重组核因子κB活化因子受体蛋白对破骨细胞活性的抑制作用

背景：核因子κB活化因子受体直接参与破骨细胞的活性及功能调节,在骨吸收类疾病发病中具有重要意义。通过基因工程得到的可溶性核因子κB活化因子受体蛋白可能为防治骨质疏松等骨吸收类疾病提供了新的有效手段。 目的：观察重组重组核因子κB活化因子受体蛋白对体外培养破骨细胞活化、吸收活性的影响。 方法：取新生24 h内SD大鼠胎鼠的四肢长骨,机械分离获得破骨细胞,采用不同浓度重组重组核因子κB活化因子受体蛋白干预后,行抗酒石酸酸性磷酸酶染色及骨吸收陷窝甲苯胺蓝染色,观察其对破骨细胞的生长情况。 结果与结论：重组核因子κB活化因子受体蛋白对破骨细胞作用3 d后,破骨细胞数量明显减少,以10-4 mol/L重组核因子κB活化因子受体蛋白最为显著。核因子κB活化因子受体蛋白作用9 d时,骨片吸收陷窝数明显减少。由此认为,在体外重组核因子κB活化因子受体蛋白可以有效抑制破骨细胞活化及骨吸收活性。     

Postischemic accumulation of lipid peroxidation products in the rat brain: immunohistochemical detection of 4-hydroxy-2-nonenal modified proteins

We report an immunohistochemical study on the distribution and alterations of 4-hydroxy-2-nonenal (HNE)-modified proteins, an indicator of lipid peroxidation, in the rat brain after 3 h of middle cerebral artery (MCA) occlusion followed by reperfusion. HNE immunoreactivity was not observed in intact neurons, but it appeared in some shrunken neurons within the infarcted zone at 3 h after reperfusion. The number of HNE-positive neurons increased with the spread of the infarcted area. The pyramidal neurons in the third layer of the frontoparietal cortex were HNE-positive and the intensity of their HNE immunoreactivity was highest at 24 h after reperfusion. At 48 h, HNE-positive neurons were observed in the medial part of the striatum, the lateral side of the frontoparietal cortex, and at the boundary between the infarcted and noninfarcted zones. In addition, strong HNE immunoreactivity was seen in microglia (identified by OX-42 immunostaining). This method seems to be useful to follow the progress of lipid peroxidation at the cellular level after ischemic injury.     

Interleukin-10 inhibits the expression of nuclear factor kappa B in rats with cerebral ischemia

BACKGROUND: Plenty of studies have demonstrated that inflammatory reaction is involved in ischemic cerebral damage, and the expression of inflammatory cytokines can be observed at the initial sites of cerebral damage at early period, including interleukin-6, interleukin-8, etc., which are all the target gene products of nuclear factor kappa B (NF-κB). The process of ischemic damage can be affected by adjusting and controlling NF-κB activity via multi-links. OBJECTIVE: To investigate the inhibitory effect of interleukin-10 on the expression of NF-κB in the ischemic sites of rats with focal cerebral ischemia in rats and its molecular mechanisms. DESIGN: A randomized and controlled animal trial. SETTING: Department of Neurology, the Affiliated Union Hospital of Fujian Medical University. MATERIALS: Thirty-two adult male Sprague-Dawley rats weighing (250±30) g were used. NF-κB p65 (RelA) rabbit anti-rat monoclonal primary antibody was the product of Neomarkers Company; Immunohistochemical kit of the SP two-step method was purchased from Beijing Zhongshan Biotechnology Co., Ltd. METHODS: The experiment was carried out in the Affiliated Union Hospital of Fujian Medical University from August 2005 to April 2006. The rats were randomly assigned into sham-operated group, middle cerebral artery occlusion (MCAO) group, vehicle-treated group and interleukin-10 treated group, 8 rats in each group. Focal cerebral ischemia was induced by occlusion of the middle cerebral artery as previously described. Rats in the MCAO group were anesthetized intraperitoneally, thyroid was bluntly dissected. Right common, external and internal carotid arteries were isolated, the trunk of external carotid artery was ligated and freed, an artery clamp was placed at the internal carotid artery, then a "V" shape incision was made at the free section of external carotid artery, filament was inserted for a depth of (18.5±0.5) mm. The rats in the sham-operated group were given the same treatments with the exception of filament insertion. After the successful model establishment for 1 hour, the rats in the interleukin-10 treated group were injected with human recombinant interleukin-10 (1 μg) via lateral ventricle, whereas those in the vehicle-treated group were injected with 5 mol/L NaP (5 μL). The rectal temperature of rats were kept at about 37 ℃ with heating lamps throughout the operation. Twenty-four hours after MCAO, the rats were examined for neurological deficits. Only those animals that scored at 1-3 points were utilized. The rats were decapitated at 24 hours postoperatively. The expression of NF-κB p65 in peri-infarct core was detected immunohistochemically. The percentage of NF-κB p65 subunit positive cells in 1 000 cells was calculated. MAIN OUTCOME MEASURES: Expression of NF-κB p65 in peri-infarct core; Percentage of NF-κB p65 subunit positive cells. RESULTS: All the 32 rats were involved in the analysis of results. NF-κB p65 expressed in cytoplasm and some nuclei. It was expressed all in cytoplasm in the sham-operated group, and partly expressed in the nucleus after cerebral ischemia. Small amounts of NF-κB p65 positive neurocytes were observed in the sham-operated group[(3.7±0.6)%], those were obviously increased in the MCAO group [(15.4±3.7)%, P < 0.01]. NF-κB p65 positive neurocytes were significantly reduced in the interleukin-10 treated group as compared with those in the vehicle-treated groups [(12.1±2.2)%, (15.5±3.6)%, P < 0.05].     

氯化锂预处理对脑出血后血肿周围神经细胞凋亡及核因子κB表达的抑制作用

目的 观察氯化锂预处理对大鼠脑出血后血肿周围神经细胞凋亡、炎症反应及核因子κB(NF-κB)表达的影响.方法 54只SD大鼠按随机数字表法分为假手术组、脑出血组和氯化锂预处理脑出血组,每组各18只.氯化锂预处理脑出血组手术前7 d起每天腹腔注射氯化锂(1mmol/kg).利用立体定向技术,将Ⅳ型胶原酶用微量进样器精确注入大鼠内囊诱导成脑出血模型.跟据术后处死动物的时间不同,各组再分别分为1、3、7 d三个亚组.分别采用TUNEL法、苏木素-伊红染色和免疫组织化学染色观察血肿周围神经细胞凋亡、炎症反应及NF-κB表达的情况.结果 在脑出血后1、3、7 d,与脑出血组(TUNEL阳性细胞数:18.32±3.75,33.24±6.37,20.49±4.87;NF-κB阳性细胞数:55.34±5.83,30.63±3.27,9.53±2.37)比较,氯化锂预处理脑出血组血肿周围区TUNEL阳性细胞数(15.84±3.12,10.88±4.75,5.83±4.39)明显减少,NF-κB阳性细胞数(29.27±3.37,16.36±3.64,7.64±2.31)明显降低,比较差异有统计学意义(P<0.05),炎症反应也明显减轻.结论 氯化锂预处理可能通过降低NF-κB的表达来减轻脑出血后的炎症反应,减少脑出血后血肿周围神经细胞凋亡,其对脑出血后脑损伤有神经保护作用.     

核转录因子κB与肌细胞的生长和运动

以往核转录因子κB的研究多限于免疫细胞,而近年来,核转录因子κB在肌细胞的研究方面备受关注。核转录因子κB的靶基因编码不同的功能蛋白,藉此,核转录因子κB既能诱发炎症加剧、蛋白降解、组织损伤,也能促进细胞生长和维持。核转录因子κB阻断剂的阻断作用与其氧化性无关。运动会激活核转录因子κB,此时,核转录因子κB的作用可能与其在病理性条件下的作用不同,是促进细胞生长发育的。文章总结并分析核转录因子κB对细胞生长的作用,及运动介导下骨骼肌中核转录因子κB的变化趋势和影响,为肌肉康复及运动适应性变化的研究提供依据。     

核因子-kВ在局部脑缺血再灌注损伤大鼠脑组织中的表达

目的　探讨局部脑动脉缺血再灌注损伤时核因子 kВ(NF kВ)和肿瘤坏死因子 α(TNF α)的动态变化。方法　采用大鼠大脑中动脉线栓动物模型 ,分别于损伤后第 2、6、12小时取脑组织行冰冻切片 ,用免疫组化方法检测NF kВ和TNF α并进行图像分析。结果　大鼠脑组织NF kВ和TNF α水平在损伤后增加 ,此二因子的水平变化具有显著相关性 (P <0 .0 5 )。结论　NF kВ和TNF α在脑动脉缺血再灌注损伤中表达增加 ,它们在脑动脉缺血和缺血再灌注损伤的病理过程中可起到重要作用     

Mercapturate metabolism of 4-hydroxy-2-nonenal in rat and human cerebrum

4-Hydroxy-2-nonenal (HNE), a potent toxin formed in the brain from oxidation of polyunsaturated fatty acids, is increased in Alzheimer disease (AD), where it is a proposed effector of amyloid beta peptide-mediated neurotoxicity. Detoxification of HNE via the mercapturic acid pathway (MAP) is the primary means by which other organs, such as liver, limit its toxic effects. Here we examined the distribution and activity of MAP detoxification for HNE in cerebrum. Our results showed that rat cerebral cortex and especially synaptosomes were less well equipped to detoxify HNE via the MAP than liver. Glutathione transferases (GSTs) catalyze the committed step in the MAP; GST-mu and GST-pi, but not OST-alpha, were detected in neurons and astrocytes in cerebrum from AD patients and controls. MAP activity in frontal cortex of AD patients was modestly but significantly increased compared to controls. These data suggest that lipid peroxidation may present a greater toxic burden to cerebrum than to other organs, and that a component of response to injury in late stage AD is a slight increase in MAP activity.     

Restraint stress modulates glucocorticoid receptors and nuclear factor kappa B in the cochlea

The regulation of glucocorticoid receptors and nuclear factor kappaB was evaluated in the spiral ganglion neurons after 4 h of restraint stress in the mouse cochlea. Immediately after restraint stress, glucocorticoid receptor protein expression was not altered in spiral ganglion neurons even though both the plasma corticosterone levels and glucocorticoid receptor nuclear translocation increased. By 24 h after restraint stress, the protein expression of glucocorticoid receptors was decreased in spiral ganglion neurons. Pre-treatment with RU486 and metyrapone prevented nuclear translocation of glucocorticoid receptors and nuclear factor kappaB. Moreover, the synthesis of nuclear factor kappaB protein (p65) and inhibitory factor kappaBalpha decreased when RU486 and metyrapone treatment was given before restraint stress. These findings suggest that restraint stress modulates glucocorticoid receptor and nuclear factor kappaB activity in the spiral ganglion neurons, resulting in an altered response to stress.     

核因子κB、基质金属蛋白酶3在退变腰椎间盘组织中的表达*

背景：对于椎间盘退变的原因一直是国内外学者研究的重点。近年来随着研究的深入,在椎间盘退变过程中多种细胞因子、黏附分子、趋化因子、炎症递质、蛋白质酶等发挥着重要作用。 目的：比较正常腰椎间盘组织及退变腰椎间盘组织中核因子κB、基质金属蛋白酶3的表达,分析两者的相关性。 方法：收集唐山市第二医院脊柱外科手术切除退变椎间盘组织68例(病变组),腰椎爆裂骨折正常椎间盘组织10例(对照组),应用苏木精-伊红染色、免疫组织化学方法和ELISA方法检测核因子κB、基质金属蛋白酶3的表达,并对两者相关性进行分析。 结果与结论：苏木精-伊红染色结果显示对照组可见纤维环和髓核结构,腰椎间盘髓核的软骨细胞表现为不规则的软骨陷窝,病变组细胞增殖明显,细胞核呈圆形或卵圆形,髓核细胞胞质呈空泡状。免疫组织化学方法显示病变组核因子κB、基质金属蛋白酶3吸光度值明显高于对照组(P < 0.05)。ELISA检测病变组核因子κB、基质金属蛋白酶3的表达均高于对照组(P < 0.05)。两者具有正相关性(r=0.643 0,P < 0.01)。     

新诊断2型糖尿病患者二甲双胍治疗后外周血单个核细胞核因子KB活性的变化

背景：IKK/核因子κB通路是巨噬细胞分泌炎症因子的关键调节通路,可启动、放大炎症反应,二甲双胍可能有潜在的抗炎作用。 目的：探讨二甲双胍对新诊断2型糖尿病患者外周血单个核细胞核因子κB活性及血清炎症指标的影响。　 设计：自身前后对照。 对象：2004-10/2006-06中山大学附属第二医院内分泌科住院和门诊收治的新诊断2型糖尿病患者29例。二甲双胍为中美上海施贵宝公司产品。 方法：29例患者经筛选进入实验后,给予二甲双胍治疗,由1.0 g/d开始,最大剂量2.0 g/d。每2周随访1次,根据血糖水平调整药物剂量,血糖控制目标为空腹血糖< 6.1 mmol/L,餐后2 h血糖< 8 mmol/L。分别于治疗前及血糖达良好控制0,2,12周时进行各项指标检查。　 主要观察指标：Western blot法检测外周血单个核细胞磷酸化核因子κB p65（Ser536）水平,高敏ELISA法检测血清炎症指标的变化。 结果：与治疗前比较,血糖达标2周时外周血单个核细胞中磷酸化核因子κB p65(Ser536)水平明显降低（P < 0.05）,至血糖达标12周时降低程度更为显著（P < 0.01）。与治疗前比较,血糖达标时（0周）及达标后2周,12周,各项炎症指标水平均明显降低（P < 0.05或0.01）,其中血糖达标12周时超敏C反应蛋白降低56%,白细胞介素6降低30%,肿瘤坏死因子α降低24%。 结论：二甲双胍可抑制新诊断2型糖尿病患者外周血单个核细胞中磷酸化核因子κB 活性,显著改善患者炎症状态。     

人睫状肌细胞核因子κB在曲伏前列腺素作用下的变化

背景：核因子κB可能与葡萄膜巩膜房水流出通道的多种细胞信号调控有关。 目的：观察前列腺素类曲伏前列腺素药物作用下，体外培养的人睫状肌细胞核因子кB及其抑制因子（inhibitor，IκB）的变化。 设计、时间及地点：对比观察实验，于2005-03/2006-11在中山眼科中心实验室完成。 材料：供体取自中山眼科医院，摘自死亡 1 h内无眼疾青年尸体眼球。患者家属对实验知情同意，并自愿捐献。 方法：在人睫状肌细胞培养基中加1 μmol/L曲伏前列腺素，根据孵育时间的不同分为4组，即0 h对照组和6，12，24 h组。 主要观察指标：采用real-time RT-PCR、免疫荧光半定量分析和ELISA法分别检测上述时间组核因子κB p65、ⅠκBα在基因和蛋白水平的表达。 结果：①与对照组比较，6，12，24 h组 核因子κB p65 mRNA表达均下降(F= 17.068,P=0.001);IκBαmRNA 6 h组、12 h组较对照组改变不明显(P > 0.05)，24 h组较对照组表达增加(F=32.742，P=0.000)。②免疫荧光半定量分析表明：核因子κB p65荧光强度6，12，24 h组均较对照组减少(F=17.216，P=0.000);IκBα6 h组较对照组没有明显改变(P=0.134)、12 h组较对照组轻微下降(P=0.032)，24 h组较对照组明显增加(F=17.346，P=0.001)。③ELISA法检测磷酸化核因子κB p65随药物作用时间延长而逐渐下降(F=15.4，P=0.001)。 结论：曲伏前列腺素作用于人睫状肌细胞后，核因子κB p65的基因表达下调，核易位抑制，IκBα的基因表达上调。