脂多糖诱导的血管外膜炎症对兔股动脉内膜增生的影响及机制

通过用脂多糖诱导兔股动脉外膜炎症,来观察其对内膜增生的影响,并用免疫组织化学法观察股动脉壁核因子κB p65的表达情况,从而探讨外膜炎症对内膜增生影响的分子机制。高脂饮食饲养新西兰大耳白兔24只,两侧股动脉按实验要求分为分为脂多糖组(n=24)和对照组(n=24)。制备用脂多糖诱导的外膜炎症模型,于术后0天(n=8)、3天(n=8)和2周(n=8)处死动物取得股动脉标本,HE染色观察形态学变化和内膜增生情况,同时用免疫组织化学方法镜下观察核因子κB p65在动脉壁的表达。结果发现3天和2周时,脂多糖组血管外膜和内膜面炎症细胞明显增多。在3天时,脂多糖组血管内皮细胞和外膜细胞在胞浆中出现核因子κB p65阳性表达。计算机图象分析发现,2周时脂多糖组内膜面积(0.93±0.14mm2)和对照组(0.75±0.15mm2)相比有明显差异(P<0.05)。结果提示脂多糖诱导的血管外膜炎症可导致兔股动脉内膜增生,而在内膜增生之前伴随着血管内皮细胞和外膜细胞核因子κBp65的阳性表达,外膜炎症可能是通过激活上述细胞中的核因子κB从而启动炎性细胞因子的转录导致内膜增生。     

核因子κB反义和诱骗寡核苷酸联合作用对培养人脐静脉内皮细胞血管细胞粘附分子1表达的影响

为探讨在人脐静脉内皮细胞中，核因子κB的反义核酸及诱骗性寡核苷酸联合作用对肿瘤坏死因子α诱导的血管细胞粘附分子1表达的影响，采用培养的人脐静脉内皮细胞株ECV304，分别和联合应用反义核酸和诱骗性寡核苷酸干预．流式细胞仪检测寡核苷酸转染效率．并用逆转录一聚合酶链反应和流式细胞仪测定其对肿瘤坏死因子α诱导的血管细胞粘附分子1的表达。结果发现．联合应用反义核酸及诱骗性寡核苷酸，可使肿瘤坏死因子α刺激的人脐静脉内皮细胞中血管细胞粘附分子1的表达明显下调，且下调幅度显著大于反义核酸或诱骗性寡核苷酸的单独作用。由此提示．核因子κB的反义核酸及诱骗性寡核苷酸可显著下调核因子κB调控的与动脉粥样硬化相关的粘附分子的表达，且联合作用效果强于单独作用，可能为核酸干预防治动脉粥样硬化提供新的思路。     

重组腺病毒IκBαM抑制高糖诱导的血管内皮细胞炎症因子的异常分泌

构建携有核因子κB抑制物IκBα的突变体IκBαM的重组腺病毒(AdIκBαM)，并感染ECV304血管内皮细胞，采用Westem blot、电泳迁移率变动分析和逆转录-聚合酶链反应等方法研究核因子κB在高浓度葡萄糖刺激的ECV304细胞分泌功能中所起的作用以及阻断其活性对内皮细胞分泌功能的影响。结果发现，高糖能诱导ECV304细胞IκBa的降解和核因子κB的激活，同时上调炎症因子细胞间粘附分子1、单核细胞趋化蛋白1和内皮素1mRNA表达水平，而感染AdlIκBαM的ECV304／IκBαM细胞则能拮抗高糖所致的IκBα降解与核因子κB激活，同时下调这些炎症因子的异常分泌水平。结果提示，核因子κB的激活在高糖所致的内皮细胞分泌功能改变中起中轴作用，抑制核因子κB的活化，有助于改善血管内皮细胞功能。     

阿魏酸通过抑制核因子κB信号途径降低肿瘤坏死因子α诱导的人血管内皮细胞氧化应激及黏附分子表达

目的 通过检测人脐静脉内皮细胞的氧化应激、细胞黏附分子和炎症相关信号分子表达,探讨阿魏酸在肿瘤坏死因子α(TNF-α)诱导的血管内皮损伤中的保护作用和机制.方法 以人脐静脉内皮细胞为研究模型,给予阿魏酸预处理,在基础或者TNF-α刺激条件下,蛋白质免疫印记法检测细胞间黏附分子1(ICAM-1)、血管细胞黏附分子1(VCAM-1)表达和炎症相关细胞信号分子;MTT法检测内皮细胞活性;Hoechst 33342细胞核染色检测细胞凋亡比率;以DHE染色检测氧自由基(ROS)生成;单核细胞和血管内皮细胞黏附实验检测血管内皮细胞的黏附功能;以蛋白质免疫印记法和细胞免疫荧光实验检测核因子κB(NF-κB)的活化.结果 TNF-α可以明显增加血管内皮细胞氧自由基生成及ICAM-1和VCAM-1的表达,并且可以明显激活NF-κB,促进其核转位.阿魏酸可以明显抑制TNF-α诱导的血管内皮细胞氧自由基生成及ICAM-1和VCAM-1表达升高,抑制NF-κB活化和核转位.结论 阿魏酸可以通过抑制NF-κB信号途径减轻TNF-α导致的血管内皮细胞氧自由基增高、黏附分子表达,抑制炎性因子导致的细胞损伤.     

川芎嗪对血管壁细胞血管细胞粘附分子1和单核细胞趋化蛋白1表达的作用及可能机制

目的观察川芎嗪对血管内皮细胞和平滑肌细胞表达血管细胞粘附分子1、单核细胞趋化蛋白1和核因子κB的影响,探讨川芎嗪抗动脉粥样硬化分子机制。方法体外培养人脐静脉内皮细胞和大鼠胸主动脉平滑肌细胞。用氧化型低密度脂蛋白、氧化型极低密度脂蛋白、血管紧张素Ⅱ和(或)川芎嗪作用于细胞后,采用免疫细胞化学和原位分子杂交检测各组细胞血管细胞粘附分子1、单核细胞趋化蛋白1和核因子κB的表达情况。用单核细胞粘附试验检测川芎嗪对单核细胞粘附于内皮细胞的影响。结果氧化型低密度脂蛋白、氧化型极低密度脂蛋白或血管紧张素Ⅱ诱导内皮细胞血管细胞粘附分子1蛋白相对表达量分别为0.552±0.008、0.460±0.006和0.486±0.025,明显高于对照组的0.365±0.019(P<0.01);诱导平滑肌细胞血管细胞粘附分子1蛋白相对表达量分别为0.564±0.007、0.513±0.021和0.524±0.008,明显高于对照组(0.416±0.013,P<0.01)。氧化型低密度脂蛋白、氧化型极低密度脂蛋白或血管紧张素Ⅱ诱导内皮细胞单核细胞趋化蛋白1蛋白相对表达量分别为0.962±0.051、0.878±0.014和0.824±0.006,明显高于对照组的0.303±0.008(P<0.01);诱导平滑肌细胞单核细胞趋化蛋白1蛋白相对表达量分别为0.877±0.011、0.845±0.023和0.881±0.009,明显高于对照组的0.362±0.018(P<0.01)。氧化型低密度脂蛋白、氧化型极低密度脂蛋白或血管紧张素Ⅱ诱导组血管细胞粘附分子1和单核细胞趋化蛋白1的mRNA表达明显高于对照组(P<0.01)。同时加入川芎嗪后血管细胞粘附分子1和单核细胞趋化蛋白1蛋白和mRNA表达明显低于相应诱导组(P<0.01)。氧化型低密度脂蛋白、氧化型极低密度脂蛋白或血管紧张素Ⅱ诱导核因子κB在核内表达,同时加入川芎嗪后核因子κB表达于胞浆,核内阴性。与氧化型低密度脂蛋白或氧化型极低密度脂蛋白诱导组(2.047±0.011和1.936±0.014)比较,加入川芎嗪后,粘附于内皮细胞的单核细胞明显减少(1.282±0.020和1.265±0.016,P<0.01)。结论川芎嗪通过抑制或阻断动脉粥硬化危险因素氧化型低密度脂蛋白、氧化型极低密度脂蛋白和血管紧张素Ⅱ诱导的核因子κB活化及核内移位,抑制血管壁细胞血管细胞粘附分子1和单核细胞趋化蛋白1表达,抑制单核细胞粘附于内皮,而发挥其抗动脉粥样硬化作用。     

罗格列酮对高糖诱导的血管内皮细胞炎症的抑制作用

目的 阐明罗格列酮对糖尿病血管并发症保护作用的分子机制.方法 用凝胶迁移分析和免疫荧光法测定不同浓度罗格列酮干预前后高糖诱导的血管内皮细胞核因子κB激活和血管细胞黏附分子1表达的改变.结果 25 mmol/L D-葡萄糖刺激30 min可诱导细胞核因子κB向核内迁移(与基础水平相比,P<0.001);5、25 μmol/L罗格列酮以剂量依赖的方式抑制了D-葡萄糖诱导这种效应,抑制率分别为25.17%(P=0.001)和51.79%(P<0.001).罗格列酮还抑制了D-葡萄糖诱导的血管细胞黏附分子1高表达.结论 罗格列酮通过抑制血管内皮细胞炎症起到直接保护血管作用,可能是其延缓和改善糖尿病血管并发症的机制之一.     

乔松素对脂多糖诱导的人脐静脉内皮细胞凋亡的影响

目的探讨蜂胶乔松素对脂多糖诱导的人脐静脉内皮细胞凋亡的影响。方法用消化灌注法收集人脐静脉内皮细胞进行体外原代培养,于培养液中给予10 mg/L脂多糖处理以建立细胞凋亡模型,乔松素处理组于模型条件培养液中分别加入不同浓度的乔松素(50、100和200 mg/L)干预,共同孵育24 h。光镜下观察细胞形态,MTT法测定细胞存活率以观察细胞增殖变化,缺口末端标记技术TUNEL法检测细胞凋亡率,免疫细胞化学法检测信号转导系统核因子κB亚基p65核易位变化。结果模型组内皮细胞凋亡率明显高于阴性对照组,不同浓度乔松素组细胞凋亡率均低于模型组,同时细胞存活率增高(P0.01)。加入不同浓度乔松素能显著降低脂多糖诱导的人脐静脉内皮细胞核因子κB p65核易位活性(P0.01)。结论乔松素可通过抑制脂多糖诱导的人脐静脉内皮细胞凋亡而保护内皮细胞功能,其机制可能与抑制核因子κB p65活化有关。     

二苯乙烯苷通过核因子κB信号通路抑制过氧化氢诱导的人脐静脉内皮细胞凋亡

目的 研究二苯乙烯苷对过氧化氢诱导人脐静脉内皮细胞凋亡的保护作用,探讨二苯乙烯苷是否通过核因子κB信号通路调节凋亡相关基因的表达来抑制细胞凋亡.方法 体外培养人脐静脉内皮细胞,实验分为对照组、过氧化氢组和二苯乙烯苷组,采用显微镜观察细胞形态,MTT法检测细胞增殖率,流式细胞术检测细胞凋亡率,Western blot检测核因子κB p65、IκB、bcl-2蛋白的表达.结果 过氧化氢能显著抑制内皮细胞增殖,增加细胞凋亡率;并增加核因子κB p65蛋白的表达,降低bcl-2和IκB蛋白的表达.二苯乙烯苷预处理后显著增加氧化损伤内皮细胞的增殖率,并降低细胞凋亡率;与过氧化氢组相比,二苯乙烯苷组核因子κB p65蛋白的表达显著降低,bcl-2和IκB蛋白的表达显著升高(P<0.01).结论 二苯乙烯苷对过氧化氢诱导人脐静脉内皮细胞凋亡具有保护作用,其机制可能与其抑制核因子κB/ IκB信号通路并上调bcl-2蛋白的表达有关.     

核因子-κB在脂多糖诱导的大鼠胰腺腺泡细胞AR42J炎性效应中的作用

目的 探讨核因子-κB(NF-κB)在急性胰腺炎体外模型中的作用.方法 以10 mg/L脂多糖刺激AR42J细胞构建急性胰腺炎的体外模型,设2 h、6 h、12 h、18 h和24 h共5个时间点,每时间点设3个复孔,逆转录-多聚酶链反应(RT-PCR)半定量法观察细胞间黏附分子-1(ICAM-1)和NF-κB p65亚单位mRNA表达的改变;链酶亲和素-生物素-过氧化物酶复合物法(SABC)检测p65蛋白在AR42J细胞中的表达;人工碘比色法观察培养液上清中淀粉酶的改变.结果 脂多糖刺激后,AR42J细胞以时间依赖方式上调ICAM-1 mRNA和p65 mRNA的表达,24 h达最高值;二者之间具有直线相关性(P＜0.01),同时p65蛋白亦呈时间依赖方式表达增强,24 h表达最强;各组间淀粉酶无明显改变(P＞0.05).结论 在脂多糖诱导的胰腺腺泡细胞AR42J炎性效应中,NF-κB调控致炎细胞因子ICAM-1的表达.     

伊贝沙坦对血管紧张素Ⅱ诱导血管内皮细胞NF-κB激活与ICAM-1表达的影响

目的：观察血管紧张素Ⅱ1型受体（AT1R）拮抗剂伊贝沙坦（irbesartan,Irb）对血管紧张素Ⅱ（AngⅡ）诱导的体外培养人脐静脉血管内皮细胞（HUVEC）核因子-κB（NF-κB）激活与细胞问粘附分子-1（ICAM-1）表达的影响。方法体外培养的第3～5代HUVEC用于实验。采用免疫组织化学分析检测细胞NF—κB弧单位p65、ICAM—1的表达程度。结果AngⅡ有效激活NF—κB并诱导HUVEC表达ICAM-1增加Irb预先孵胄2h能抑制NF—κB并减轻AngⅡ诱导的HUVEC ICAM-1表达。结论AngⅡ通过激活NF-κB使ICAM-1表达增加损伤血管内皮功能Irb能抑制NF-κB激活降低HUVEC ICAM-1表达,从而保护血管内皮功能,这有可能减轻心血管疾病损伤的进展。     

Cytochrome 1A1 and 1B1 gene diversity in the Zanzibar islands

Amodiaquine (AQ) is a 4‐aminoquinoline widely used in the treatment of malaria as part of the artemisinin combination therapy (ACT). AQ is metabolised towards its main metabolite desethylamodiaquine mainly by cytochrome P450 2C8 (CYP2C8). CYP1A1 and CYP1B1 play a minor role in the metabolism but they seem to be significantly involved in the formation of the short‐lived quinine‐imine. To complete the genetic variation picture of the main genes involved in AQ metabolism in the Zanzibar population, previously characterised for CYP2C8, we analysed in this study CYP1A1 and CYP1B1 main genetic polymorphisms. The results obtained show a low frequency of the CYP1A1*2B/C allele (2.4%) and a high frequency of CYP1B1*6 (approximately 42%) followed by CYP1B1*2 (approximately 27%) in Zanzibar islands. Genotype data for CYP1A1 and CYP1B1 show a low incidence of fast metabolisers, revealing a relatively safe genetic background in Zanzibar’s population regarding the appearance of adverse effects.     

CYP1A1 , GSTM1 , GSTT1 and NQO1 polymorphisms and colorectal adenomas in Japanese men

AIM: To investigate the role of functional genetic poly-morphisms of metabolic enzymes of tobacco carcinogens in the development of colorectal adenomas. METHODS: The study subjects were 455 patients with colorectal adenomas and 1052 controls with no polyps who underwent total colonoscopy in a preretirement health examination at two Self Defense Forces hospitals. The genetic polymorphisms studied wereCYP1A1*2A (rs 4646903), CYP1A1*2C (rs 1048943), GSTM1 (null or non-null genotype), GSTT1 (null or non-null genotype) and NQO1 C609T (rs 1800566). Genotypes were determined by the polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR method using genomic DNA extracted from the buffy coat. Cigarette smoking and other life-style factors were ascertained by a self-administered questionnaire. The associations of the polymorphisms with colorectal adenomas were examined by means of OR and 95%CI, which were derived from logistic regression analysis. Statistical adjustment was made for smoking, alcohol use, body mass index and other factors. The gene-gene interaction and effect modification of smoking were evaluated by the likelihood ratio test. RESULTS: None of the five polymorphisms showed a significant association with colorectal adenomas, nor was the combination of GSTM1 and GSTT1 . A borderline significant interaction was observed for the combination of CYP1A1*2C and NQO1 (P = 0.051). The OR associated with CYP1A1*2C was significantly lower than unity among individuals with the NQO1 609CC genotype. The adjusted OR for the combination of the CYP1A1*2C allele and NQO1 609CC genotype was 0.61 (95%CI: 0.42-0.91). Although the interaction was not statistically significant (P = 0.24), the OR for individuals carrying the CYP1A1*2C allele and GSTT1 null genotype decreased significantly compared with those who had neither CYP1A1*2C allele nor GSTT1 null genotype (adjusted OR: 0.69, 95%CI: 0.49-0.97). Smoking did not modify the associations of the individual polymorphisms with colorectal adenomas. There w     

Inhibition of procarcinogen-bioactivating human CYP1A1, CYP1A2 and CYP1B1 enzymes by melatonin

Abstract:  Administration of melatonin to rodents decreases the incidence of tumorigenesis initiated by benzo[ a ]pyrene or 7,12-dimethylbenz[ a ]anthracene, which requires bioactivation by cytochrome P450 enzymes, such as CYP1A1, CYP1A2 and CYP1B1, to produce carcinogenic metabolites. The present study tested the hypothesis that melatonin is a modulator of human CYP1 catalytic activity and gene expression. As a comparison, we also investigated the effect of melatonin on the catalytic activity of CYP2A6, which is also a procarcinogen-bioactivating enzyme. Melatonin (3–300 μ m ) decreased 7-ethoxyresorufin O -dealkylation catalyzed by human hepatic microsomes and recombinant CYP1A1, CYP1A2 and CYP1B1, whereas it did not affect coumarin 7-hydroxylation catalyzed by hepatic microsomes or recombinant CYP2A6. Melatonin inhibited CYP1 enzymes by mixed inhibition, with apparent K _i values (mean ± S.E.M.) of 59 ± 1 (CYP1A1), 12 ± 1 (CYP1A2), 14 ± 2 (CYP1B1) and 46 ± 8 μ m (hepatic microsomes). Additional experiments indicated that melatonin decreased benzo[ a ]pyrene hydroxylation catalyzed by hepatic microsomes and CYP1A2 but not by CYP1A1 or CYP1B1. Treatment of MCF-10A human mammary epithelial cells with melatonin (up to 300 μ m ) did not affect basal or benzo[ a ]pyrene-inducible CYP1A1 or CYP1B1 gene expression. Consistent with this finding, melatonin did not influence reporter activity in aryl hydrocarbon receptor-dependent pGudluc6.1-transfected MCF-10A cells treated with or without benzo[ a ]pyrene, as assessed in an in vitro cell-based luciferase reporter gene assay. Overall, melatonin is an in vitro inhibitor of human CYP1 catalytic activity, and it may be useful to develop potent analogues of melatonin as potential cancer chemopreventive agents that block CYP1-mediated chemical carcinogenesis.     

The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1)

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.     

Interleukin 1 (IL 1)

Interleukin 1 is an essential factor of macrophage dependent T cell activation and has a large quantity of other biological activities. This paper gives a review of present knowledge of Interleukin 1. In addition to biochemical properties, the IL 1 production and IL 1 activities, methods for determining of IL 1 and inhibitory factors of IL 1 induced T cell proliferation are described.     

Pandemic H1N1 influenza

The 2009 H1N1 influenza A virus that has targeted not only those with chronic medical illness, the very young and old, but also a large segment of the patient population that has previously been afforded relative protection - those who are young, generally healthy, and immune naive. The illness is mild in most, but results in hospitalization and severe ARDS in an important minority. Among those who become critically ill, 20-40% will die, predominantly of severe hypoxic respiratory failure. However, and potentially in part due to the young age of those affected, intensive care with aggressive oxygenation support will allow most people to recover. The volume of patients infected and with critical illness placed substantial strain on the capacity of the health care system and critical care most specifically. Despite this, the 2009 pandemic has engaged our specialty and highlighted its importance like no other. Thus far, the national and global critical care response has been brisk, collaborative and helpful - not only for this pandemic, but for subsequent challenges in years ahead.