银杏黄酮苷元对氧化低密度脂蛋白所致内皮细胞炎症损伤的影响

目的：观察银杏黄酮苷元（ GA）对氧化低密度脂蛋白（ ox－LDL）诱导人主动脉内皮细胞（HAECs）细胞间粘附分子－1（ICAM－1）、血管细胞粘附分子－1（VCAM－1）、单核细胞趋化蛋白－1（MCP－1）的影响及核转录因子－κB（NF－κB）在其中的调控作用。方法体外培养HAECs,建立HAECs的ox－LDL损伤模型前,用不同浓度GA预处理细胞1 h,用四甲基噻唑兰法检测内皮细胞活性,免疫荧光法检测细胞核内NF－κB的激活,细胞酶联免疫吸附法检测MCP－1、ICAM－1及VCAM－1水平。结果30～60 mg · L－1 GA预处理组显著抑制ox－LDL诱导的内皮细胞NF－κB激活（ P＜0.05）,下调VCAM－1、ICAM－1及MCP－1表达（ P＜0.05）。结论 GA能够有效抑制ox－LDL诱导HAECs炎症因子ICAM－1、VCAM－1、MCP－1表达。     

远志对Aβ25—35诱导AD模型大鼠海马区NF—κB活化研究

目的：观察远志对双侧海马注射Aβ25—35建立的AD模型大鼠海马区NF—κB p65的活化、P-IκB、IκB表达的影响,从NF—κB信号转导通路调控机制探讨远志对AD可能的防治作用途径。方法：将96只大鼠随机分为正常对照组,假手术组,模型组,远志低、中、高剂量组共6组,每组16只。采用双侧海马注射β淀粉样多肽25—35片断（Aβ25—35）制作大鼠AD模型;连续给药4周后采用免疫荧光法检测各组大鼠NF—κB p65活化;免疫组化法观察P—IκB、IκB在大鼠海马区的表达。结果：远志能抑制AD模型大鼠海马区NF—κB p65活化。结论：远志能够抑制AD模型大鼠海马区NFκB p65活化,这种作用主要是通过抑制IκBa磷酸化降解而不是通过刺激IκBa蛋白合成增加来实现的。     

磷脂酰肌醇3—激酶调控白介素—18诱导核因子—κB活化

目的：研究磷脂酰肌醇（PI）3－激酶在白介素18（IL－18）诱导核因子－κB（NF－κB）活化中的作用。方法：Lipofectin介导反义PI 3－激酶寡核苷酸转染HepG2细胞。用逆转录PCR法检测PI 3－激酶mRNA表达水平,以Sandwich ELISA法检测NF－κB的活化。结果：1）反义PI 3－激酶寡核苷酸抑制PI 3－激酶mRNA表达。（2）IL－18诱导NF－κB活化。（3）反义PI 3－激酶寡核苷酸呈时间（5－24h)和浓度（1－8mg/L)依赖性地抑制IL－18诱导的NF－κB活化。结论：PI 3－激酶调控白介素－18诱导的NF－κB活化。     

大黄素对心肌梗死后小鼠心肌组织TNF-α、IL-10基因表达和NF—κB活性的影响

目的：研究大黄素对小鼠缺血后心肌局部细胞因子TNF—α和IL-10表达及NF—κB活性的影响。方法：持续性结扎小鼠冠状动脉左前降支造成心肌缺血损伤模型,用大黄素进行干预。实时定量PCR检测干预后心肌组织的TNF—α和IL-10的mRNA,ELISA法检测TNF—α和IL-10蛋白水平,EMSA测定NF—κB的活性。结果：经大黄素干预后,心肌组织TNF—α的表达明显减少（P〈O．01）,IL-10的表达增加（P〈0．01）,IL-10／TNF—α的比值显著性升高,NF—κB的活性明显减弱（P〈0．01）。结论：大黄素显著抑制干预后心肌组织促炎性细胞因子的表达,抑制NF—κB的活性。     

粉防己碱对脑缺血／再灌注导致的中性粒细胞募集反应的作用

目的：探讨粉防己碱对脑缺血／再灌注（I／R）后中性粒细胞募集反应的作用及分子机制。方法：大鼠（♂）脑中动脉线栓闭塞（2h)／再通法建立局灶性脑缺血／再灌注模型。采用干湿重法、^51Cr标记中性粒细胞法、逆转录聚合酶链反应和凝胶迁移电泳分别检测脑组织含水量、中性粒细胞募集、细胞间粘附分子－1（ICAM－1）mRNA转录的水平以及细胞核内激活的核因子－κB（NF－κB）的水平。结果：脑含水量和中性粒细胞募集数量从再灌注3h到24h平行增加,在再灌注1h可检测到ICAM－1 mRNA的表达,12h达到最高值,24h下降到3h的水平。在再灌注0.5h可检测到NF－κB的激活,6h达到最高值,24h下降到1h的水平。在再灌注6h、12h和24h,粉防己碱（10和20mg/kg)可抑制这些变化。结论：粉防己碱可抑制脑缺血／再灌注后中性粒细胞的募集,ICAM－1 mRNA的转录和NF－κB的激活。     

丹皮酚通过抑制NF-κB信号通路诱导Tca8113细胞凋亡

目的 研究丹皮酚对核因子κB（NF-κB）信号通路的影响,探讨其诱导肿瘤细胞凋亡的分子机制。方法 将丹皮酚以不同浓度及不同时间作用于人舌鳞癌Tca8113细胞,应用MTT法观察不同浓度丹皮酚在不同作用时间下对Tca8113细胞增殖的抑制作用,Hoechst 33342荧光染色及流式细胞仪检测细胞凋亡情况,Western blot法检测凋亡相关蛋白Bax和Bcl-2,以及NF-κB信号通路组分中IκBα和p-IκBα的表达情况,并采用凝胶电泳迁移率检测（EMSA）对NF-κB活性进行测定。结果 经丹皮酚作用后,Tca8113细胞生长受到明显抑制,并呈明显的时间、剂量依赖效应关系。丹皮酚有明显诱导Tca8113细胞凋亡的作用,Western Blot 以及EMSA检测结果显示,丹皮酚上调Tca8113细胞中Bax与IκBα的表达,下调Bcl-2的表达,并能下调IκBα磷酸化及NF κB活性。结论 丹皮酚能够抑制NF-κB信号转导通路,进而抑制Tca8113细胞的增殖并诱导其凋亡。     

姜黄素通过阻断NF-κB减少IL-1β诱导的内皮脂酶表达

目的观察姜黄素对培养的人血管内皮细胞内皮脂酶表达的影响,并探讨其可能的作用机制。方法不同浓度的姜黄素处理HUVEC-12细胞。RT-PCR检测内皮酯酶(endo-thelial lipase,EL)mRNA的表达;Western blot检测核因子-κB抑制因子-α(inhibitor of nuclear factor-κB-α,IκB-α)蛋白的表达;间接免疫荧光检测核因子-κB(nuclear factor-κB,NF-κB)蛋白的活化。结果IL-1β处理HUVEC-12细胞可以明显上调EL mRNA的表达,同时降低胞质蛋白IκB-α的表达水平,激活核转录因子NF-κB,增加胞核蛋白NF-κB的水平。姜黄素预处理HUVEC-12细胞可以抑制IL-1β对EL的上调作用,同时逆转IL-1β诱导的IκB-α蛋白降解和NF-κB活化。结论姜黄素可以通过阻断NF-κB活化减少IL-1β诱导的人血管内皮细胞EL的表达。     

阿托伐他汀对LPS诱导人血管内皮细胞IκBα表达的影响

目的通过观察阿托伐他汀对LPS诱导的人血管内皮细胞IκBα表达的影响来探明其抑制核因子κB活性的机制。方法将培养的人血管内皮细胞株ECV304,分为对照组、LPS组、阿托伐他汀低、中、高3个浓度组。阿托伐他汀3组加入不同阿托伐他汀孵育后与LPS组均加入LPS刺激30min。用免疫荧光法观察核因子κB活化情况,用逆转录-聚合酶链反应观察IκBα mRNA的表达情况,用蛋白印迹法观察IκBα及p-IκBα蛋白含量的变化。结果阿托伐他汀3组能够抑制p65由胞质转移至核内、能够剂量依赖性的降低pIκBα蛋白表达、减少IκBα的降解;阿托伐他汀高浓度组IκBα mRNA的表达增加。结论阿托伐他汀可以通过影响IκBα的表达和降解来抑制核因子κB活性。     

脂氧素A4、保护素D1、ResolvinD1抑制多种激动剂引起的NFκB的活化

目的探讨脂质小分子脂氧素A4(LXA4)、保护素D1(ProD1)、ResolvinD1(RvD1)对核因子κB(NFκB)活性的影响及作用机制。方法稳定表达NFκB荧光素酶报告基因的中国仓鼠卵巢细胞分别由100 nmol/L LXA4、ProD1、RvD1预处理30 min后,细胞被激动剂LPS、HSP70、HMGB1或S100A4刺激。通过检测荧光素酶活性以评价脂质小分子对激动剂激活NFκB活性的作用。细胞培养上清中TNFα的含量由ELISA检测,胞核中NFκB的含量由Western印迹检测。结果 LPS、HSP70、HMGB1和S100A4显著上调NFκB的活性,增加细胞分泌的TNFα的量。LXA4、ProD1、RvD1显著抑制NFκB激活,降低细胞分泌的TNFα含量,减少NFκB的入核。结论 LXA4、ProD1、RvD1显著抑制多种激动剂活化NFκB,其作用机制可能与其能降低NFκB的入核有关,这几个脂质小分子在研制新型抗炎药物方面具有进一步开发和研究的潜力。     

葛根素联合门冬胰岛素对妊娠期糖尿病患者 TLR4／NF－κB 炎症信号通路及脂肪因子的影响

目的：观察葛根素联合门冬胰岛素对妊娠期糖尿病（ GDM）患者外周血单核细胞Toll样受体4／核因子－κB（ TLR4／NF－κB）炎症信号通路及脂肪因子的影响。方法选取2013年6月至2015年12月诊治的GDM患者100例,随机分为对照组和观察组,每组50例。对照组给予门冬胰岛素治疗,观察组在对照组基础上加用葛根素注射液治疗。比较2组外周血单核细胞（ PBMC） TLR4、NF－κB、IκB mRNA和蛋白表达及血清白细胞介素6（ IL－6）、白细胞介素12（IL－12）、肿瘤坏死因子α（TNF－α）、C－反应蛋白（CRP）、网膜素（chemerin）、脂联素（APN）、内脂素（visfatin）变化。结果2组治疗后TLR4、NF－κB mRNA和蛋白表达、IL－6、IL－12、TNF－α、CRP、visfatin水平较治疗前显著降低,IκB mR－NA和蛋白表达、chemerin、APN水平较治疗前显著升高（ P ＜0．05）,但观察组以上指标改善程度优于对照组,差异有统计学意义（ P ＜0．05）。结论葛根素联合门冬胰岛素能够明显抑制TLR4／NF－κB炎症信号通路活化引起的炎性因子的释放,并调控脂肪因子分泌。     

Ethyl pyruvate modulates acute inflammatory reactions in human endothelial cells in relation to the NF-kappaB pathway

BACKGROUND AND PURPOSE: Endothelial cell activation plays a critical role in regulating leukocyte recruitment during inflammation and infection. Ethanol (EtOH) reduces host defence systems, including cell adhesion. However, well-known side effects of EtOH limit its clinical use as an anti-inflammatory drug. Instead, ethyl pyruvate (EtP) may represent a better alternative. Here, we compared effects of EtP and EtOH on neutrophil recruitment and activation of human umbilical vein endothelial cells (HUVECs). EXPERIMENTAL APPROACH: Adhesion of neutrophils to HUVEC monolayers, surface expression of intercellular cell adhesion molecule, E-selectin, vascular cell adhesion molecule, release of interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF) from HUVECs were assessed as well as translocation of interleukin-1 receptor-associated kinase (IRAK-1), the nuclear factor-kappa B (NF-kappaB) subunits p50, p65 and IkappaB-alpha. NF-kappaB activation was analysed with a luciferase reporter plasmid. Cells were stimulated with IL-1beta, lipopolysaccharide (LPS) or tumour necrosis factor-alpha.Key results:EtP was several-fold more potent than EtOH in reducing adhesion of neutrophils to activated HUVECs, generation of IL-8 or G-CSF and surface expression of the adhesion molecules. This last reaction was decreased by EtP even when added after cytokines or LPS. Translocation of IRAK-1, IkappaBalpha and the NF-kappaB p65 subunit to the HUVEC nucleus was inhibited by EtP for all stimuli, whereas the diminished p50 translocation was stimulus specific. When p65 was constitutively expressed in Cos7 cells, stimulation of an NF-kappaB-dependent reporter gene was not affected by EtP, suggesting that EtP acted upstream of gene activation. CONCLUSIONS AND IMPLICATIONS: EtP impedes adhesive, secretory and signalling events typical of the early inflammatory response in endothelial cells, suggesting EtP as a possible treatment for acute inflammatory conditions.     

Deguelin inhibits expression of IκBα protein in Raji and U937 cells

AIM: To determine whether deguelin can regulate the expression of nuclear factor kappa B (NF-kappaB) binding protein (IkappaBalpha) in U937 human leukemia cells and Raji human B lymphoma cells. METHODS: The localization of IkappaBalpha protein was investigated by using an immunofluorescence method. The expression of IkappaBalpha and NF-kappaB /p65 proteins in Raji and U937 cells were investigated by using Western blotting. Apoptosis was detected through annexin V/PI double-labeled cytometry. RESULTS: IkappaBalpha localized in the cytoplasm in untreated and deguelin-treated cells. After treatment with tumor necrosis factor alpha (TNF-alpha) or deguelin plus TNF-alpha for 15 min, there was a substantial reduction in the amount of IkappaBalpha protein. The expression of IkappaBalpha was downregulated by deguelin in Raji and U937 cells. Deguelin induced apoptosis in U937 cells. CONCLUSION: Deguelin inhibited the expression of IkappaBalpha protein in U937 and Raji cells. The anti-proliferative activity of deguelin is related to the signal pathway of NF-kappaB.     

Inhibition of the ERK phosphorylation plays a role in terbinafine-induced p21 up-regulation and DNA synthesis inhibition in human vascular endothelial cells

Previously, we showed that terbinafine (TB) induces cell-cycle arrest in cultured human umbilical vein endothelial cells (HUVEC) through an up-regulation of the p21 protein. The aim of this study is to delineate the molecular mechanisms underlying TB-induced increase of p21 protein. RT-PCR analysis demonstrated that the mRNA levels of p21 and p53 were increased in the TB-treated HUVEC. The p21 promoter activity was also increased by TB treatment. Transfection of HUVEC with p53 dominant negative (DN) abolished the TB-induced increases of p21 promoter activity and protein level, suggesting that the TB-induced increase of p21 is p53-dependent. Western blot analysis demonstrated that TB decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK). Over-expression of mitogen-activated protein kinase (MEK)-1, the immediate upstream activator kinase of ERK, abolished the TB-induced increases of p21 and p53 protein and decrease of thymidine incorporation. The ERK inhibitor (PD98059) enhanced the TB-induced inhibition of thymidine incorporation into HUVEC. Taken together, these data suggest that the decrease of ERK activity plays a role in the TB-induced up-regulation of p21 in HUVEC. On the other hand, pretreatment of the cells with geranylgeraniol (GGOH), farnesol (FOH), or Ras inhibitor peptide did not affect the TB-induced decrease of thymidine incorporation. Taken together, our results suggest that TB might cause a decrease of MEK, which in turn up-regulates p53 through the inhibition of ERK phosphorylation, and finally causes an increase of p21 expression and cell-cycle arrest.     

前列腺素A1通过NF—κB介导抑制大鼠心脏微血管内皮细胞凋亡

目的:研究前列腺素Al(PGAl)对心脏微血管内皮细胞凋亡的影响.方法:培养分离大鼠心脏微血管内皮细胞,建立缺氧再给氧模型,通过原位缺口末端标记观察PGAl对内皮细胞凋亡的作用;通过凝胶电泳迁移率测定NF-κB的活性;用Western blot法测定Bcl-2和Bax蛋白表达;用Northern blot法测定bcl-2 mRNA的表达.结果:PGAl能明显减少缺氧再给氧内皮细胞的凋亡,抑制NF-κB的活性,升高Bcl-2蛋白及bcl-2 mRNA的表达,不改变Bax蛋白的表达,导致Bcl-2/Bax增加.结论:PGAl通过NF-κB介导抑制大鼠心脏微血管内皮细胞的凋亡.     

Protection from experimental colitis by theaflavin-3,3'-digallate correlates with inhibition of IKK and NF-kappaB activation

BACKGROUND AND PURPOSE: Inflammatory bowel disease (IBD) is associated with activation of nuclear factor kappa B (NF-kappaB) involved in regulating the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokine genes. As theaflavin-3,3'-digallate (TFDG), the most potent anti-oxidant polyphenol of black tea, down-regulates NF-kappaB activation, we investigated if TFDG is beneficial in colonic inflammation by suppressing iNOS and proinflammatory cytokines. EXPERIMENTAL APPROACH: The in vivo efficacy of TFDG was assessed in mice with trinitrobenzene sulfonic acid (TNBS)-induced colitis. Both mRNA and protein levels of proinflammatory cytokines and iNOS were analyzed in colon tissue treated with or without TFDG. NF-kappaB activation was determined by electrophoretic mobility shift assay and levels of NF-kappaB inhibitory protein (IkappaBalpha) were analyzed by Western blotting. KEY RESULTS: Oral administration of TFDG (5 mg kg(-1) daily i.g.) significantly improved TNBS-induced colitis associated with decreased mRNA and protein levels of TNF-alpha, IL-12, IFN-gamma and iNOS in colonic mucosa. DNA binding and Western blotting revealed increase in NF-kappaB activation and IkappaBalpha depletion in TNBS-treated mice from Day 2 through Day 8 with a maximum at Day 4, which resulted from increased phosphorylation of IkappaBalpha and higher activity of IkappaB kinase (IKK). Pretreatment with TFDG markedly inhibited TNBS-induced increases in nuclear localization of NF-kappaB, cytosolic IKK activity and preserved IkappaBalpha in colon tissue. CONCLUSIONS AND IMPLICATIONS: TFDG exerts protective effects in experimental colitis and inhibits production of inflammatory mediators through a mechanism that, at least in part, involves inhibition of NF-kappaB activation.