核因子-κB在HUVEC细胞凋亡信号通路中的作用

目的：探讨核因子-κB（NF-κB）在人脐静脉内皮细胞凋亡信号通路中的作用。方法：体外培养人脐静脉内皮细胞系（HUVEC）,实验分为正常对照组、AngⅡ组和Gliotoxin干预组。应用改良MTF法,观察0．01μmol／L、0．1μmol／L、μmol／L和10μmol／L4种浓度的AngⅡ在不同时间对HUVEC细胞活性的影响。应用DNA凝胶电泳和流式细胞术检测AngⅡ作用于细胞后引起细胞凋亡的情况。应用免疫细胞化学技术检测NF-κB p65的核移位,评价NF-KB活化情况。结果：10μmol／L AngⅡ作用于细胞24h时,细胞活性下降,DNA凝胶电泳和流式细胞结果提示细胞发生凋亡,凋亡细胞率明显高于正常对照组,差异具有统计学意义（P〈0．05）,0．1mg／L Gliotoxin可拮抗AngⅡ的细胞抑制活性作用;免疫细胞化学技术显示,HUVEC细胞经AugⅡ诱导后,NF-κB出现明显核移位现象,提示NF-κB发生活化;Gliotoxin明显抑制NF-κB活化,与AngⅡ组相比,差异有统计学意义（P〈0．05）。结论：①ArcⅡ可引起HU—VEC细胞发生凋亡;而NF-κB特异性抑制剂Ghotoxin能够拮抗AngⅡ对HUVEC细胞的作用;②NF-κB可能是AngⅡ调控HUVEC细胞生存／凋亡通路中的重要信号转导分子。     

双荧光素酶报告质粒检测小鼠巨噬细胞热休克转录因子1调控核转录因子kappa B活性的研究

目的：观察烧伤血清刺激后小鼠巨噬细胞NF-κB活性的变化,以及HSF1对NF-κB可能的调控作用.方法：制作15%TBSA Ⅲ°烧伤小鼠模型,提取烧伤血清.通过表达质粒与报告质粒共转染,检测烧伤血清诱导下NF-κB活性的变化以及过表达HSFI后NF-κB活性的变化规律.结果：对比正常血清,烧伤血清刺激后相对荧光素酶活性早期即明显增加（P〈0.05）,这种变化在诱导后2 h即达到高峰,12 h后逐渐下降;过表达HSF1可以显著抑制烧伤血清引起这种活性变化（P〈0.05）.结论：烧伤后NF-κB早期即活化,热休克反应可能通过HSF1途径抑制NF-κB的活性.     

NF-κB和TNF-α在暴发性肝衰竭肝组织中的表达及其意义

目的探讨NF-κB,TNF-α与暴发性肝衰竭发生、发展的关系及其意义。方法雄性Wistar大鼠腹腔注射D-GalN＋LPS制造暴发性肝衰竭模型,采用免疫组化方法动态观察肝组织中NF-κB p65蛋白和TNF-α表达情况。结果①动态观察了暴发性肝衰竭病理变化过程。②模型组NF-κB p65蛋白表达阳性细胞主要为肝细胞、枯否细胞。造模后4h、8h、12h时核阳性表达的细胞数逐渐增多,明显高于对照组。2h组主要是非实质细胞的胞质着色。而4h、8h、12h、24h组以肝细胞胞核着色为主。对照组不同时间点NF-κB p65蛋白表达无明显差异（P〉0．05）。模型组则随着肝细胞坏死程度的增加而增加（r=0．694,P〈0．01）。③随着肝细胞炎症坏死程度的增加,模型组肝细胞质TNF-α表达逐渐增加（r=0．896,P〈0．01）,以肝细胞表达为主。对照组肝细胞质TNF-α表达较弱（P〉0．05）。④肝组织中NF-κB活化与TNF-α表达呈密切正相关（r=0．852,P〈0．01）。结论NF-κB和TNF-α参与了暴发性肝衰竭的发生发展。NF-κB可能是暴发性肝衰竭肝细胞炎症坏死过程中的关键环节。     

白介素-17在牙龈上皮细胞中调控趋化因子的机制研究

目的：有研究发现白介素-17（IL-17）作为炎症反应的重要标志物在炎症反应中具有重要作用,并且其表达水平的高低与牙周疾病的严重程度有着正相关性,但是目前为止白介素-17对于牙龈上皮细胞的趋化因子生成的影响尚没有研究,所以本课题主要探索在牙龈上皮细胞（human gingival epithelial cells,HGECs）中白介素-17如何通过趋化因子调控炎症反应,并进一步探究其作用机制。方法：酶联免疫吸附剂测定法（enzyme linked immunosorbent assay,ELISA）观察细胞中相关趋化因子分泌,同时蛋白印迹法（western blot）观察细胞中核转录因子κB的变化。结果：无IL-17刺激组与IL-17刺激组比较,IL-17刺激组的趋化因子白介素8（CXCL8）分泌量显著性增加而另一趋化因子单核细胞趋化蛋白-1（CCL2）却没有显著变化,并且通过磷酸化IκB的表达量显著性增加提示NF-κB被激活（P〈0.05）;同时IL-17刺激组与IL-17＋NF-κB抑制剂组比较,IL-17＋NF-κB抑制剂组的CXCL8分泌量显著降低,而通过磷酸化IκB的表达量显著减少提示NF-κB的活性被抑制（P〈0.05）。结论：NF-κB对IL-17刺激下的牙龈上皮细胞趋化因子的表达调控具有关键性作用,同时可能也为将来治疗IL-17诱导的牙周炎症性疾病提供了新的靶点。     

川芎嗪对平滑肌细胞NF-κB激活、骨形成蛋白-2表达的影响

目的　观察川芎嗪对血管平滑肌细胞核转录因子 kappaB (NuclearFactor kappaB ,NF κB)的激活与骨形成蛋白 - 2 (bonemorphogenicprotein 2 ,BMP 2 )表达的影响。方法　贴块法培养血管平滑肌细胞 ,分为正常对照组 ,血管紧张素Ⅱ (AngiotensinⅡ ,AgⅡ )刺激组和川芎干预组。各取 15、 30、 6 0min测NF κB激活情况 ,6、 12、 2 4h测BMP 2表达变化。采用免疫组化及原位杂交法测蛋白表达和mRNA转录水平。结果　 (1)AgⅡ刺激 15min即有NF κBp6 5核转移 ,30min达高峰 (P <0 0 1) ,1h后减退。川芎干预组NF κB激活与正常组无差异。 (2 )AgⅡ刺激 6hBMP 2表达增强 (P <0 0 5 ) ,12h减弱 (P <0 0 1) ,2 4h更弱。川芎干预组 6hBMP 2表达亦增强 ,12h与 2 4h保持正常水平。结论　川芎嗪抑制AgⅡ诱导的NF κB激活与BMP 2表达降低 ,表明它在抗动脉粥样硬化方面意义重大。     

TNF-α刺激大鼠骨髓间充质干细胞的机制研究

目的：研究肿瘤坏死因子-α（Tumor necrosis factor-α,TNF-α）刺激大鼠骨髓间充质干细胞（marrow-derived mesenchymalstem cells,MSCs）的作用机制。方法：采取大鼠骨髓,以密度梯度离心分离出单个核细胞（MNCs）,于体外培养并由牛垂体提取物（PEX）诱导扩增传代培养出骨髓间充质干细胞（MSCs）。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,用TNF-α刺激骨髓间充质干细胞（MSCs）,通过酶联免疫吸附剂测定法（enzyme linked immunosorbent assay,ELISA）观察比较不同组别细胞的生长因子分泌和蛋白印迹法（western blot）来观察细胞中蛋白的变化。结果：①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞的培养成功。②无TNF-α刺激组与TNF-α刺激组比较,TNF-α刺激组的生长因子分泌显著性增加,而通过磷酸化IκB的表达量显著性增加提示NF-κB被激活（P〈0.05）;同时TNF-α刺激组与TNF-α＋NF-κB抑制剂组比较,TNF-α＋NF-κB抑制剂组的生长因子分泌显著降低,而通过磷酸化IκB的表达量显著减少提示NF-κB的活性被抑制（P〈0.05）。结论：NF-κB对TNF-α刺激下的骨髓间充质干细胞分泌生长因子有关键性作用。     

核因子-κB在脂多糖诱导大鼠心肌血红素加氧酶-1基因表达中的作用

目的：观察核因子-κB（NF-κB）在脂多糖（LPS）诱导大鼠心肌血红素加氧酶-1（HO-1）表达中的作用,探讨其对内毒素休克（ES）的影响。方法：用生理多导仪监测大鼠静脉注射LPS（8mg／Kg）后12h平均动脉压（MAP）变化：用免疫组化方法检测心肌组织NF-κBp65和HO-1蛋白表达的变化;用逆转录多聚酶链反应（RT-PCR）检测心肌组织HO-1基因表达的变化。结果：①LPS组MAP较对照组快速持续降低（P〈0．01）：②LPS可诱导大鼠心肌间质血管内皮细胞和心肌细胞NF-κB阳性表达增强,1／2h和2h表达明显升高,6h和12h逐渐降低;③LPS可诱导大鼠心肌HO-1基因表达上调,2h开始增加,6h达到高峰,12h表达下调;LPS组大鼠心肌间质血管内皮细胞和心肌细胞HO-1蛋白表达在6h明显增强,12h表达减弱。④应用PDTC可明显减轻ES大鼠心肌损伤,并抑制心肌HO-1蛋白和基因表达。结论：LPS活化的心肌NF-κB参与LPS诱导心肌HO-1蛋白和基因高表达的信号转导,可能是导致ES顽固性低血压的机制之一。     

乳酸调控大鼠肠黏膜微血管内皮细胞的NF-κB信号通路

目的探讨内毒素（LPS）刺激大鼠肠黏膜微血管内皮细胞（RIMMVECs）后,乳酸（LA）调控NF-κB信号通路中磷酸化IκBα和NF-κB p65蛋白表达情况,肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）mRNA表达情况,阐明乳酸发挥作用的最佳时间及其调控NF-κB信号通路的部位。方法提取RIMMVECs总蛋白和总RNA,用Western blotting检测NF-κB p65、IκBα及p-IκBα蛋白表达水平,用real-time PCR对TNF-α和IL-6 mRNA进行定量检测。结果乳酸能降低LPS诱导RIMMVECs分泌的TNF-α和IL-6 mRNA表达水平,并分别于24 h和3 h下调效果最明显;乳酸能抑制IκBα磷酸化及NF-κB转录活性,并于4～8 h达到最佳效果;乳酸发挥作用部位是抑制信号通路中IκBα磷酸化。结论乳酸通过抑制IκBα磷酸化而阻断NF-κB的激活,抑制下游炎性因子表达,进而发挥出很好的预防炎症效果。     

坏死细胞释放IL-1β对血管平滑肌细胞侵袭和迁移的影响及机制研究

目的研究坏死血管平滑肌细胞释放IL-1β对周围正常细胞侵袭和迁移的影响,并探讨其可能的机制。方法无血清低糖低氧条件下培养细胞,建立细胞坏死模型,收集坏死细胞培养上清液,用于干预正常血管平滑肌细胞。实验设坏死上清组（NCS）、IL-1β组、NCS＋IL-1RA组和对照组（Control）。Tran-swell小室实验和细胞划痕实验检测各组细胞侵袭和迁徙能力,RT-PCR和Western印迹检测各组MMP-2和MMP-9的表达,Western印迹检测各组NF-κB的激活情况。结果NCS组和IL-1β组细胞侵袭和迁移能力均显著高于对照组（P〈0．05）,NCS＋IL-1RA组细胞侵袭和迁移能力显著低于NCS组和IL-1组（P〈0．05）;对照组和NCS＋IL．1RA组NF-κB、MMP-2和MMP-9均显著低于NCS组和IL-1β组（P〈0．05）,IκB显著高于NCS组和IL-1β组（P〈0．05）。使用NF-κB特异性抑制剂处理后,NCS组和IL-1β组细胞侵袭和迁移能力显著性降低（P〈0．01）;MMP-2和MMP-9的表达也显著性减少（P〈0．01）。结论坏死血管平滑肌细胞能够上调MMP-2和MMP-9的表达,促进正常细胞的侵袭和迁移,且这种促进作用与IL-1β的大量释放诱导NF-κB的活化有关。     

Anti-proliferative effect of rosiglitazone on angiotensin II-induced vascular smooth muscle cell proliferation is mediated by the mTOR pathway

VSMC (vascular smooth muscle cell) proliferation contributes significantly to intimal thickening in atherosclerosis, restenosis and venous bypass graft diseases. Ang II (angiotensin II) has been implicated in VSMC proliferation though the activation of multiple growth-promoting signals. Although TZDs (thiazolidinediones) can inhibit VSMC proliferation and reduce Ang II-induced fibrosis, the mechanism underlying the inhibition of VSMC proliferation and fibrosis needs elucidation. We have used primary cultured rat aortic VSMCs and specific antibodies to investigate the inhibitory mechanism of rosiglitazone on Ang II-induced VSMC proliferation. Rosiglitazone treatment significantly inhibited Ang II-induced rat aortic VSMC proliferation in a dose-dependent manner. Western blot analysis showed that rosiglitazone significantly lowered phosphorylated ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt (also known as protein kinase B), mTOR (mammalian target of rapamycin), p70S6K (70 kDa S6 kinase) and 4EBP1 (eukaryotic initiation factor 4E-binding protein) levels in Ang II-treated VSMCs. In addition, PPAR-γ (peroxisome-proliferator-activated receptor γ) mRNA increased significantly and CTGF (connective tissue growth factor), Fn (fibronectin) and Col III (collagen III) levels decreased significantly. The results demonstrate that the rosiglitazone directly inhibits the pro-atherosclerotic effect of Ang II on rat aortic VSMCs. It also attenuates Ang II-induced ECM (extracellular matrix) molecules and CTGF production in rat aortic VSMCs, reducing fibrosis. Importantly, PPAR-γ activation mediates these effects, in part, through the mTOR-p70S6K and -4EBP1 system.     

YS 49, 1-(alpha-naphtylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, regulates angiotensin II-stimulated ROS production, JNK phosphorylation and vascular smooth muscle cell proliferation via the induction of heme oxygenase-1

Overexpression of the gene for heme oxygenase (HO)-1 leads to a reduction in pressor responsiveness to angiotensin II (Ang II) in experimental animals. Using rat vascular smooth muscle cells (VSMCs), we tested whether YS 49 [1-(alpha-naphtylmethyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline] inhibits Ang II-stimulated proliferation of VSMCs via induction of HO-1. YS 49 induced HO-1 protein production in a dose-and time-dependent manner in VSMCs. Treatment with YS 49 significantly and dose-dependently inhibited Ang II-induced VSMC proliferation, ROS production, and phosphorylation of JNK, but not P38 MAP kinase or ERK1/2. The antiproliferation effect of YS 49 was reversed by pretreatment with the HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX), or with hemoglobin, a carbon monoxide (CO) scavenger. Similarly, VSMC proliferation, ROS production and phosphorylation of JNK by Ang II were significantly inhibited in VSMCs transfected with the HO-1 gene. Thus, HO-1 and the HO-1 product CO play, at least in part, a crucial role in Ang II-stimulated VSMC proliferation through the regulation of ROS production and JNK phosphorylation. Therefore, YS 49 has potential as a therapeutic strategy for the pathogenesis of Ang II-related vascular diseases such as hypertension and atherosclerosis, via the induction of HO-1 gene activity.     

Rosiglitzone Suppresses Angiotensin II-Induced Production of KLF5 and Cell Proliferation in Rat Vascular Smooth Muscle Cells

Krüppel-like factor (KLF) 5, which initiates vascular smooth muscle cell (VSMC) proliferation, also participates in Angiotensin (Ang) II-induced vascular remodeling. The protective effect of rosiglitazone on vascular remodeling may be due to their impact on VSMC proliferation. However, the underlying mechanisms involved remain unclear. This study was designed to investigate whether the antiproliferation effects of rosiglitazone are mediated by regulating Ang II/KLF5 response. We found that, in aortas of Ang II-infused rats, vascular remodeling and KLF5 expression were markedly increased, and its target gene cyclin D1 was overexpressed. Co-treatment with rosiglitazone diminished these changes. In growth-arrested VSMCs, PPAR-γ agonists (rosiglitazone and 15d-PGJ2) dose-dependently inhibited Ang II-induced cell proliferation and expression of KLF5 and cyclin D1. Moreover, these effects were attenuated by the PPAR-γ antagonists GW9662, bisphenol A diglycidyl ether and PPAR-γ specific siRNA. Furthermore, rosiglitazone inhibited Ang II-induced phosphorylation of protein kinase C (PKC) ζ and extracellular signal-regulated kinase (ERK) 1/2 and activation of early growth response protein (Egr). In conclusion, in Ang II-stimulated VSMCs, rosiglitazone might have an antiproliferative effect through mechanisms that include reducing KLF5 expression, and a crosstalk between PPAR-γ and PKCζ/ERK_1/2/Egr may be involved in. These findings not only provide a previously unrecognized mechanism by which PPAR-γ agonists inhibit VSMC proliferation, but also document a novel evidence for the beneficial vascular effect of PPAR-γ activation.     

Down-regulation by antisense oligonucleotides establishes a role for the proline-rich tyrosine kinase PYK2 in angiotensin ii-induced signaling in vascular smooth muscle

Abnormal vascular smooth muscle cell (VSMC) growth plays a key role in the pathogenesis of hypertension and atherosclerosis. Angiotensin II (Ang II) elicits a hypertrophic growth response characterized by an increase in protein synthesis in the absence of DNA synthesis and cell proliferation. Intracellular signaling mechanisms linking angiotensin type I receptor activation to protein synthesis in VSMC have not been fully characterized. The present study investigates the role of the nonreceptor proline-rich tyrosine kinase 2 (PYK2) in Ang II-induced VSMC protein synthesis and in the regulation of two signaling pathways that have been implicated in the control of protein synthesis, the extracellular signal-regulated kinase (ERK1/2) and the phosphatidylinositol 3-kinase/Akt pathways. PYK2 antisense oligonucleotides were used to down-regulate PYK2 expression in cultured VSMC. An 80% down-regulation in PYK2 expression resulted in an approximately 80% inhibition of ERK1/2 (3.8 +/- 1.3 versus 16.6 +/- 1.8), p70S6 kinase (1.03 +/- 0.03 versus 3.8 +/- 0.5), and Akt activation (3.0 +/- 0.8 versus 16.0 +/- 1.0) by Ang II. Furthermore, PYK2 down-regulation resulted in a complete inhibition of Ang II-induced VSMC protein synthesis. These data conclusively identify PYK2 as an upstream regulator of both the ERK1/2 and the phosphatidylinositol 3-kinase/Akt pathways that are involved in Ang II-induced VSMC protein synthesis.     

Dimethylarginine dimethylaminohydrolase-1 mediates inhibitory effect of interleukin-10 on angiotensin II-induced hypertensive effects in vascular smooth muscle cells of spontaneously hypertensive rats

In hypertension studies, anti-inflammatory cytokine interleukin-10 (IL-10) has been shown to prevent angiotensin II (Ang II)-induced vasoconstriction and regulate vascular function by down-regulating pro-inflammatory cytokine and superoxide production in vascular cells. However, little is known about the mechanism behind the down-regulatory effect of IL-10 on Ang II-induced hypertensive mediators. In this study, we demonstrated the effects of IL-10 on expression of dimethylarginine dimethylaminohydrolase (DDAH)-1, a regulator of NO bioavailability, as well as the down-regulatory mechanism of action of IL-10 in relation to Ang II-induced hypertensive mediator expression and cell proliferation in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR). IL-10 increased DDAH-1 but not DDAH-2 expression and increased DDAH activity. Additionally, IL-10 attenuated Ang II-induced DDAH-1 inhibition in SHR VSMCs. Increased DDAH activity due to IL-10 was mediated mainly through Ang II subtype II receptor (AT₂ R) and AMP-activated protein kinase (AMPK) activation. DDAH-1 induced by IL-10 partially mediated the inhibitory action of IL-10 on Ang II-induced 12-lipoxygenase (LO) and endothelin (ET)-1 expression in SHR VSMCs. In addition, the inhibitory effect of IL-10 on proliferation of Ang II-induced VSMCs was mediated partially via DDAH-1 activity. These results suggest that DDAH-1 plays a potentially important role in the anti-hypertensive activity of IL-10 during Ang II-induced hypertension.     

The effects of thiazolidinediones on vascular smooth muscle cell activation by angiotensin II

Angiotensin II (Ang II) stimulates the activation of extracellular signal-regulated kinase (ERK), a subgroup of the mitogen-activated protein kinase (MAPK) family, in cultured vascular smooth muscle cells (VSMC). This ERK activation was recently shown to be a critical regulatory factor for Ang II-mediated migration and growth. It has been demonstrated that the thiazolidinedione troglitazone (TRO) blocked Ang II-induced DNA synthesis and migration in VSMC. Here we provide evidence for TRO to inhibit Ang II-induced ERK activation which was suggested to constitute the mechanism by which this agent blocks Ang II-induced VSMC growth and migration. We have found that pretreatment with PD98059, which selectively blocks the activity of ERK pathway at the level of MAPK kinase, decreased Ang II-induced AP-1 activation and that TRO is capable of inhibiting Ang II-induced AP-1 activation. On the other hand, the other thiazolidinediones pioglitazone (PIO) and rosiglitazone (ROSI) had little effect on Ang II-induced activation of ERK or AP-1, suggesting the inhibitory effects of TRO on VSMC activation by Ang II be independent of the peroxisome proliferator-activated receptor-gamma (PPARgamma) for which thiazolidinediones are ligands. Ang II-induced ERK activation was inhibited by protein kinase C (PKC)-specific inhibitor GF109203X, while TRO was also able to block PKC activator phorbol 12 myristate 13-acetate (PMA)-induced ERK activation. Accordingly, TRO may inhibit Ang II-induced MAPK activation at least partly by an inhibition of PKC. These results support the assumption that by targeting MAPK activation, TRO may inhibits the critical signaling steps leading to restenosis and atherosclerosis that may result in part from dysregulated VSMC growth and migration induced by Ang II.