细胞应激反应、炎症反应、免疫应答和细胞抗凋亡等相关的基因的转录。NF-κB活化的失调参与多种疾病的发生、发展过程,例如感染性休克(脓毒症)、多脏器功能衰竭等烧伤临床上常见,目前尚缺乏特异的治疗方法的并发症,而NF-κB在这些并发症的发生发展过程中起着非常重要的作用;其他如缺血再灌注损伤、类风湿关节炎、支气管哮喘、动脉粥样硬化、溃疡性结肠炎、老年性痴呆、肿瘤细胞抗凋亡及艾滋病(AIDS)等的发生和发展也都与NF-κB的过度活化直接相关。NF-κB是一个进化高度保守的在许多生命过程中发挥重要作用并主要对环境变化产生应答的转录因子,主要是以p50/p65异二聚体形式普遍存在于细胞浆中的一种快反应转录因子,与NF-κB抑制蛋白(IκB)结合而呈非活性状态。研究表明它可以被多种刺激剂,如内毒素(LPS)、TNF-α(tumor necrosis factor-alpha )、IL-1(interleukin 1)、神经生长因子、蛋白激酶C(Protein Kinase C,PKC)激活剂、有丝分裂原、氧化剂、细菌、病毒、免疫刺激剂、自由基以及紫外线等激活。激活的NF-κB与IκB解离后转位入核与靶基因启动子/增强子上的κB位点结合,从而调节许多靶基因的表达,因此被誉为控制早期基因表达的开关。通过靶基因表达产物,NF-κB参与了感染、炎症、免疫反应、细胞凋亡和肿瘤等病理过程以及细胞周期调控与细胞分化等。调节和控制NF-κB活性是缓解组织损伤的一种有力策略,因此深入研究NF-κB信号转导通路,探索细胞内NF-κB活化阻断剂,可能为临床治疗开辟新途径。
近年来,研究发现NF-κB信号系统受到许多金属元素的影响。如金、锌、铜等不同化合物均可通过抑制IKK(sIκB kinases, IκB激酶)的活性来阻断NF-κB的活化;金精三羧酸主要通过强烈抑制NF-κB与DNA结合来控制艾滋病毒HIV活性;许多金属如金、钯、镍及汞亦可影响NF-κB与DNA结合从而调节靶基因的激活。
镧是一种化学性质十分活跃的稀土金属,研究证实稀土具有抗菌、促进创面愈合、调节细胞免疫功能等作用。临床上,稀土已经在磁共振、时间分辨检测、放射性同位素诊断中用作诊断试剂,铈浴法还用于治疗烧伤患者;2004年10月,碳酸镧被美国FDA批准用于临床治疗磷酸水平高的血透患者。我们的前期研究工作证明稀土化合物氯化镧对LPS引起的体内效应有明显的拮抗作用。实验表明镧可降低内毒素血症小鼠炎症介质水平,减轻内毒素对机体主要脏器的损伤,对致死量内毒素攻击小鼠具有明显的保护作用。本课题拟通过分析氯化镧对多种细胞内NF-κB上游相关信号分子以及NF-κB系统内部的信号分子表达及活化的影响,并观察氯化镧对LPS诱导NF-κB所调控的下游炎症反应介质表达的影响,探索镧抑制NF-κB活化的可能机制,为临床感染性休克(脓毒症)、多脏器功能衰竭等疾病的治疗提供新思路,并为开发稀土的药用价值提供实验依据。
第I部分氯化镧对内毒素诱导NF-κB上游信号分子的作用目的分析氯化镧对LPS诱导NF-κB上游信号分子的表达和活化的影响。
方法:
1.细胞培养及分组:RAW264.7巨噬细胞:以含5%胎牛血清的DMEM/F12液体培养基稀释细胞密度为1×106个/ml,分别接种于去热原培养瓶中及预先放置无菌去热源盖玻片的24孔培养板中,于5%CO2、37℃恒温培养,实验随机分组如下:①LPS组:以含1μg/ml LPS的无血清DMEM/F12液体培养基培养RAW264.7细胞。②氯化镧+LPS组:以含氯化镧2.5μmol/L(经预实验证明该浓度可以有效抑制NF-κB的活化)的无血清培养基培养RAW264.7细胞4h后,弃培养基并以无热原生理盐水漂洗细胞三遍,再加入含1μg/ml LPS的培养基继续培养。③空白对照组:培养基为不含血清的DMEM/F12,不加氯化镧和LPS。④氯化镧对照组:以含氯化镧2.5μmol/L的无血清DMEM/F12培养基培养RAW264.7细胞。按检测指标要求分别于不同时间段收集培养上清、细胞及细胞爬片待检。
2.检测指标:
(1)观察氯化镧对LPS诱导RAW264.7细胞表面Toll样受体4(TLR4)基因表达(转录和翻译)的作用:利用流式细胞术及逆转录多聚酶链式反应(RT-PCR)技术分析TLR4基因表达情况;
(2)氯化镧对LPS活化NF-κB上游部分信号通路的影响:
①观察氯化镧对LPS诱导RAW264.7细胞p38MAPK蛋白表达和磷酸化的作用:采用细胞免疫荧光染色分析p38MAPK/p-p38MAPK在巨噬细胞细胞中的分布与表达;免疫印迹技术(Western blot)测定巨噬细胞内p38MAPK蛋白表达和磷酸化水平的改变。
②观察氯化镧对LPS诱导RAW264.7巨噬细胞中PKC蛋白表达和磷酸化的作用:采用细胞免疫荧光染色分析PKC在巨噬细胞的表达和磷酸化转位情况; Western blot测定巨噬细胞内PKC蛋白表达和磷酸化水平的改变。
结果:
(1)氯化镧对NF-κB上游信号膜受体(TLR4)表达的作用:利用流式细胞术及逆转录多聚酶链式反应(RT-PCR)技术分析1μg/ml LPS刺激后巨噬细胞表面TLR4的表达及氯化镧的影响,结果表明LPS能够显著上调巨噬细胞TLR4基因和胞膜TLR4蛋白的表达,尽管氯化镧(2.5μmol/L)本身不上调巨噬细胞TLR4基因的表达,氯化镧亦不能抑制LPS诱导巨噬细胞表面受体TLR4的基因的表达。
(2)氯化镧对LPS活化NF-κB上游部分信号通路的影响:
①p38 MAPK信号通路:1μg/ml LPS能够有效激活RAW264.7巨噬细胞的p38MAPK信号通路,氯化镧(2.5μmol/L)自身不激活该信号通路同时也不能阻断LPS介导的p38蛋白的表达和磷酸化。
②PKC信号系统:1μg/ml LPS同样能够有效激活RAW264.7巨噬细胞的PKC信号,令PKC蛋白表达和磷酸化水平显著升高,并从胞浆转位至胞膜。2.5μmol/L氯化镧即能够有效阻断LPS诱导的PKC蛋白表达和磷酸化水平升高及膜易位。结论:氯化镧通过阻断NF-κB上游PKC信号通路,从而可能部分抑制NF-κB信号通路的活化。
第II部分氯化镧对NF-κB信号通路中关键分子活化作用的分析
目的观察不同激活机制下(激活物分别为LPS及TNF-α,靶细胞分别为RAW264.7及Hela细胞及NF-κB组成性活化细胞株U266),氯化镧对各细胞中NF-κB信号通路关键分子的蛋白表达和磷酸化及NF-κB/p65亚基与靶基因结合能力的影响,探索氯化镧抑制NF-κB活化的可能作用位点。
方法
1.细胞培养及分组:
(1)RAW264.7细胞:(同上)
(2)Hela细胞:以1×106个/ml接种细胞于含10%胎牛血清的RMPI 1640液体培养基中,于5%CO2、37℃恒温培养,随机分组如下:①TNF-α组:以含TNF-α20ng/ml的无血清RMPI 1640液体培养基培养Hela细胞。②氯化镧+TNF-α组:分别以含氯化镧5 , 25, 50和100μmol/L的RMPI 1640培养基培养Hela细胞4h后弃培养基并以无热原生理盐水漂洗细胞三遍,再加入含TNF-α20ng/ml的培养基继续培养。③空白对照组:培养基为不含血清的RMPI 1640,不加氯化镧和TNF-α。④氯化镧对照组:以含氯化镧100μmol/L的RMPI 1640培养基培养Hela细胞。各组细胞根据检测方法的要求分别培养于24孔培养板(孔中预先放置无菌盖玻片)或培养瓶中,于30min后,收集培养上清、细胞及盖玻片待检。
(3) U266细胞:以1×106个/ml接种细胞于含15%胎牛血清的RMPI 1640液体培养基的培养瓶中,于5%CO2、37℃恒温培养,细胞悬浮生长,隔天半量换液,实验分组如下:①空白对照组:培养基为不含血清的RMPI 1640,不给予任何干预和刺激。②氯化镧对照组:分别以含氯化镧2.5, 5 ,25,50和100μmol/L的无血清培养基培养U266细胞。各组细胞根据检测方法的要求分别培养于培养瓶中,30min后收集培养细胞待检。
2.检测指标:
(1)Western blot技术测定IκB上游激酶IKKα, IKKβ的表达和磷酸化情况;
(2)Western blot技术测定胞浆提取物中IκBα蛋白表达和磷酸化水平;
(3)细胞免疫荧光染色技术分析NF-κB/p65蛋白在细胞中的表达和分布;
(4)Western blot技术测定胞核提取物中NF-κB /p65蛋白表达水平;
(5)Transcription Factor Assay kit试剂盒(酶联免疫吸附技术(ELISA))测定胞核提取物中p65蛋白与DNA靶序列结合能力。
结果
1.氯化镧对LPS诱导的巨噬细胞NF-κB信号通路中关键分子表达和/或活化的影响:
(1)氯化镧对LPS诱导的NF-κB活化的抑制作用:氯化镧(2.5μmol/L)能够显著抑制LPS诱导NF-κB/p65自胞浆转位到细胞核,并且显著减弱NF-κB/p65与靶DNA结合活性。
(2)氯化镧对LPS诱导的IκBα降解的抑制作用:氯化镧显著抑制LPS诱导的胞浆IκBα降解,从而抑制了NF-κB/p65自胞浆转位到细胞核。
(3)氯化镧对LPS诱导的IκBα磷酸化的抑制作用:氯化镧不能阻断LPS介导的RAW264.7巨噬细胞中IκBα的磷酸化。
(4)氯化镧对LPS诱导的IKKs活性的影响:氯化镧不影响RAW264.7巨噬细胞中IKKα/β表达和磷酸化水平,从而不能阻断LPS介导的IκBα的磷酸化。
在RAW264.7细胞株中,NF-κB激活剂LPS诱导IKKβ及IκBα磷酸化,令IκBα降解,最终使NF-κB/p65转位入核与靶基因结合。氯化镧虽然不能阻断IKKβ及IκBα磷酸化,但是能够阻止RAW264.7细胞中IκBα的降解,并抑制NF-κB/p65转位入核并抑制NF-κB/p65与靶基因的结合活性,最终阻断NF-κB信号转导通路。
2.氯化镧对TNF-α诱导的Hela细胞NF-κB信号通路中关键分子表达和/或活化的影响:
(1)氯化镧对TNF-α诱导的NF-κB活化的抑制作用:25-100μmol/L氯化镧能够显著抑制TNF-α诱导NF-κB/p65蛋白表达水平并抑制其自胞浆转位到细胞核,5-100μmol/L氯化镧还能显著减弱Hela细胞中NF-κB/p65与靶DNA结合活性,上述抑制效应呈剂量依赖关系。
(2)氯化镧对TNF-α诱导的IκBα磷酸化和降解的抑制作用:TNF-α能够诱导Hela细胞中IκBα的磷酸化和降解。随着氯化镧处理的浓度不断升高,IκBα表达水平逐步升高,而磷酸化水平不断降低。表明氯化镧能够抑制TNF-α诱导的IκBα的磷酸化和表达水平的降低,且该效应随着随着氯化镧浓度的升高而逐步增强。
(3)氯化镧对LPS诱导的IKKs活性的影响:氯化镧不影响Hela细胞中IKKα/IKKβ蛋白的表达水平。静息状态的Hela细胞几乎不表达p-IKKβ,而在TNF-α诱导下p-IKKβ水平则明显升高,随着氯化镧处理浓度的逐渐升高,p-IKKβ水平逐步下降,50-100μmol/L氯化镧能够显著抑制TNF-α诱导Hela细胞中IKKβ的磷酸化。
NF-κB的另一个激活剂TNF-α,亦通过磷酸化IKKβ及IκBα,导致IκBα降解,最终亦使NF-κB/p65转位入核与靶基因结合,启动相关基因的表达。在Hela细胞株中,氯化镧不仅能够抑制IκBα的降解和NF-κB/p65转位入核,阻断NF-κB/p65与靶基因的结合,还能够阻断IKKβ及IκBα磷酸化。可见在不同细胞及激活条件下,氯化镧抑制NF-κB信号通路的靶点不完全相同。
3.氯化镧对U266细胞中NF-κB的组成性活化(constitutive activation)的影响:
(1)氯化镧对NF-κB组成性活化的作用:0-100μmol/L的氯化镧不能抑制NF-κB的组成性活化,逆转p65蛋白的移位。
(2)氯化镧对U266细胞中IκBα蛋白的表达和磷酸化的作用:0-100μmol/L氯化镧亦不能影响IκBα蛋白的表达和磷酸化水平。
(3)氯化镧对U266细胞中IKKs活性的影响:0-100μmol/L的氯化镧对U266细胞中IKKα/IKKβ蛋白的表达和磷酸化水平影响不明显。
(4)氯化镧在胞外条件下,对U266细胞核提取物中NF-κB/p65与DNA的结合能力的影响:实验表明在细胞外条件下,5μmol/L氯化镧即能够显著抑制p65蛋白与靶DNA结合活性,并且该抑制效应随着氯化镧浓度的增高不断增强,100μmol/L氯化镧能够完全抑制胞核提取物中NF-κB/p65与靶DNA的结合。研究结果提示:对于NF-κB组成性活化U266细胞,氯化镧不影响IKKβ的磷酸化及IκBα的磷酸化和降解,并无法逆转NF-κB转位,但能够在细胞外条件下阻断NF-κB/p65与靶DNA的结合。
第III部分氯化镧对LPS诱导NF-κB调控的炎症介质表达的影响目的通过观察氯化镧对LPS诱导的炎性因子TNF-α、NO及诱导型一氧化氮合成酶(iNOS)表达水平及活性的作用,了解氯化镧对LPS诱导NF-κB下游靶基因表达的影响。
方法
(1)实验分组:(同第I部分)。
(2)逆转录-多聚酶链式反应(RT-PCR)测定TNF-α及iNOS基因表达水平。
(3)酶联免疫吸附技术(ELISA)测定细胞上清中TNF-α含量。
(4)细胞免疫荧光染色技术分析iNOS蛋白在细胞中的表达和分布。
(5) Western blot技术测定细胞中iNOS蛋白表达水平。
(6)硝酸还原酶法检测培养上清NO含量。
结果
(1)氯化镧对LPS诱导的NF-κB下游炎症介质TNF-α表达和释放的抑制作用:2.5μmol/L氯化镧不仅能够下调静息状态下巨噬细胞TNF-α基因的表达和多肽的释放还能够显著抑制LPS上调TNF-α基因表达和多肽释放。
(2)氯化镧对LPS诱导NF-κB下游基因iNOS表达的抑制作用:2.5μmol/L氯化镧能明显下调iNOSmRNA的表达水平,减少iNOS蛋白的生成,显著降低产物NO的生成。研究结果提示氯化镧可通过抑制iNOS基因及蛋白的表达水平而控制NO的生成。
结论:本研究结果(第I-第III部分)提示,氯化镧可通过阻断NF-κB上游PKC信号通路,从而部分抑制NF-κB信号通路的活化;氯化镧还能阻断IKKβ及IκBα磷酸化,抑制IκBα的降解和NF-κB/p65转位入核,并抑制NF-κB/p65与靶基因的结合,最终阻断NF-κB信号转导,从而下调炎性介质基因的表达和释放。
Background
Nuclear factor-κB (NF-κB) is a ubiquitous rapid response transcription factor that controls the expression of genes involved in immune responses, apoptosis, and cell cycle. Incorrect regulation of NF-κB,which plays an important role in the development of complications such as septic shock and multiple organ dysfunction syndrome, may cause inflammatory and autoimmune diseases, viral infection and cancer. NF-κB involves apparently in a variety of human diseases including ischaemia-reperfusion injury , rheumatoid arthritis , bronchial asthma ,artherosclerosis, colitis gravis,alzheimer's disease and Acquired Immune Deficiency Syndrome. NF-κB family members have been identified to share a highly conserved Rel homology domain, responsible for their dimerization and binding to DNA and IκB (inhibitor of NF-κB). The transcription factor NF-κB works only when two members form a dimer. The most abundant activated form consists of a p50 or p52 subunit and a p65 subunit. NF-κB can be activated by a variety of stimuli, including bacterial LPS (Lipopolysaccharide), cytokines (such as TNF-α and IL-1β), nerve growth factor, activators of PKC (Protein Kinase C,PKC), T and B cell mitogens, viral proteins, and stress inducers (such as reactive oxygen species or UV radiation). In the cytoplasm, NF- κB is inhibited by IκB. Upstream activating signal may cause phosphorylation of IκB by IKK (IκB kinase). This triggers the degradation of IκB through the ubiquitin system, where the target molecule is masked by a chain of ubiquitins for degradation by the 26S protesome. The free NF-κB can then translocate to the nucleus, and regulate expression of a large number of genes that are critical for the regulation of apoptosis, viral replication, tumorigenesis, inflammation, and various autoimmune diseases. The activation of NF-κB is thought to be part of a stress response as it is activated by a variety of stimuli that include growth factors, cytokines, lymphokines, UV, pharmacological agents, and stress. Therefore, NF-κB takes part in pathology process such as infection, inflammation, immune reaction, cell apoptosis and cancer. Therapeutic interventions aimed at limiting NF-κB activation could prove to be a powerful strategy for attenuating tissue injury.
NF-κB signaling pathway has been reported to be affected by various metal elements recently. Gold, zinc and copper compounds can block the activation of NF-κB by inhibiting the activity of IKKs. It was reported that Aurine tricarboxylic acid attenuating the viability of AIDS virus by decreasing DNA binding activity of NF-κB intensively. Metals such as gold, palladium, nickel and mercury all regulate the activation of target gene by inhibiting the NF-κB–DNA binding activity.
Lanthanum, a representative of lanthanides with extremely active physical and chemical property, evidenced to possess antibacterial effect and regulating cellular immunity, has lower toxicity than synthetic drug and some ove-metals.
Lanthanides have already been employed as diagnostic reagent in clinical examination such as magnetic resonance, Time-resolved Fluorescence Immunoassay and radio active isotope. Lanthanides compounds are also applied in the treatment of various diseases. For example, Lanthanum carbonate is well tolerated and is effective for the long-term maintenance of serum phosphorus control in patients with end-stage renal disease. Studies also show the greater efficacy of sulfadiazine combined with cerium nitrate in the treatment of burns patients. We have previously reported that lanthanum chloride is able to bind LPS and reduce its toxicity, inhibit LPS-induced apoptosis of thymocyte and damage of liver and lungs, markedly decrease the level of TNF-αin plasma and TNF-αmRNA expression in mice challenged with LPS,thus protect mice from lethal dose of LPS challenge. The present study was designed to elucidate the mechanism of lanthanum on activation of NF-κB and its related signaling molecules and pathways as well as the expression of its down-stream inflammatory media in various cells .
Part I The effects of lanthanum on the LPS mediated NF-κB related upstream signaling molecules and pathways
Object To analyze the effects of lanthanum on the LPS mediated NF-κB related upstream signaling molecules and pathways.
Methods
1. Cell culture and Sample treatment: The RAW264.7 macrophages were maintained in DMEM/F12 supplemented with 5% heat-inactivated FBS at 37℃in a humidified 5% CO2 atmosphere. To prevent interference from FBS (FBS may bind with LPS and/or lanthanum chloride), cells were washed triplicate with PBS (phosphate buffered saline, PH7.4) before incubation with LaCl3 or LPS, fresh DMEM/F12, rather than DMEM/F12 containing 5% FBS, was added to the cells; and the cells were divided at random into 4 groups: control group, LPS group, LaCl3 group and LaCl3 + LPS group. In control group, the cells were cultured with DMEM/F12; the cells from LPS group were incubated with DMEM/F12 containing 1μg/ml LPS; In LaCl3 group, cells were incubated with 2.5μmol/L of LaCl3 for indicated time; Post lanthanum exposure, the cells from LaCl3 + LPS group were washed triplicate with PBS to remove LaCl3 and treated with subsequent LPS (1μg/ml) activation. Then the cells and culture supernatants were collected to analyze the expression and activation of NF-κB related singaling molecules and pathways at various time points accordingly.
2. Content and Methods:
(1) The expression of Toll-like receptor 4 (TLR4): TLR4 mRNA level in RAW264.7 cells were examined by RT-PCR (reverse transcription polymerase chain reaction). Membrane TLR4 expression was detected by flow cytometry (FCM).
(2) Expression and phosphorylation of p38 MAPK: Expression and location of p38 MAPK and phosphorated p38 MAPK were detected by IF (immunofluorescence) and western blot.
(3) Expression and phosphorylation of PKC: Expression and location of PKC and phosphorated PKC were detected by IF (immunofluorescence) and western blot.
Results
(1) The effects of Lanthanum on the expression of TLR4: RT-PCR and FCM results showed that LPS upregulated TLR4 expression, 2.5μmol/L LaCl3 had no effect on the expression itself , it had no effect on LPS- induced TLR4 expression, either.
(2) The effects of Lanthanum on the LPS-mediated activation of upstream signaling pathways of NF-κB:
①p38 MAPK signaling pathway: p38 MAPK signaling pathway of RAW264.7 cells was activated by 1μg/ml LPS. 2.5μmol/L LaCl3 had no effect on the expression and phosphorylation of p38MAPK itself , it had no effect on LPS- induced expression and phosphorylation of p38MAPK, either.
②PKC signaling pathway: PKC signaling pathway of RAW264.7 cells was also activated in RAW264.7 cells by 1μg/ml LPS. LPS-mediated PKC expression and phosphorylation as well as membrane traslocation was significantly inhibited by 2.5μmol/L of LaCl3.
Conclusion:
Lantanum may block NF-κB signaling partly via PKC signaling pathway.
Part II The effects of lanthanum on key molecules of NF-κB pathway Object To explore the possible inhinitory sites of lanthanum on NF-κB activation by detecting the expression and phosphorylation of key molecules of NF-κB and the NF-κB/p65-DNA binding activity in various stimuli-actived cell lines.
Methods
1. Cell culture and Sample treatment:
(1) The RAW264.7 macrophages:(The same as above)
(2) Hela cervical carcinoma cells: the cells were maintained in RMPI 1640 supplemented with 10% heat-inactivated FBS at 37℃in a humidified 5% CO2 atmosphere. To prevent interference from FBS (FBS may bind with lanthanum chloride), cells were washed triplicate with PBS before incubation with LaCl3/TNF-α, fresh RMPI 1640, rather than RMPI 1640 containing 10% FBS, was added to the cells. The cells were divided at random into 4 groups: control group, TNF-αgroup, LaCl3 group and LaCl3 + TNF-αgroup. In control group, the cells were cultured with RMPI 1640; the cells from TNF-αgroup were incubated with 20ng/ml TNF-α; in LaCl3 group, cells were incubated with 100μmol/L of LaCl3 for 30min; Post 2h 5, 25, 50 and 100μmol/L lanthanum exposure, the cells from LaCl3 +TNF-αgroup were washed triplicate with PBS to remove LaCl3 and treated with subsequent TNF-α(20ng/ml) activation for 30 min. Then the cells were collected to analyze the expression and activation of key singaling molecules and the NF-κB/p65-DNA binding activity of NF-κB pathway.
(3) Multiple myeloma u266 cells: the cells were maintained in RMPI 1640 supplemented with 15% heat-inactivated FBS at 37℃in a humidified 5% CO2 atmosphere. To prevent interference from FBS, cells were washed triplicate with PBS before incubation with LaCl3, fresh RMPI 1640, rather than RMPI 1640 containing 15% FBS, was added to the cells. The cells were divided at random into 2 groups: control group, and LaCl3 group. In control group, the cells were cultured with RMPI 1640; in LaCl3 group, cells were incubated with 2.5, 5 ,25,50 and 100μmol/L of LaCl3 for 2h, respectively. Then the cells were collected to analyze the expression and activation of key singaling molecules and the NF-κB/p65-DNA binding activity of NF-κB pathway.
2. Content and Methods:
(1) The expression and phosphorylation of IκB upstream kianse IKKα, IKKβwere detected by western blot;
(2) The expression and phosphorylation of IκBαwere also detected by western blot;
(3) Immunofluorescence was employed in detecting the expression and localization of NF-κB /p65;
(4) NF-κB /p65 in the nuclear extract was detected by western blot.
(5) NF-κB /p65-DNA binding activity of nuclear extract was detected by Transcription Factor Assay kit
Results and conclusion
1. The effects of lanthanum on expression and/or activation of key signaling molecules in NF-κB pathway of LPS- induced macrophages:
(1) NF-κB activation: LaCl3 (2.5μmol/L) can inhibit LPS-mediated NF-κB/p65 overexpression, translocation and decrease the NF-κB/p65-DNA binding activity significantly.
(2) Degradation of IκBα: LaCl3 (2.5μmol/L) can inhibit LPS-mediated IκBαdegradation, thus inhibited the nuclear translocation of NF-κB/p65.
(3) Phosphorylation of IκBα: LaCl3 (2.5μmol/L) can not inhibit LPS-mediated IκBαphosphorylation.
(4) Phosphorylation of IKKs: LaCl3 (2.5μmol/L) can not inhibit LPS-mediated expression and phosphorylation of IKKα/β.
LPS, an activator of NF-κB, induces IKKβand IκBαphosphorylation, causes degradation of IκBαand leads to the nuclear translocation of NF-κB/p65 protein, which ultimately bind with target DNA. Although lanthanum can not block the phosphorylation of IKKβand IκBα, it can inhibit IκBαdegradation, NF-κB/p65 protein translocation and its DNA binding activity, thus blocks NF-κB signaling pathway in microphages.
2. The effects of lanthanum on expression and/or activation of key signaling molecules in NF-κB pathway of TNF-α- induced Hela cervical carcinoma cells:
(1) NF-κB activation: LaCl3 (25-100μmol/L) can inhibit TNF-α-mediated NF-κB/p65 expression and translocation remarkably. 5-100μmol/L LaCl3 decrease the NF-κB/p65-DNA binding activity significantly. The inhibitory effects are dose-dependent.
(2) Phosphorylation and degradation of IκBα: Phosphorylation and degradation of IκBαwas induced by TNF-α, LaCl3 can inhibit TNF-α-mediated IκBαphosphrylation and degradation dose-dependently.
(3) Phosphorylation of IKKs: IKKαand IKKβexpression was not affected by TNF-αand /or LaCl3 treatment. TNF-α-induced IKKβphosphorylation was attenuated by LaCl3 dose-dependently. 50-100μmol/L LaCl3 decreased TNF-α-mediated IKKβphosphorylation significantly.
Another activator of NF-κB, TNF-α, induces IKKβand IκBαphosphorylation, causes degradation of IκBαand leads to the nuclear translocation of NF-κB/p65 protein, which ultimately bind with target DNA in Hela cells. Lanthanum can not only block the phosphorylation of IKKβand IκBαbut also inhibit NF-κB/p65 protein translocation and its DNA binding activity; thus blocks NF-κB signaling pathway in Hela cells.
3. The effects of lanthanum on constitutive activation of key signaling molecules in NF-κB pathway of multiple myeloma u266 cells.
(1) NF-κB activation: LaCl3 (0-100μmol/L) can not inhibit constitutive NF-κB/p65 expression and translocation of p65 subunit from the nuclear back into cytoplasm.
(2) Phosphorylation and degradation of IκBα: LaCl3 (0-100μmol/L) can not inhibit IκBαphosphrylation and degradation
(3) Phosphorylation of IKKs: IKKαand IKKβexpression and phosphorylation was not affected by LaCl3 (0-100μmol/L) treatment.
(4) At the presence of 5μmol/L LaCl3, DNA binding activity of NF-κB/p65 from nuclear extract of U266 cells decreased significantly. With increasing LaCl3 dosage, the inhibitory effects progressively increased and the binding activity was completely abolished at the highest dose (100μmol/L).
The rescults show that in NF-κB constitutive activation U266 cells, lanthanum can not inhibit phosphorylation of IKKβand IκBα, degradation of IκBαand the nuclear translocation of NF-κB/p65 protein. However, Lanthanum can block the DNA binding activity of NF-κB/p65 from the nuclear extract of U266 cells in a dose-dependent manner.
Part III The effects of lanthanum on the expression of NF-κB regulated inflammatory media
Object To explore the effects of lanthuanum on LPS-induced NF-κB -dependent gene expression in RAW264.7 cells, level of inflammatory media (TNF-αand NO) was detected and the gene expression of TNF-αand inducible nitric oxide synthase (iNOS) was measured.
Methods
(1) Cell culture and Sample treatment: The same as part I, briefly, RAW264.7 macrophages were maintained in DMEM/F12 supplemented with 5% heat-inactivated FBS at 37℃in a humidified 5% CO2 atmosphere. The cells were divided at random into 4 groups: control group, LPS group, LaCl3 group and LaCl3 + LPS group.
(2) The expression of TNF-αand iNOS mRNA: RT-PCR was employed to detect TNF-αand iNOS mRNA level in RAW264.7 cells.
(3) TNF-αin the culture supernatants was detected by ELISA (Enzym kinked immunosorbent assay).
(4) Expression and localization of iNOS: Expression and location of iNOS was detected by both IF (immunofluorescence) and western blot.
(5) NO production in culture supernatant was determinded by nitrate reductase assay. Results and conclusion
(1) TNF-αexpression: LaCl3 at low concentration (2.5μmol/L) can remarkably inhibit the expression of TNF-α, one of LPS-induced NF-κB-dependent inflammatory media, in both rest and LPS activated RAW264.7 cells.
(2) iNOS expression : Immunofluorescence analysis and western blotting detection showed LaCl3 (2.5μmol/L) can remarkably inhibit the expression of iNOS protein in LPS-induced RAW264.7 macrophages. The results of RT-PCR showed the LPS-induced expression of iNOS mRNA can be decreased by LaCl3 significantly. NO production assay indicated LaCl3 can also reduce the NO production significantly in macrophages induced by LPS.
In summary (PartI-III), NF-κB signal pathway can be blocked by lanthanum chloride via blocking the PKC signal pathway partly; And lanthanum chloride can inhibit the phosphorylation of IKKβand IκBα, suppress IκBαdegradation and nuclear translocation of NF-κB/p65 as well as attenuat NF-κB/p65-DNA binding activitaty, all these result in blockage of NF-κB signial pathway and inhibition of inflammatory media expression and release.
近年来,研究发现NF-κB信号系统受到许多金属元素的影响。如金、锌、铜等不同化合物均可通过抑制IKK(sIκB kinases, IκB激酶)的活性来阻断NF-κB的活化;金精三羧酸主要通过强烈抑制NF-κB与DNA结合来控制艾滋病毒HIV活性;许多金属如金、钯、镍及汞亦可影响NF-κB与DNA结合从而调节靶基因的激活。
镧是一种化学性质十分活跃的稀土金属,研究证实稀土具有抗菌、促进创面愈合、调节细胞免疫功能等作用。临床上,稀土已经在磁共振、时间分辨检测、放射性同位素诊断中用作诊断试剂,铈浴法还用于治疗烧伤患者;2004年10月,碳酸镧被美国FDA批准用于临床治疗磷酸水平高的血透患者。我们的前期研究工作证明稀土化合物氯化镧对LPS引起的体内效应有明显的拮抗作用。实验表明镧可降低内毒素血症小鼠炎症介质水平,减轻内毒素对机体主要脏器的损伤,对致死量内毒素攻击小鼠具有明显的保护作用。本课题拟通过分析氯化镧对多种细胞内NF-κB上游相关信号分子以及NF-κB系统内部的信号分子表达及活化的影响,并观察氯化镧对LPS诱导NF-κB所调控的下游炎症反应介质表达的影响,探索镧抑制NF-κB活化的可能机制,为临床感染性休克(脓毒症)、多脏器功能衰竭等疾病的治疗提供新思路,并为开发稀土的药用价值提供实验依据。
第I部分氯化镧对内毒素诱导NF-κB上游信号分子的作用目的分析氯化镧对LPS诱导NF-κB上游信号分子的表达和活化的影响。
方法:
1.细胞培养及分组:RAW264.7巨噬细胞:以含5%胎牛血清的DMEM/F12液体培养基稀释细胞密度为1×106个/ml,分别接种于去热原培养瓶中及预先放置无菌去热源盖玻片的24孔培养板中,于5%CO2、37℃恒温培养,实验随机分组如下:①LPS组:以含1μg/ml LPS的无血清DMEM/F12液体培养基培养RAW264.7细胞。②氯化镧+LPS组:以含氯化镧2.5μmol/L(经预实验证明该浓度可以有效抑制NF-κB的活化)的无血清培养基培养RAW264.7细胞4h后,弃培养基并以无热原生理盐水漂洗细胞三遍,再加入含1μg/ml LPS的培养基继续培养。③空白对照组:培养基为不含血清的DMEM/F12,不加氯化镧和LPS。④氯化镧对照组:以含氯化镧2.5μmol/L的无血清DMEM/F12培养基培养RAW264.7细胞。按检测指标要求分别于不同时间段收集培养上清、细胞及细胞爬片待检。
2.检测指标:
(1)观察氯化镧对LPS诱导RAW264.7细胞表面Toll样受体4(TLR4)基因表达(转录和翻译)的作用:利用流式细胞术及逆转录多聚酶链式反应(RT-PCR)技术分析TLR4基因表达情况;
(2)氯化镧对LPS活化NF-κB上游部分信号通路的影响:
①观察氯化镧对LPS诱导RAW264.7细胞p38MAPK蛋白表达和磷酸化的作用:采用细胞免疫荧光染色分析p38MAPK/p-p38MAPK在巨噬细胞细胞中的分布与表达;免疫印迹技术(Western blot)测定巨噬细胞内p38MAPK蛋白表达和磷酸化水平的改变。
②观察氯化镧对LPS诱导RAW264.7巨噬细胞中PKC蛋白表达和磷酸化的作用:采用细胞免疫荧光染色分析PKC在巨噬细胞的表达和磷酸化转位情况; Western blot测定巨噬细胞内PKC蛋白表达和磷酸化水平的改变。
结果:
(1)氯化镧对NF-κB上游信号膜受体(TLR4)表达的作用:利用流式细胞术及逆转录多聚酶链式反应(RT-PCR)技术分析1μg/ml LPS刺激后巨噬细胞表面TLR4的表达及氯化镧的影响,结果表明LPS能够显著上调巨噬细胞TLR4基因和胞膜TLR4蛋白的表达,尽管氯化镧(2.5μmol/L)本身不上调巨噬细胞TLR4基因的表达,氯化镧亦不能抑制LPS诱导巨噬细胞表面受体TLR4的基因的表达。
(2)氯化镧对LPS活化NF-κB上游部分信号通路的影响:
①p38 MAPK信号通路:1μg/ml LPS能够有效激活RAW264.7巨噬细胞的p38MAPK信号通路,氯化镧(2.5μmol/L)自身不激活该信号通路同时也不能阻断LPS介导的p38蛋白的表达和磷酸化。
②PKC信号系统:1μg/ml LPS同样能够有效激活RAW264.7巨噬细胞的PKC信号,令PKC蛋白表达和磷酸化水平显著升高,并从胞浆转位至胞膜。2.5μmol/L氯化镧即能够有效阻断LPS诱导的PKC蛋白表达和磷酸化水平升高及膜易位。结论:氯化镧通过阻断NF-κB上游PKC信号通路,从而可能部分抑制NF-κB信号通路的活化。
第II部分氯化镧对NF-κB信号通路中关键分子活化作用的分析
目的观察不同激活机制下(激活物分别为LPS及TNF-α,靶细胞分别为RAW264.7及Hela细胞及NF-κB组成性活化细胞株U266),氯化镧对各细胞中NF-κB信号通路关键分子的蛋白表达和磷酸化及NF-κB/p65亚基与靶基因结合能力的影响,探索氯化镧抑制NF-κB活化的可能作用位点。
方法
1.细胞培养及分组:
(1)RAW264.7细胞:(同上)
(2)Hela细胞:以1×106个/ml接种细胞于含10%胎牛血清的RMPI 1640液体培养基中,于5%CO2、37℃恒温培养,随机分组如下:①TNF-α组:以含TNF-α20ng/ml的无血清RMPI 1640液体培养基培养Hela细胞。②氯化镧+TNF-α组:分别以含氯化镧5 , 25, 50和100μmol/L的RMPI 1640培养基培养Hela细胞4h后弃培养基并以无热原生理盐水漂洗细胞三遍,再加入含TNF-α20ng/ml的培养基继续培养。③空白对照组:培养基为不含血清的RMPI 1640,不加氯化镧和TNF-α。④氯化镧对照组:以含氯化镧100μmol/L的RMPI 1640培养基培养Hela细胞。各组细胞根据检测方法的要求分别培养于24孔培养板(孔中预先放置无菌盖玻片)或培养瓶中,于30min后,收集培养上清、细胞及盖玻片待检。
(3) U266细胞:以1×106个/ml接种细胞于含15%胎牛血清的RMPI 1640液体培养基的培养瓶中,于5%CO2、37℃恒温培养,细胞悬浮生长,隔天半量换液,实验分组如下:①空白对照组:培养基为不含血清的RMPI 1640,不给予任何干预和刺激。②氯化镧对照组:分别以含氯化镧2.5, 5 ,25,50和100μmol/L的无血清培养基培养U266细胞。各组细胞根据检测方法的要求分别培养于培养瓶中,30min后收集培养细胞待检。
2.检测指标:
(1)Western blot技术测定IκB上游激酶IKKα, IKKβ的表达和磷酸化情况;
(2)Western blot技术测定胞浆提取物中IκBα蛋白表达和磷酸化水平;
(3)细胞免疫荧光染色技术分析NF-κB/p65蛋白在细胞中的表达和分布;
(4)Western blot技术测定胞核提取物中NF-κB /p65蛋白表达水平;
(5)Transcription Factor Assay kit试剂盒(酶联免疫吸附技术(ELISA))测定胞核提取物中p65蛋白与DNA靶序列结合能力。
结果
1.氯化镧对LPS诱导的巨噬细胞NF-κB信号通路中关键分子表达和/或活化的影响:
(1)氯化镧对LPS诱导的NF-κB活化的抑制作用:氯化镧(2.5μmol/L)能够显著抑制LPS诱导NF-κB/p65自胞浆转位到细胞核,并且显著减弱NF-κB/p65与靶DNA结合活性。
(2)氯化镧对LPS诱导的IκBα降解的抑制作用:氯化镧显著抑制LPS诱导的胞浆IκBα降解,从而抑制了NF-κB/p65自胞浆转位到细胞核。
(3)氯化镧对LPS诱导的IκBα磷酸化的抑制作用:氯化镧不能阻断LPS介导的RAW264.7巨噬细胞中IκBα的磷酸化。
(4)氯化镧对LPS诱导的IKKs活性的影响:氯化镧不影响RAW264.7巨噬细胞中IKKα/β表达和磷酸化水平,从而不能阻断LPS介导的IκBα的磷酸化。
在RAW264.7细胞株中,NF-κB激活剂LPS诱导IKKβ及IκBα磷酸化,令IκBα降解,最终使NF-κB/p65转位入核与靶基因结合。氯化镧虽然不能阻断IKKβ及IκBα磷酸化,但是能够阻止RAW264.7细胞中IκBα的降解,并抑制NF-κB/p65转位入核并抑制NF-κB/p65与靶基因的结合活性,最终阻断NF-κB信号转导通路。
2.氯化镧对TNF-α诱导的Hela细胞NF-κB信号通路中关键分子表达和/或活化的影响:
(1)氯化镧对TNF-α诱导的NF-κB活化的抑制作用:25-100μmol/L氯化镧能够显著抑制TNF-α诱导NF-κB/p65蛋白表达水平并抑制其自胞浆转位到细胞核,5-100μmol/L氯化镧还能显著减弱Hela细胞中NF-κB/p65与靶DNA结合活性,上述抑制效应呈剂量依赖关系。
(2)氯化镧对TNF-α诱导的IκBα磷酸化和降解的抑制作用:TNF-α能够诱导Hela细胞中IκBα的磷酸化和降解。随着氯化镧处理的浓度不断升高,IκBα表达水平逐步升高,而磷酸化水平不断降低。表明氯化镧能够抑制TNF-α诱导的IκBα的磷酸化和表达水平的降低,且该效应随着随着氯化镧浓度的升高而逐步增强。
(3)氯化镧对LPS诱导的IKKs活性的影响:氯化镧不影响Hela细胞中IKKα/IKKβ蛋白的表达水平。静息状态的Hela细胞几乎不表达p-IKKβ,而在TNF-α诱导下p-IKKβ水平则明显升高,随着氯化镧处理浓度的逐渐升高,p-IKKβ水平逐步下降,50-100μmol/L氯化镧能够显著抑制TNF-α诱导Hela细胞中IKKβ的磷酸化。
NF-κB的另一个激活剂TNF-α,亦通过磷酸化IKKβ及IκBα,导致IκBα降解,最终亦使NF-κB/p65转位入核与靶基因结合,启动相关基因的表达。在Hela细胞株中,氯化镧不仅能够抑制IκBα的降解和NF-κB/p65转位入核,阻断NF-κB/p65与靶基因的结合,还能够阻断IKKβ及IκBα磷酸化。可见在不同细胞及激活条件下,氯化镧抑制NF-κB信号通路的靶点不完全相同。
3.氯化镧对U266细胞中NF-κB的组成性活化(constitutive activation)的影响:
(1)氯化镧对NF-κB组成性活化的作用:0-100μmol/L的氯化镧不能抑制NF-κB的组成性活化,逆转p65蛋白的移位。
(2)氯化镧对U266细胞中IκBα蛋白的表达和磷酸化的作用:0-100μmol/L氯化镧亦不能影响IκBα蛋白的表达和磷酸化水平。
(3)氯化镧对U266细胞中IKKs活性的影响:0-100μmol/L的氯化镧对U266细胞中IKKα/IKKβ蛋白的表达和磷酸化水平影响不明显。
(4)氯化镧在胞外条件下,对U266细胞核提取物中NF-κB/p65与DNA的结合能力的影响:实验表明在细胞外条件下,5μmol/L氯化镧即能够显著抑制p65蛋白与靶DNA结合活性,并且该抑制效应随着氯化镧浓度的增高不断增强,100μmol/L氯化镧能够完全抑制胞核提取物中NF-κB/p65与靶DNA的结合。研究结果提示:对于NF-κB组成性活化U266细胞,氯化镧不影响IKKβ的磷酸化及IκBα的磷酸化和降解,并无法逆转NF-κB转位,但能够在细胞外条件下阻断NF-κB/p65与靶DNA的结合。
第III部分氯化镧对LPS诱导NF-κB调控的炎症介质表达的影响目的通过观察氯化镧对LPS诱导的炎性因子TNF-α、NO及诱导型一氧化氮合成酶(iNOS)表达水平及活性的作用,了解氯化镧对LPS诱导NF-κB下游靶基因表达的影响。
方法
(1)实验分组:(同第I部分)。
(2)逆转录-多聚酶链式反应(RT-PCR)测定TNF-α及iNOS基因表达水平。
(3)酶联免疫吸附技术(ELISA)测定细胞上清中TNF-α含量。
(4)细胞免疫荧光染色技术分析iNOS蛋白在细胞中的表达和分布。
(5) Western blot技术测定细胞中iNOS蛋白表达水平。
(6)硝酸还原酶法检测培养上清NO含量。
结果
(1)氯化镧对LPS诱导的NF-κB下游炎症介质TNF-α表达和释放的抑制作用:2.5μmol/L氯化镧不仅能够下调静息状态下巨噬细胞TNF-α基因的表达和多肽的释放还能够显著抑制LPS上调TNF-α基因表达和多肽释放。
(2)氯化镧对LPS诱导NF-κB下游基因iNOS表达的抑制作用:2.5μmol/L氯化镧能明显下调iNOSmRNA的表达水平,减少iNOS蛋白的生成,显著降低产物NO的生成。研究结果提示氯化镧可通过抑制iNOS基因及蛋白的表达水平而控制NO的生成。
结论:本研究结果(第I-第III部分)提示,氯化镧可通过阻断NF-κB上游PKC信号通路,从而部分抑制NF-κB信号通路的活化;氯化镧还能阻断IKKβ及IκBα磷酸化,抑制IκBα的降解和NF-κB/p65转位入核,并抑制NF-κB/p65与靶基因的结合,最终阻断NF-κB信号转导,从而下调炎性介质基因的表达和释放。
Background
Nuclear factor-κB (NF-κB) is a ubiquitous rapid response transcription factor that controls the expression of genes involved in immune responses, apoptosis, and cell cycle. Incorrect regulation of NF-κB,which plays an important role in the development of complications such as septic shock and multiple organ dysfunction syndrome, may cause inflammatory and autoimmune diseases, viral infection and cancer. NF-κB involves apparently in a variety of human diseases including ischaemia-reperfusion injury , rheumatoid arthritis , bronchial asthma ,artherosclerosis, colitis gravis,alzheimer's disease and Acquired Immune Deficiency Syndrome. NF-κB family members have been identified to share a highly conserved Rel homology domain, responsible for their dimerization and binding to DNA and IκB (inhibitor of NF-κB). The transcription factor NF-κB works only when two members form a dimer. The most abundant activated form consists of a p50 or p52 subunit and a p65 subunit. NF-κB can be activated by a variety of stimuli, including bacterial LPS (Lipopolysaccharide), cytokines (such as TNF-α and IL-1β), nerve growth factor, activators of PKC (Protein Kinase C,PKC), T and B cell mitogens, viral proteins, and stress inducers (such as reactive oxygen species or UV radiation). In the cytoplasm, NF- κB is inhibited by IκB. Upstream activating signal may cause phosphorylation of IκB by IKK (IκB kinase). This triggers the degradation of IκB through the ubiquitin system, where the target molecule is masked by a chain of ubiquitins for degradation by the 26S protesome. The free NF-κB can then translocate to the nucleus, and regulate expression of a large number of genes that are critical for the regulation of apoptosis, viral replication, tumorigenesis, inflammation, and various autoimmune diseases. The activation of NF-κB is thought to be part of a stress response as it is activated by a variety of stimuli that include growth factors, cytokines, lymphokines, UV, pharmacological agents, and stress. Therefore, NF-κB takes part in pathology process such as infection, inflammation, immune reaction, cell apoptosis and cancer. Therapeutic interventions aimed at limiting NF-κB activation could prove to be a powerful strategy for attenuating tissue injury.
NF-κB signaling pathway has been reported to be affected by various metal elements recently. Gold, zinc and copper compounds can block the activation of NF-κB by inhibiting the activity of IKKs. It was reported that Aurine tricarboxylic acid attenuating the viability of AIDS virus by decreasing DNA binding activity of NF-κB intensively. Metals such as gold, palladium, nickel and mercury all regulate the activation of target gene by inhibiting the NF-κB–DNA binding activity.
Lanthanum, a representative of lanthanides with extremely active physical and chemical property, evidenced to possess antibacterial effect and regulating cellular immunity, has lower toxicity than synthetic drug and some ove-metals.
Lanthanides have already been employed as diagnostic reagent in clinical examination such as magnetic resonance, Time-resolved Fluorescence Immunoassay and radio active isotope. Lanthanides compounds are also applied in the treatment of various diseases. For example, Lanthanum carbonate is well tolerated and is effective for the long-term maintenance of serum phosphorus control in patients with end-stage renal disease. Studies also show the greater efficacy of sulfadiazine combined with cerium nitrate in the treatment of burns patients. We have previously reported that lanthanum chloride is able to bind LPS and reduce its toxicity, inhibit LPS-induced apoptosis of thymocyte and damage of liver and lungs, markedly decrease the level of TNF-αin plasma and TNF-αmRNA expression in mice challenged with LPS,thus protect mice from lethal dose of LPS challenge. The present study was designed to elucidate the mechanism of lanthanum on activation of NF-κB and its related signaling molecules and pathways as well as the expression of its down-stream inflammatory media in various cells .
Part I The effects of lanthanum on the LPS mediated NF-κB related upstream signaling molecules and pathways
Object To analyze the effects of lanthanum on the LPS mediated NF-κB related upstream signaling molecules and pathways.
Methods
1. Cell culture and Sample treatment: The RAW264.7 macrophages were maintained in DMEM/F12 supplemented with 5% heat-inactivated FBS at 37℃in a humidified 5% CO2 atmosphere. To prevent interference from FBS (FBS may bind with LPS and/or lanthanum chloride), cells were washed triplicate with PBS (phosphate buffered saline, PH7.4) before incubation with LaCl3 or LPS, fresh DMEM/F12, rather than DMEM/F12 containing 5% FBS, was added to the cells; and the cells were divided at random into 4 groups: control group, LPS group, LaCl3 group and LaCl3 + LPS group. In control group, the cells were cultured with DMEM/F12; the cells from LPS group were incubated with DMEM/F12 containing 1μg/ml LPS; In LaCl3 group, cells were incubated with 2.5μmol/L of LaCl3 for indicated time; Post lanthanum exposure, the cells from LaCl3 + LPS group were washed triplicate with PBS to remove LaCl3 and treated with subsequent LPS (1μg/ml) activation. Then the cells and culture supernatants were collected to analyze the expression and activation of NF-κB related singaling molecules and pathways at various time points accordingly.
2. Content and Methods:
(1) The expression of Toll-like receptor 4 (TLR4): TLR4 mRNA level in RAW264.7 cells were examined by RT-PCR (reverse transcription polymerase chain reaction). Membrane TLR4 expression was detected by flow cytometry (FCM).
(2) Expression and phosphorylation of p38 MAPK: Expression and location of p38 MAPK and phosphorated p38 MAPK were detected by IF (immunofluorescence) and western blot.
(3) Expression and phosphorylation of PKC: Expression and location of PKC and phosphorated PKC were detected by IF (immunofluorescence) and western blot.
Results
(1) The effects of Lanthanum on the expression of TLR4: RT-PCR and FCM results showed that LPS upregulated TLR4 expression, 2.5μmol/L LaCl3 had no effect on the expression itself , it had no effect on LPS- induced TLR4 expression, either.
(2) The effects of Lanthanum on the LPS-mediated activation of upstream signaling pathways of NF-κB:
①p38 MAPK signaling pathway: p38 MAPK signaling pathway of RAW264.7 cells was activated by 1μg/ml LPS. 2.5μmol/L LaCl3 had no effect on the expression and phosphorylation of p38MAPK itself , it had no effect on LPS- induced expression and phosphorylation of p38MAPK, either.
②PKC signaling pathway: PKC signaling pathway of RAW264.7 cells was also activated in RAW264.7 cells by 1μg/ml LPS. LPS-mediated PKC expression and phosphorylation as well as membrane traslocation was significantly inhibited by 2.5μmol/L of LaCl3.
Conclusion:
Lantanum may block NF-κB signaling partly via PKC signaling pathway.
Part II The effects of lanthanum on key molecules of NF-κB pathway Object To explore the possible inhinitory sites of lanthanum on NF-κB activation by detecting the expression and phosphorylation of key molecules of NF-κB and the NF-κB/p65-DNA binding activity in various stimuli-actived cell lines.
Methods
1. Cell culture and Sample treatment:
(1) The RAW264.7 macrophages:(The same as above)
(2) Hela cervical carcinoma cells: the cells were maintained in RMPI 1640 supplemented with 10% heat-inactivated FBS at 37℃in a humidified 5% CO2 atmosphere. To prevent interference from FBS (FBS may bind with lanthanum chloride), cells were washed triplicate with PBS before incubation with LaCl3/TNF-α, fresh RMPI 1640, rather than RMPI 1640 containing 10% FBS, was added to the cells. The cells were divided at random into 4 groups: control group, TNF-αgroup, LaCl3 group and LaCl3 + TNF-αgroup. In control group, the cells were cultured with RMPI 1640; the cells from TNF-αgroup were incubated with 20ng/ml TNF-α; in LaCl3 group, cells were incubated with 100μmol/L of LaCl3 for 30min; Post 2h 5, 25, 50 and 100μmol/L lanthanum exposure, the cells from LaCl3 +TNF-αgroup were washed triplicate with PBS to remove LaCl3 and treated with subsequent TNF-α(20ng/ml) activation for 30 min. Then the cells were collected to analyze the expression and activation of key singaling molecules and the NF-κB/p65-DNA binding activity of NF-κB pathway.
(3) Multiple myeloma u266 cells: the cells were maintained in RMPI 1640 supplemented with 15% heat-inactivated FBS at 37℃in a humidified 5% CO2 atmosphere. To prevent interference from FBS, cells were washed triplicate with PBS before incubation with LaCl3, fresh RMPI 1640, rather than RMPI 1640 containing 15% FBS, was added to the cells. The cells were divided at random into 2 groups: control group, and LaCl3 group. In control group, the cells were cultured with RMPI 1640; in LaCl3 group, cells were incubated with 2.5, 5 ,25,50 and 100μmol/L of LaCl3 for 2h, respectively. Then the cells were collected to analyze the expression and activation of key singaling molecules and the NF-κB/p65-DNA binding activity of NF-κB pathway.
2. Content and Methods:
(1) The expression and phosphorylation of IκB upstream kianse IKKα, IKKβwere detected by western blot;
(2) The expression and phosphorylation of IκBαwere also detected by western blot;
(3) Immunofluorescence was employed in detecting the expression and localization of NF-κB /p65;
(4) NF-κB /p65 in the nuclear extract was detected by western blot.
(5) NF-κB /p65-DNA binding activity of nuclear extract was detected by Transcription Factor Assay kit
Results and conclusion
1. The effects of lanthanum on expression and/or activation of key signaling molecules in NF-κB pathway of LPS- induced macrophages:
(1) NF-κB activation: LaCl3 (2.5μmol/L) can inhibit LPS-mediated NF-κB/p65 overexpression, translocation and decrease the NF-κB/p65-DNA binding activity significantly.
(2) Degradation of IκBα: LaCl3 (2.5μmol/L) can inhibit LPS-mediated IκBαdegradation, thus inhibited the nuclear translocation of NF-κB/p65.
(3) Phosphorylation of IκBα: LaCl3 (2.5μmol/L) can not inhibit LPS-mediated IκBαphosphorylation.
(4) Phosphorylation of IKKs: LaCl3 (2.5μmol/L) can not inhibit LPS-mediated expression and phosphorylation of IKKα/β.
LPS, an activator of NF-κB, induces IKKβand IκBαphosphorylation, causes degradation of IκBαand leads to the nuclear translocation of NF-κB/p65 protein, which ultimately bind with target DNA. Although lanthanum can not block the phosphorylation of IKKβand IκBα, it can inhibit IκBαdegradation, NF-κB/p65 protein translocation and its DNA binding activity, thus blocks NF-κB signaling pathway in microphages.
2. The effects of lanthanum on expression and/or activation of key signaling molecules in NF-κB pathway of TNF-α- induced Hela cervical carcinoma cells:
(1) NF-κB activation: LaCl3 (25-100μmol/L) can inhibit TNF-α-mediated NF-κB/p65 expression and translocation remarkably. 5-100μmol/L LaCl3 decrease the NF-κB/p65-DNA binding activity significantly. The inhibitory effects are dose-dependent.
(2) Phosphorylation and degradation of IκBα: Phosphorylation and degradation of IκBαwas induced by TNF-α, LaCl3 can inhibit TNF-α-mediated IκBαphosphrylation and degradation dose-dependently.
(3) Phosphorylation of IKKs: IKKαand IKKβexpression was not affected by TNF-αand /or LaCl3 treatment. TNF-α-induced IKKβphosphorylation was attenuated by LaCl3 dose-dependently. 50-100μmol/L LaCl3 decreased TNF-α-mediated IKKβphosphorylation significantly.
Another activator of NF-κB, TNF-α, induces IKKβand IκBαphosphorylation, causes degradation of IκBαand leads to the nuclear translocation of NF-κB/p65 protein, which ultimately bind with target DNA in Hela cells. Lanthanum can not only block the phosphorylation of IKKβand IκBαbut also inhibit NF-κB/p65 protein translocation and its DNA binding activity; thus blocks NF-κB signaling pathway in Hela cells.
3. The effects of lanthanum on constitutive activation of key signaling molecules in NF-κB pathway of multiple myeloma u266 cells.
(1) NF-κB activation: LaCl3 (0-100μmol/L) can not inhibit constitutive NF-κB/p65 expression and translocation of p65 subunit from the nuclear back into cytoplasm.
(2) Phosphorylation and degradation of IκBα: LaCl3 (0-100μmol/L) can not inhibit IκBαphosphrylation and degradation
(3) Phosphorylation of IKKs: IKKαand IKKβexpression and phosphorylation was not affected by LaCl3 (0-100μmol/L) treatment.
(4) At the presence of 5μmol/L LaCl3, DNA binding activity of NF-κB/p65 from nuclear extract of U266 cells decreased significantly. With increasing LaCl3 dosage, the inhibitory effects progressively increased and the binding activity was completely abolished at the highest dose (100μmol/L).
The rescults show that in NF-κB constitutive activation U266 cells, lanthanum can not inhibit phosphorylation of IKKβand IκBα, degradation of IκBαand the nuclear translocation of NF-κB/p65 protein. However, Lanthanum can block the DNA binding activity of NF-κB/p65 from the nuclear extract of U266 cells in a dose-dependent manner.
Part III The effects of lanthanum on the expression of NF-κB regulated inflammatory media
Object To explore the effects of lanthuanum on LPS-induced NF-κB -dependent gene expression in RAW264.7 cells, level of inflammatory media (TNF-αand NO) was detected and the gene expression of TNF-αand inducible nitric oxide synthase (iNOS) was measured.
Methods
(1) Cell culture and Sample treatment: The same as part I, briefly, RAW264.7 macrophages were maintained in DMEM/F12 supplemented with 5% heat-inactivated FBS at 37℃in a humidified 5% CO2 atmosphere. The cells were divided at random into 4 groups: control group, LPS group, LaCl3 group and LaCl3 + LPS group.
(2) The expression of TNF-αand iNOS mRNA: RT-PCR was employed to detect TNF-αand iNOS mRNA level in RAW264.7 cells.
(3) TNF-αin the culture supernatants was detected by ELISA (Enzym kinked immunosorbent assay).
(4) Expression and localization of iNOS: Expression and location of iNOS was detected by both IF (immunofluorescence) and western blot.
(5) NO production in culture supernatant was determinded by nitrate reductase assay. Results and conclusion
(1) TNF-αexpression: LaCl3 at low concentration (2.5μmol/L) can remarkably inhibit the expression of TNF-α, one of LPS-induced NF-κB-dependent inflammatory media, in both rest and LPS activated RAW264.7 cells.
(2) iNOS expression : Immunofluorescence analysis and western blotting detection showed LaCl3 (2.5μmol/L) can remarkably inhibit the expression of iNOS protein in LPS-induced RAW264.7 macrophages. The results of RT-PCR showed the LPS-induced expression of iNOS mRNA can be decreased by LaCl3 significantly. NO production assay indicated LaCl3 can also reduce the NO production significantly in macrophages induced by LPS.
In summary (PartI-III), NF-κB signal pathway can be blocked by lanthanum chloride via blocking the PKC signal pathway partly; And lanthanum chloride can inhibit the phosphorylation of IKKβand IκBα, suppress IκBαdegradation and nuclear translocation of NF-κB/p65 as well as attenuat NF-κB/p65-DNA binding activitaty, all these result in blockage of NF-κB signial pathway and inhibition of inflammatory media expression and release.
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