Role of Nuclear Factor kappaB in Intestine Injury Induced by Hepatic Ischemia Reperfusion

Summary: The role of nuclear factor kappaB in intestine injury induced by hepatic ischemia reperfusion was investigated. Eighteen male Wistar rats were divided into 3 groups randomly: sham operation group (group A), hepatic ischemia reperfusion group (group B) and hepatic ischemia reperfusion plus pyrrolidine dithiocarbamate (PDTC) group (group C). The rats in group A were only subjected to laparotomy, those in group B underwent partial hepatic ischemia reperfusion (ischemia for 1 h and reperfusion for 2 h) and those in group C underwent the same procedure as that of group B but received PDTC 200 mg/kg i.v. before and after ischemia. After reperfusion, tissues of jejunum and venous blood were obtained for measurement of TNF-α, MDA and MPO. The levels of TNF-α in jejunum and venous blood, the levels of MPO in jejunum in group B were significantly higher than those in group A and group C (P<0.05). There was no significant different in the levels of MDA between group B and group C. The severity of histological intestinal injury in group B and group C was similar. Hepatic ischemia reperfusion caused intestine injury, NF-kappaB may play an important role in this course and the targeting of upstream components of the inflammatory response, such as NF-kappaB, may have important therapeutic applications.

Fruits extracts of Hovenia dulcis Thunb.suppresses lipopolysaccharide—stimulated inflammatory responses through nuclear factor—kappaB pathway in Raw 264.7 cells

Objective:To investigate the anti-inflammatory effects and the action mechanism of the fruits of Horenia dulcis(H.dulcis) in lipopolysaccharide(LPS)-induced mouse macrophage Raw 264.7cells.Methods:The extract of H.dulcis fruits(EHDF) were extracted with 70%ethanol.Mouse macrophages were treated with different concentrations of EHDF in the presence and absence of LPS(1 μg/mL).To demonstrate the inflammatory mediators including nitric oxide,inducible nitric oxide synthase and cyclooxygenase(COX)-2 expression levels were analyzed by usingin vitro assay systems.COX-derived pro-inflammatory cytokines including interleukin-1 β.tumor necrosis factor- α and prostaglandin F_2 were determined using ELISA kits.Cell viability,heme oxygenase-1 expression,nuclear factor-kappaB and nuclear factor F.2-related factors 2 translocation were also investigated.Results:EHDF potently inhibited the LPS-stimulated nitric oxide,inducible nitric oxide synthase.COX-2,interleukin-1 β and tumor necrosis factor- α expression in a dose-dependent manner.EHDF suppressed the phosphorylation of inhibited kappaB-alpha and p65 nuclear translocation.Treatment of macrophage cells with EHDF alone induced the heme oxygenase-1 and nuclear translocation of nuclear factor E2-reIated factor 2.Conclusions:These results suggest that the ethanol extract of H.dulcis fruit exerts its anti-inflammatory effects by inhibiting inhibited kappaBalpha phorylation and nuclear translocation of nuclear factor-kappaB.

Influence of edaravone on Notch1 and nuclear factor-kappaB in rats with cerebral ischemia/reperfusion injury

BACKGROUND： It has been demonstrated that edaravone has a a neuroprotective role, inhibits free radical increase, and reduces celt apoptosis. The Notch pathway is a key factor in neurogenesis and cellular apoptosis The proinflammatory transcription factor nuclear factor-kappaB （NF-κB） plays an important role in inflammation and oxidation. OBJECTIVE： To observe the influence of edaravone on Notchl and NF-κB mRNA and protein expression in rats with focal cerebral ischemia/reperfusion injury. DESIGN, TIME AND SETTING： This randomized controlled neural and molecular biology experiment was performed at the Department of Neurology, the First Affiliated Hospital of Chongqing Medical University, and the Chongqing Key Laboratory of Neurology between July 2007 and May 2008. MATERIALS： Thirty female Wistar rats were used. Edaravone was purchased from Jiangsu Xiansheng Pharmaceutical Limited Company, China. METHODS： Wistar rats were randomly divided into five groups （n = 6）. Thread was inserted into the internal carotid artery of the sham operation group but the middle cerebral artery was not ligated. A focal cerebral ischemia/reperfusion model was established by inserting thread into the right middle cerebral artery. The model rats in the edaravone groups were given tail vein injections of edaravone at 3 mg/kg body weight after ischemia for 2 hours and reperfusion for 12 or 24 hours. Ischemia/reperfusion groups （model group） received intravenous infusion of normal saline at the same volume as the edaravone groups after ischemia for 2 hours and reperfusion for 12 or 24 hours. MAIN OUTCOME MEASURES： The volume of the ischemic region was measured by 2,3,5-triphenyltetrazolium chloride staining. Notchl and NF-κB protein and mRNA expression were measured by immunohistochemistry and RT-PCR. Protein expression was represented by the absorbance value. RESULTS： Edaravone greatly reduced the focal infarct volume. Notchl and NF-κB protein and mRNA expression were rapidly upregulated following cerebral ischemia/reperfusion injury in model and edaravone groups compared with the sham operation group （P 〈 0.01 ）. In addition, edaravone treatment significantly upregulated Notchl expression but down-regulated NF-κB expression compared with model groups （P 〈 0.01）. CONCLUSION： Edaravone possibly protects brain tissue from ischemia/reperfusion injury by upregulating Notchl expression and regulating NF-κB expression.

Interon-gamma Enhances the Antitumor Effect of All-trans Retinoic Acid on Hepatocellular Carcinoma Cells by Inhibiting the Expression of Nuclear Factor-kappaB

Objective： To explore the combination effects of all-trans retinoic acid （ATRA） with interferon-gamma （IFN-γ） on human hepatocarcinoma cell line SMMC-7721 and the mechanism of action. Methods： SMMC-7721 cells were divided into treated group and control group. The cells were treated with ATRA or ATRA＋ IFN-γ in the former and added with PBS in the latter. The inhibition rate of SMMC-7721 cell proliferation was detected by MTT, the cell change in morphology was observed by electron microscope. The apoptosis was detected by flow cytometry and the expression changes of nuclear factor-kappaB （NF-κB） was analyzed by Western blotting when the SMMC-7721 cells were treated with ATRA and IFN-γ. Results： The SMMC-7721 cell proliferation was suppressed and apoptosis was induced after the cells were treated with ATRA treatment, and these effects were enhanced when ATRA was combined with IFN-γ. The expression of NF-κB was reduced after SMMC-7721 cell was treated with ATRA, and reduced significantly when the cells were treated with the combination of ATRA and IFN-γ. Conclusion： IFN-γ can enhance the inhibiting effects of ATRA on cell proliferation and inducing apoptosis on SMMC-7721 cell and these effects might be mediated by inhibiting the expression of NF-κB.

Effect of Nuclear Factor-κB on Airway Remodeling in Asthmatic Rats

In order to investigate the effect of nuclear factor κB (NF κB) on airway remodeling in asthmatic rats, 18 Wistar rats were divided into three groups: asthmatic group; pyrrolidine dithiocarbamate (PDTC) group, in which rats were injected intraperitoneally with NF κB specific inhibitor PDTC (100 mg/kg) before ovalbumin (OVA) challenge; control group. The NF κB activity and the expression of inhibitory protein κBα (I κBα) in airway were detected by electrophoretic mobility shift assay (EMSA), Western blot and immunohistochemistry respectively. The infiltration of inflammatory cells, the number of Goblet cells, the area of collagen and smooth muscle in airway were measured by means of image analysis system. The results showed that with the up regulation of airway NF κB activity in asthmatic group, the number of goblet cells (3.08±0.86/100 μm basement membrane (BM)), the area of collagen (24.71±4.24 μm 2/μm BM) and smooth muscle (13.81±2.11 μm 2/μm BM) in airway were significantly increased ( P <0.05) as compared with control group (0.14±0.05/100 μm BM, 14.31 ±3.16 μm 2/μm BM and 7.67±2.35 μm 2/μm BM respectively) and PDTC group (0.33±0.14/100 μm BM, 18.16±2.85 μm 2/μm BM and 8.95±2.16 μm 2/μm BM respectively). However, there was no significant difference between PDTC group and control group ( P >0.05). It was concluded that the activity of NF κB is increased in airway of asthmatic rats. Inhibition of NF κB activation can attenuate constructional changes in asthma airway, suggesting NF κB may contribute to asthmatic airway remodeling.

Inhibitory Activity of Nuclear Factor-κB Potentiates Cisplatin-induced Apoptosis in A549 Cells

Whether inhibiting the activity of nuclear factor （NF）-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated. The recombinant plasmid pcDNA3.1（＋）/IκBα expressing IκBα was constructed. The in vitro cultured A549 cells were transfected with pcDNA3.1 （＋）/IκBα alone, or pcDNA3.1（＋）/IκBα combined with cisplatin. The mitochondrial membrane potential （△ψm） was determined by rhodamine 123, the activity of caspase-3 was tested by colorimetric assay, and cell apoptosis was detected by flow cytometry with the annexin V/propidium iodide assay. The results showed that the activity of NF-κB in A549 cells was inhibited by transfecting pcDNA3.1（＋）/IκBα. Transfection of pcDNA3.1（＋）/IκBα alone did not promote apoptosis. Treatment of cisplatin alone had a little effect on cell apoptosis. Transfection of pcDNA3.1（＋）/IκBα combined with cisplatin treatment significantly induced apoptosis of A549 ceils. It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.

葛根素对脑缺血再灌注后核因子kappaB表达的影响

目的:研究大鼠局灶性脑缺血再灌注后核因子kappaB(NF-κB)在时间和空间上表达的变化过程及葛根素对其的影响;方法:线栓法建立右侧大脑中动脉闭塞大鼠模型,分别于缺血90 m in后再灌注2,6,12,24,72 h,缺血前1 h及随后每间隔6 h腹腔内给药。大鼠在各时间点断头取脑,测算脑梗死体积,免疫组织化学方法和免疫印迹法检测脑组织内NF-κB的表达。结果:大鼠缺血再灌注后,免疫组化分析提示NF-κB从细胞浆向细胞内移位,核内表达水平在6 h开始明显升高,在24 h达到高峰,72 h下降,其脑梗死体积随着再灌注时间延长而增加。葛根素明显降低NF-κB在缺血再灌注后24,72 h的表达及减小脑梗死体积。结论:脑缺血再灌注后,脑梗死周边区域出现NF-κB从胞浆至胞核的转位并伴表达增加,葛根素可能通过抑制NF-κB的表达,减轻缺血再灌注损伤。

运动训练对核转录因子kappaB信号通路及炎性基因影响的研究进展

NF-κB(核转录因子-kappaB)信号通路在机体的免疫应答、细胞增殖、凋亡和生长发育中发挥重要作用。运动训练过程中机体产生的活性氧以及运动性肌肉损伤激活了NF-κB信号通路,对NF-κB活性的影响与运动训练的持续时间、频率和强度有关。急性剧烈运动导致了NF-κB活性一过性提高;长期有规律的运动训练能够降低由于衰老和慢性炎症反应而上调的NF-κB的活性;长期剧烈的运动训练导致了NF-κB的慢性持续激活,使通路上各指标的表达发生变化,细胞核中聚集NF-κB的亚基p65浓度增多,转录靶基因,从而使炎性基因的表达大幅度升高,一方面放大机体固有免疫系统对抗运动性应激,另一方面参与了骨骼肌运动性慢性炎症的形成。

Homocysteine-induced Enhanced Expression of Tissue Factor in Human Vascular Smooth Muscle Cells

The homocysteine （Hcy）-induced tissue factor （TF） expression in human vascular smooth muscle cells （VSMCs） and the effect of Hcy on the activity of nuclear factor-kappaB （NF-кB） and the expression of inducible nitric oxide synthase （iNOS） were investigated. Human umbilical artery VSMCs were cultured by tissue explanting method, identified by α-actin immunohistochemistry, and incubated with different concentrations of Hcy/PTDC （NF-кB inhibitor）. Semi-quantitative RT-PCR was performed to detect the expression of TF mRNA in VSMCs. Flow cytometry was used to assay the expression of TF protein on the surface of VSMCs and the expression of iNOS in VSMCs. Western blot was carried out to detect the expression of NF-кB protein in nuclei. The results showed that Hcy could induce VSMCs expressing TF mRNA significantly after the VSMCs were incubated with Hcy at concentrations of 10, 100, 500 μmol/L respectively. There was low expression level of TF protein on the surface of the resting VSMCs and Hcy could also induce VSMCs expressing TF pro- tein on the cell surface in different concentrations. Additionally, Hcy could rapidly induce the activation of NF-кB and this effect could be significantly inhibited by PDTC. Hcy alone could not induce the expression of iNOS in VSMCs. It was concluded that Hcy could significantly induce the expression of TF in VSMCs and enhance the activation of NF-ΚB, subsequently mediate TF gene expression and protein synthesis. NF-кB-mediated expression of TF in VSMCs might be the important mechanism of atherosclerosis and thrombosis induced by Hcy.

曲克芦丁调控核因子κB信号通路抑制脑梗死模型大鼠脑损伤及神经元凋亡

背景:目前已有研究发现曲克芦丁对脑部疾病有明显的改善作用,但是关于曲克芦丁对脑梗死治疗及神经元细胞影响的研究较少。目的:探究曲克芦丁调控核因子κB信号通路对脑梗死大鼠脑损伤及神经元凋亡的作用机制。方法:选取清洁级大鼠50只,将其随机分为健康组、模型组、曲克芦丁+核因子κB激动剂组、曲克芦丁组、核因子κB抑制剂组,后4组均采用动脉结扎法建立脑梗死大鼠模型。健康组及模型组采用等量生理盐水灌胃处理,曲克芦丁+核因子κB激动剂组采用72 mg/kg曲克芦丁灌胃+20 mg/kg RANK腹腔注射干预,曲克芦丁组采用72 mg/kg曲克芦丁灌胃处理,核因子κB抑制剂组采用120 mg/kg核因子κB抑制剂二硫代氨基甲酸吡咯烷进行腹腔注射,各组给药均1次/d,均连续干预30 d。药物干预完成后24 h,采用Zea-longa检测大鼠神经功能损伤,苏木精-伊红染色观察脑组织病理改变,TUNEL法检测神经元凋亡,采用免疫印迹法及PCR检测核因子κB p65、核因子κB p50蛋白及mRNA表达水平。结果与结论:①与健康组比较,模型组大鼠神经功能评分、神经元凋亡率、核因子κB p65、核因子κB p50 mRNA及蛋白表达水平升高(P<0.05);②与模型组比较,曲克芦丁+核因子κB激动剂组神经功能评分、神经元凋亡率、核因子κB p65、核因子κB p50 mRNA及蛋白表达水平降低(P<0.05);③与曲克芦丁+核因子κB激动剂组比较,曲克芦丁组、核因子κB抑制剂组神经功能评分、神经元凋亡率、核因子κB p65、核因子κB p50 mRNA及蛋白表达水平降低(P<0.05),且曲克芦丁组与核因子κB抑制剂组比较无差异(P>0.05);④模型组大鼠脑组织有大量神经元细胞胞质空泡化,明显水肿和坏死现象及大量炎性细胞浸润现象;曲克芦丁+核因子κB激动剂组脑组织肿胀减轻,网状结构和固缩细胞减少,仍伴有部分水肿;曲克芦丁组及核因子κB抑制剂组脑组织肿胀和神经元水肿变性、细胞胞质空泡化及神经元细胞核固缩减少,炎症细胞浸润明显降低;⑤提示曲克芦丁能够降低脑梗死大鼠神经功能损伤表达、抑制神经元凋亡及改善其脑组织病理损伤,其作用机制可能与调控核因子κB及相关信号通路的表达相关。

猪NF-kappaB p65/p50基因克隆、序列鉴定及实时荧光定量PCR检测方法的建立

NF-κB信号通路在病毒感染、免疫损伤性疾病、肿瘤疾病中发挥重要作用,其活化水平常作为机体免疫应答水平、疾病发展进程的检测指标。为获得猪NF-κB p65/p50基因序列特征并建立体外定量检测其表达水平的实时荧光定量PCR方法,作者对猪NF-κB p65ORF和成熟p50编码区进行RT-PCR扩增、克隆、测序及生物信息学分析;设计特异性荧光定量引物,建立检测p65、p50实时荧光定量PCR方法。结果显示:所扩增的猪NF-κB p65ORF长1 662bp,编码553aa,成熟p50编码区长1 653bp,编码551aa;与不同动物的p65、p50序列相似性均较高;56.5%p65位于细胞核内,78.3%p50存在于细胞质中,p65、p50均无信号肽及跨膜区。荧光定量结果表明:起始模板与Ct值之间线性关系好,相关系数达到0.999,扩增效率均在95%左右;敏感性高,初始模板的检出下限达到101 copies.μL-1;特异性强,扩增产物形成单一的特异性熔解峰;重复性好,组内变异系数均小于5‰。本试验为进一步研究猪NF-κB p65/p50生物学功能及其在猪相关疾病发生发展中表达量变化奠定了基础。

氯化甲基汞对发育阶段大鼠脑核因子kappaBDNA结合活性的影响

为探讨氯化甲基汞 (MMC)对发育大鼠脑组织核因子 kappa B(NF- k B) DNA结合活性的影响 ,采用凝胶移位法可见发育大鼠的大、小脑核蛋白提取物与 k B探针结合 ,对照和实验组均有 2条结合带。出生后 3天大鼠的大、小脑核蛋白提取物中 NF- k B DNA结合活性高于出生后 7天者。提示在发育大鼠脑中 NF- k B对发育起一定作用。宫内接触 MMC后出生 3和 7天的大脑核蛋白提取物中 NF- k B 和 NF- k B DNA结合活性低于对照组 ,而小脑中的 NF- k B 和 NF- k B DNA结合活性则高于对照组。在体外结合反应体系中 ,随着 MMC剂量的增高可增强大、小脑核蛋白提取物中 NF- k B DNA结合活性。提示接触MMC后大、小脑神经细胞反应能力不同 ,小脑中 NF- k B被激活。

苦豆碱通过抑制TLR4/NF-κB/NLRP3通路改善香烟烟雾诱导的人支气管上皮细胞损伤

目的探究苦豆碱(Alo)对香烟烟雾诱导的人支气管上皮细胞损伤的作用及其可能的作用机制。方法16HBE人支气管上皮细胞经100 mL/L香烟烟雾提取物(CSE)和(50、100、200)μmol/L Alo共处理后,CCK-8法检测细胞活力,试剂盒检测乳酸脱氢酶(LDH)活性;原位末端转移酶标记技术(TUNEL)、Western blot法检测细胞凋亡,ELISA检测炎性因子水平;2′,7′-二氯二氢荧光素二乙酸酯(DCFH-DA)荧光探针和相关试剂盒检测氧化应激水平;Western blot法检测Toll样受体4(TLR4)/核因子κB(NF-κB)/含pyrin结构域核苷酸结合寡聚结构域样受体家族蛋白3(NLRP3)通路相关蛋白表达水平。16HBE细胞经100 mL/L CSE和200μmol/L Alo共处理后,采用上述方法检测过表达TLR4对TLR4/NF-κB/NLRP3通路、细胞LDH活性、凋亡、炎症反应及氧化应激的影响。结果CSE暴露可降低16HBE细胞活力,增加LDH释放和细胞凋亡,增强炎症反应和氧化应激水平,且激活TLR4/NF-κB/NLRP3通路;经Alo处理后,细胞活性升高,LDH释放减少、凋亡降低、炎症减轻、氧化应激水平下降,且TLR4/NF-κB/NLRP3通路失活;TLR4过表达可逆转Alo处理对CSE诱导的16HBE细胞损伤的保护作用。结论Alo可通过抑制TLR4/NF-κB/NLRP3通路减轻CSE诱导的人支气管上皮细胞损伤。

平喘宁通过TRPM8/PKC/NF-κB信号通路缓解冷刺激加重的人支气管上皮细胞炎症作用机制研究

目的探讨平喘宁(PCN)通过瞬时受体电位melastatin家族成员8/蛋白激酶C/核因子-κB(TRPM8/PKC/NF-κB)信号通路对缓解冷刺激加重的人支气管上皮细胞炎症的作用机制,以此阐发平喘宁治疗寒哮的科学内涵。方法选用人支气管上皮细胞16HBE作为研究对象,用流式细胞术检测18℃培养不同时间的16HBE细胞的Ca^(2+)表达水平,选择Ca^(2+)水平较高的时间段组作为模型组;采用细胞增殖与活性检测试剂盒(CCK-8)法检测TRPM8离子通道的特异性抑制剂(BCTC)和PCN冻干粉对16HBE细胞的活性影响,以筛选出作用于16HBE细胞的适宜条件的浓度和时间。将细胞分为对照组、18℃冷刺激组(模型组)、18℃+BCTC组(BCTC组)、18℃+PCN冻干粉组(PCN组)、18℃+BCTC+PCN冻干粉组(BCTC+PCN组)。流式细胞术测量各组细胞内Ca^(2+)浓度变化;qRT-PCR测定各组TRPM8、PKC、NF-κB mRNA的表达水平;免疫荧光标记法检测各组磷酸化PKC、磷酸化NF-κB抑制因子(IκB)的蛋白含量;ELISA法检测各组细胞炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素-13(IL-13)的含量变化。结果与对照组比较,模型组的Ca^(2+)浓度显著升高(P<0.01),TRPM8、PKC、NF-κB p65 mRNA表达水平显著升高(P<0.01),磷酸化IκB、磷酸化PKC表达水平显著升高(P<0.01),促炎因子TNF-α、IL-13表达水平显著升高(P<0.01)。与模型组比较,PCN组的Ca^(2+)浓度显著降低(P<0.01),TRPM8、PKC、NF-κB p65 mRNA表达水平显著降低(P<0.01),磷酸化IκB、磷酸化PKC表达水平显著降低(P<0.01),促炎因子TNF-α、IL-13表达水平显著降低(P<0.01)。与BCTC组相比,PCN组的Ca^(2+)浓度均显著降低(P<0.01),NF-κB p65 mRNA表达水平显著降低(P<0.05),TRPM8、PKC mRNA表达水平降低,但差异无统计学意义(P>0.05),磷酸化IκB、磷酸化PKC表达水平显著降低(P<0.01),促炎因子TNF-α、IL-13表达水平显著降低(P<0.01);BCTC+PCN组的Ca^(2+)浓度均显著降低(P<0.01),TRPM8、PKC、NF-κB p65 mRNA表达水平显著降低(P<0.01),磷酸化IκB、磷酸化PKC表达水平显著降低(P<0.01),促炎因子TNF-α、IL-13表达水平显著降低(P<0.01)。结论平喘宁可能通过调节TRPM8/PKC/NF-κB信号通路减轻冷刺激加重的人支气管上皮细胞炎症。

姜黄素对磨损颗粒诱导的体外破骨细胞的作用及机制实验研究

目的:探讨姜黄素(Cur)对磨损颗粒诱导的体外破骨细胞的作用及机制。方法:将小鼠RAW264.7巨噬细胞分为五组培养,即空白组(完全培养基)、模型组[空白组+钛(Ti)颗粒0.1 mg/ml]、低浓度Cur组(模型组+Cur 1μmol/L)、中浓度Cur组(模型组+Cur 10μmol/L)和高浓度Cur组(模型组+Cur 15μmol/L)。CCK-8法检测细胞增殖活性。抗酒石酸酸性磷酸酶(TRAP)染色法和鬼笔环肽染色法检测成熟破骨细胞数量。酶联免疫吸附(ELISA)法检测细胞上清液中肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和基质金属蛋白酶(MMP)-9的表达。Western blot法检测p-p65、磷酸化核因子(NF)-κB抑制蛋白α(p-IκBα)蛋白水平。PCR法检TNF-α、IL-1β、活化T细胞核因子(NFATc)1和TRAP mRNA的表达水平。结果:Cur浓度小于15μmol/L对RAW264.7巨噬细胞增殖活性的影响与空白组比较差异无统计学意义(均P>0.05),因此采用不高于15μmol/L Cur进行后续实验。中、高浓度Cur组TRAP阳性成熟破骨细胞数量低于模型组(均P<0.05)。与模型组比较,不同浓度Cur组TNF-α、IL-1β和MMP-9表达,p-p65与p-IκBα蛋白表达,TRAP、TNF-α、IL-1β和NFATc1 mRNA表达降低(均P<0.05)。结论:Cur对体外磨损颗粒诱导的破骨细胞生成和破骨细胞功能有抑制作用,其机制可能是抑制NF-κB信号通路。

TLR4/NF-κB p65高表达对皮肤鳞状细胞癌增殖、侵袭和转移的影响

目的探讨与分析Toll样受体4(Toll-like recepTor 4,TLR4)/核因子KappaB(NF-κB)p65高表达对皮肤鳞状细胞癌增殖、侵袭和转移的影响。方法对数生长期的人皮肤鳞状细胞癌A431细胞株分为三组,空白组、对照组与实验组,分别转染DMEM培养基、pc3.0空载体、pc3.0-TLR4NF-κB p65载体,检测皮肤鳞状细胞癌增殖、侵袭和转移表达情况。结果转染24 h、48 h后,实验组的TLR4、NF-κB p65 RNA表达水平显著高于空白组与对照组,差异有统计学意义(P<0.05),空白组与对照组比较,差异无统计学意义(P>0.05)。实验组的细胞增殖指数显著低于空白组与对照组,差异有统计学意义(P<0.05),对照组与空白组比较,差异无统计学意义(P>0.05)。实验组的细胞侵袭指数与细胞转移指数都明显低于空白组与对照组,差异有统计学意义(P<0.05),对照组与空白组比较,差异无统计学意义(P>0.05)。实验组的Caspase-9/Myc蛋白表达水平显著高于空白组与对照组,差异有统计学意义(P<0.05),空白组与对照组比较,差异无统计学意义(P>0.05)。结论TLR4/NF-κB p65高表达能抑制皮肤鳞状细胞癌细胞的增殖、转移与侵袭,也可促进Caspase-9/Myc蛋白的表达,从而发挥促进肿瘤细胞凋亡的作用。

慢病毒介导的解整合素-金属蛋白酶17RNA干扰对气道上皮细胞MMP-9表达及NF-κB活性的影响

目的探讨脂多糖(LPS)诱导的气道上皮细胞基质金属蛋白酶9(MMP-9)表达的TNF-α/NF-κB信号转导机制及慢病毒介导的解整合素-金属蛋白酶17(ADAM17)RNA干扰(RNAi)对MMP-9表达的影响。方法构建ADAM17 siRNA慢病毒载体、包装重组慢病毒。以NF-κB抑制剂(pyrrolidine dithiocarbamate,PDTC)或TNF-α拮抗剂(etanercept)预处理HBE4-E6/E7细胞,以LPS刺激HBE4-E6/E7细胞24 h。以重组慢病毒感染HBE4-E6/E7细胞72 h后,以LPS或TNF-α刺激HBE4-E6/E7细胞24 h。以半定量RT-PCR检测MMP-9 mRNA表达;以酶联免疫吸附试验检测TNF-α蛋白含量;以Western blot检测MMP-9蛋白表达;以凝胶阻滞分析实验检测NF-κB活性。结果 LPS或TNF-α刺激均明显增加HBE4-E6/E7细胞MMP-9 mRNA和蛋白表达及NF-κB活性(P<0.05);etanercept和PDTC均明显抑制LPS诱导的MMP-9表达及NF-κB活性(P<0.05)。慢病毒介导的ADAM17 RNAi明显降低LPS诱导的HBE4-E6/E7细胞上清液中TNF-α蛋白含量(P<0.05),亦明显降低MMP-9 mRNA和蛋白表达及NF-κB活性(P<0.05),但不能降低TNF-α诱导的MMP-9mRNA和蛋白表达及NF-κB活性(P>0.05)。PDTC明显抑制TNF-α诱导的MMP-9 mRNA和蛋白表达及NF-κB活性(P<0.05)。结论 TNF-α/NF-κB信号通路参与调控LPS诱导的气道上皮细胞MMP-9的表达,ADAM17通过调节TNF-α释放在其信号通路上游起到重要作用。

和厚朴酚对脂多糖诱导的急性肺损伤小鼠的保护作用

目的探讨和厚朴酚(HNK)对脂多糖(LPS)诱导的急性肺损伤小鼠的保护作用。方法选取SPF级BALB/c小鼠40只,随机分为5组,每组8只,即正常组,模型组,HNK高、低剂量组,地塞米松(DEX)组,利用LPS诱导小鼠急性肺损伤(ALI)模型,和厚朴酚腹腔注射后检测肺干湿比值(W/D)、进行支气管肺泡灌洗液(BALF)中性粒细胞计数、检测血气分析、计算肺泡液体清除率(AFC)等。ELISA法检测各组小鼠血清中TNF-α和细胞因子IL-1β表达的变化,Western blot法和Real time PCR检测各组小鼠肺组织MMP-9和NF-κB表达。结果模型组中,小鼠BALF中中性粒细胞计数、肺W/D比值、TNF-α、IL-1β、MMP-9以及NF-κB水平明显增高,与正常组比较,差异有统计学意义(P<0.05);小鼠PaO_2、酸碱度(pH)、AFC显著降低,与正常组比较,差异有统计学意义(P<0.05)。与模型组比较,HNK高、低剂量组,小鼠BALF中中性粒细胞计数、肺W/D比值、TNF-α、IL-1β、MMP-9以及NF-κB表达水平明显下降,PaO_2、p H、AFC显著上升,差异均有统计学意义(P<0.05)。结论和厚朴酚通过抑制NF-κB、MMP-9对LPS诱导的急性肺损伤发挥重要的保护作用。

急性期川崎病患儿外周血单个核细胞NF-κB、MMP-9表达变化及意义

目的探讨核细胞核因子-κB(NF-κB)和基质金属蛋白酶9(MMP-9)在川崎病发生、发展中的作用及其机制。方法研究对象为川崎病患儿21例(川崎病组)、呼吸道感染患者18例(阳性对照组)、正常查体者17例(正常对照组)。采用密度梯度离心法无菌分离各组外周血PBMCs自然培养,采用RT-PCR法和Westen blot法测定细胞中NF-κB p65 mRNA和蛋白,采用ELISA方法测定细胞上清液中MMP-9蛋白。将川崎病组外周血PBMCs体外培养并分为川崎病1、2、3组,川崎病1组继续自然培养,川崎病2组加入终浓度为20 nmol/L的NF-κB激动剂PMA,川崎病3组同时加入PMA(浓度同上)及终浓度为100μmol/L的NF-κB抑制剂PDTC,孵育2 h后收集上清液和细胞,分别采用RT-PCR法和Westen blot法测定NF-κB p65 mRNA和蛋白表达,采用ELISA法测定细胞上清液中MMP-9蛋白水平。结果川崎病组PBMCs中NF-κB p65 mRNA、蛋白及上清液MMP-9表达均高于阳性对照组和健康对照组(P均<0.05)。川崎病1、3组PBMCs中NF-κB p65蛋白及上清液中MMP-9表达水平均低于川崎病2组(P均<0.05)。结论急性期川崎病患儿外周血PBMCs中NF-κB和细胞上清液中MMP-9表达增强;二者可能参与了川崎病的发生发展过程。。