小白菊内酯抑制紫杉醇诱导的肿瘤细胞凋亡

目的　探讨NF κB在紫杉醇诱导肿瘤细胞凋亡中的作用及小白菊内酯对紫杉醇诱导凋亡的影响。方法　以人乳癌BCap37细胞和人表皮KB细胞为研究对象 ,用 5,1 0和 2 0 μmol·L-1 小白菊内酯预处理细胞 ,以DNA凋亡梯状条带、DNA含量、MTT、细胞甩片及电泳迁移率变动分析 (EMSA)法检测它对紫杉醇诱导细胞凋亡的影响并探索其作用靶点。结果　通过检测DNA凋亡梯状条带、DNA含量和细胞生存率 ,2 0μmol·L-1 小白菊内酯能显著抑制紫杉醇所诱导的BCap37和KB细胞凋亡 ,但不影响紫杉醇诱导肿瘤细胞G2 /M期阻滞。EMSA实验表明小白菊内酯能抑制紫杉醇诱导激活NF κB。结论　小白菊内酯通过抑制NF κB的激活来抑制紫杉醇所诱导的肿瘤细胞凋亡 ,而紫杉醇诱导肿瘤细胞凋亡的过程可能与G2 /M期阻滞无关     

胃癌中NF-кB与COX-2表达的研究

目的探讨NF-кB、COX-2的表达与胃癌的关系及临床意义。方法采用免疫组织化学方法(S-P法)对胃癌组织、正常胃黏膜中NF-кB p65及COX-2蛋白表达进行检测。结果 NF-кB p65蛋白和COX-2蛋白在胃癌组织中的阳性表达率分别为67%和60%,正常胃黏膜中的阳性表达率分别为21%和18%,2者比较均有显著性差异(P均<0.01)。在低分化组的阳性表达率分别为79%和72%,高/中分化组的阳性表达率分别为53%和47%,2者比较均有显著性差异(P均<0.05);在浸润深度为T3+T4组的阳性表达率分别为73%和66%,T1+T2组的阳性表达率分别为22%和22%,2者比较均有显著性差异(P<0.01);在临床Ⅲ～Ⅳ期组的阳性表达率分别为78%和74%,Ⅰ～Ⅱ期组的阳性表达率分别为48%和37%,2者比较均有显著性差异(P均<0.01)。不同性别、年龄、肿瘤部位及大体分型胃癌组织中NF-кB p65和COX-2蛋白的表达无显著性差异(P均>0.05)。胃癌组织中NF-кB p65和COX-2蛋白的表达呈明显正相关(rs=0.539,P<0.01)。结论胃癌组织中的NF-кB和COX-2表达增高,提示其与胃癌的发生、发展、侵袭、淋巴转移密切相关。     

丙型肝炎病毒核心蛋白细胞内信号传导途径的探讨

目的：研究丙型肝炎病毒核心蛋白致病作用的细胞内信号传导途径（STPs)。方法：在前期工作已证实HCV核心蛋白激活NF－κB介导的细胞内STPs这一结论的基础上，对核心蛋白活化NF－κB的机制和作用位点进行了测定。用0．4μg pCXN2-core与pNF-κB共转染Hela细胞3h后分别加入5mmol IKKβ的抑制剂乙酰水杨酸和25μmol消炎痛（作为对照）各0．5ml，继续转染33h，测定荧光素酶比活性，以此检测乙酰水杨酸对NF－κB介导的STPs的抑制作用。以pCXN2-core转染Cos-7细胞36h，收获细胞溶解液，用Western印迹分析检测IκBα表达，确定IκBα的降解情况。用pCXN2-core、pMEKKΔ（表达非活性MEKK的质粒）、pNF-κB共转Hela细胞36h，测定荧光素酶比活性，确定HCV核心蛋白是否通过MEKK活化NF－κB介导的STPs。结果：（1）HCV核心蛋白活化NF－κB介导的STPs被乙酰水杨酸抑制；（2）pCXN2-core转染后的细胞溶 解液中IκBα的量较pCXN2转染后的少；（3）HCV核心蛋白活化NF－κB介导的STPs的作用不被MEKKΔ阻断。结论：（1）乙酰水杨酸通过抑制KK的IκBα磷酸化作用，抑制IκB降解，阻断NF－κB的活化，表明核心蛋白作用位点可能在IKK或其上游某个部位；（2）在表达HCV核心蛋白的细胞存在IκBα降解的增加，造成NF－κB的活化；（3）核心蛋白的作用位点处于MEKK作用点下游。（4）HCV核心蛋白可能的细胞内信号传导途径是：HCV核心蛋白直接作用于IKK→IKK磷酸化IκBα→IκBα降解→NF－κB脱离抑制从胞浆转入胞核，与DNA结合发挥作用。     

Assay of DNA-binding proteins with a dsDNA-coupled plate

OBJECTIVES: This paper fabricated a cost-effective dsDNA-coupled plate (dcPlate) and applied it to measure the abundance and DNA-binding activity of a DNA-binding protein (DBP). DESIGN AND METHODS: The dcPlate was manufactured by covalently immobilizing an amino-modified oligonucleotide in wells of the plate coated with N-oxysuccinimide esters. The dcPlate was applied to measure the abundance of DNA-binding activity of a DBP in the same four steps, including protein incubation, primary antibody binding, enzyme-linked secondary antibody binding, and colorimetric development. RESULTS: The detections of three purified DBPs including NF-kappaB, AP1 and SP1, and HeLa cell nuclear extract and assays of DNA-binding activity of NF-kappaB p50 to five various DNA sequences demonstrated that dcPlate can be used to measure the abundance of DBPs quantitatively and assay DNA-binding activity of DBPs in high throughputs format. CONCLUSIONS: The homemade cost-effective dcPlate provides a simple and versatile platform for studying DBPs.     

膀胱移行细胞癌组织中NF-κB p65、uPA、Bcl-2的表达变化及其意义

目的观察膀胱移行细胞癌（下称膀胱癌）组织中NF—κBp65和尿激酶型纤溶酶原激活物（uPA）及Bcl-2的表达变化,并探讨其相关性。方法采用免疫组化SP法对40例膀胱癌组织和10例正常膀胱组织中NF—κBp65、uPA、Bcl-2进行测定,分析三者与膀胱癌分级、分期、侵袭转移及复发的关系及NF-κBp65表达与uPA、Bcl-2的相关性。结果NF-κBp65、uPA、Bcl-2在膀胱癌组织中的表达均明显高于正常膀胱组织（P〈0．05）;NF—κBp65和uPA与膀胱癌病理分级、分期、淋巴结转移有关（P均〈0．05）,Bcl-2与膀胱癌病理分级、分期有关（P〈0．05）,与淋巴结转移无关（P〉0．05）;膀胱癌组织中,NF—κBp65表达与uPA、Bcl-2表达呈正相关（r分别为0．388、0．462,P均〈0．05）。结论在膀胱癌组织中NF-κBp65、uPA、Bcl-2均呈高表达,并与膀胱癌的临床病理学特征有关;NF—κBp65可能通过调控uPA、Bcl-2的表达促进膀胱癌的进展;NF—κBp65、uPA、Bcl-2可以作为判断膀胱癌侵袭性、转移及预后的生物学标志物。     

实验性结肠炎大鼠内毒素-Toll样受体4-核因子κB信号通路的变化及益生菌的作用

目的探讨和分析实验性结肠炎大鼠血浆内毒素水平的变化及结肠Toll样受体4（TLR4）和核因子κB（NF-κB）表达及意义,并分析益生菌的作用。方法30只雄性W istar大鼠均分为正常对照组（NC组）、模型对照组（UC组）和益生菌治疗组（PC组）;UC和PC组建立2,4,6-三硝基苯磺酸（TNBS）实验性结肠炎大鼠模型,PC组大鼠给予双歧三联活菌悬液（2.2×10^9CFU/只）治疗,1次/d,共4周。光镜下观察肠黏膜炎性反应并评分;鲎试验检测各组大鼠血浆内毒素水平;W estern印迹法和实时荧光定量PCR法检测大鼠结肠TLR4和NF-κB p65蛋白及mRNA的表达,分析其变化及益生菌的作用。结果PC组炎性反应评分较UC组明显降低（P〈0.05）,但高于NC组（P〈0.01）;PC组血浆内毒素明显低于UC组（P〈0.05）,但高于NC组（P〈0.01）;UC组TLR4和NF-κB p65的蛋白及mRNA表达明显高于PC组和NC组（P〈0.05、0.01）,PC组高于NC组（P〈0.01）。结论内毒素-TLR4-NF-κB信号通路参与了大鼠实验性结肠炎的发生和发展,减少内毒素的产生、降低大鼠结肠TLR4和NF-κB表达可能是益生菌减轻大鼠结肠炎症的机制之一。     

Effects of Huogu I Formula (活骨I方) on correlated factors of bone regeneration in chickens with steroid-induced necrosis of femoral head

Objective:To study the mechanism of HuoguⅠFormula（活骨Ⅰ方） in treating osteonecrosis of femoral head.Methods:Forty-eight healthy female Leghorn chickens were randomly divided into control group,model group and HuoguⅠgroup,and each group consisted of 16 chickens.At the meantime of model establishment,chickens of the HuoguⅠgroup were administrated with decoction,while the model and control group with distilled water by gavage.At the 8th and 16th week after medication,blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations.Specifically, expressions of bone morphogenetic protein-2（BMP2）,transforming growth factor betal（TGFβ₁）,Smad4 and Smad7 were evaluated by immunohistochemistry,while expression of osteoprotegerin/receptor activator of nuclear factor kappaB ligand（OPG/RANKL） mRNA was detected by in situ hybridization.Results:Compared with the control group,serum levels of total cholesterol（TC）,triglyceride（TG） and low-density lipoprotein cholesterol （LDL-C） in the model group rose significantly.Positive cell counting of BMP2,TGFβ₁,Smad4 and OPG in femoral head of the model group dropped prominently.Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group,levels of TC,TG and LDL-C in Huogu I group reduced significantly.Positive cell counting of BMP2,TGFβ₁,Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly.Especially at the 8th week,these variations were more significant.Conclusion:HuoguⅠFormula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2,TGFβ₁,Smads and OPG/RANKL of osteoclast in femoral head.     

Caffeic acid phenethyl ester-mediated Nrf2 activation and IκB kinase inhibition are involved in NFκB inhibitory effect: Structural analysis for NFκB inhibition

Caffeic acid phenethyl ester (CAPE) is an active component of propolis from honeybee. We investigated potential molecular mechanisms underlying CAPE-mediated nuclear factor kappa beta (NFκB) inhibition and analyzed structure of CAPE for its biological effect. CAPE attenuated expression of NFκB dependent luciferase stimulated with TNF-α or LPS and suppressed LPS-mediated induction of iNOS, a target gene product of NFκB. In HCT116 cells, CAPE interfered with TNF-α dependent IκBα degradation and subsequent nuclear accumulation of p65, which occurred by direct inhibition of inhibitory protein kappaB kinase (IKK). CAPE increased the expression of Nrf2-dependent luciferase and heme oxygenase-1, a target gene of Nrf2, and elevated the nuclear level of Nrf2 protein, indicating that CAPE activated the Nrf2 pathway. In HCT116 cells with stable expression of Nrf2 shRNA, CAPE elicited a reduced inhibitory effect on TNF-α-activated NFкB compared to scramble RNA expressing control cells. On the other hand, the NFκB inhibitory effect of CAPE was diminished by removal or modification of the Michael reaction acceptor, catechol or phenethyl moiety in CAPE. These data suggest that CAPE inhibits TNF-α-dependent NFκB activation via direct inhibition of IKK as well as activation of Nrf2 pathway, in which the functional groups in CAPE may be involved.