Study on the nuclear translocation mechanism in the inhibition of nuclear factor-κB activation in bacterial lipoprotein-tolerant THP-1 cells

Objective To approach the nuclear factor-κB (NF-κB) nuclear translocation mechanism in bacterial lipoprotein (BLP) tolerance. Methods Human monocytic THP-1 cells were first pretreated with 10, 100, 1 000 ng/ml BLP for 20 hours to induce BLP tolerance. Then THP-1 cells without BLP pretreatment (control group) or with BLP pretreatment (tolerance group) were stimulated with 0, 10, 100,1 000 ng/ml BLP again for 6 hours. The tumor necrosis factor-α (TNF-α) content in culture medium was measured by enzyme linked immunosorbent assay (ELISA) in order to determine the most suitable BLP pretreatment and stimulation concentration. Western blotting was used to detect the protein level, nuclear translocation and phosphorylation of NF-ΚB p50 and p65 in the cells of control and tolerance groups treated with respective conditions for 0, 0.5, 1, 2 and 6 hours. Results In control group BLP stimulation (10,100, 1 000 ng/ml) could induce THP-1 activation and TNF-α production (pg/ml: 184.86 ± 32. 51,3 215. 88±167. 09, 6 042. 96±245. 37) in a dose-dependent manner. In tolerance group, 100 ng/ml BLP pretreatment resulted in almost complete inhibition of TNF-α production as induced by 10-1 000 ng/ml BLP stimulation. Therefore, 100 ng/ml BLP pretreatment and 1 000 ng/ml stimulation were selected for following cell treatment. Western blotting analysts showed that there was an increase of p50 protein level in BLP-tolerant cells comparing with control group (0 hour: 542. 9±15. 6 vs. 272. 8±13. 2, 0. 5 hour: 558. 0±16. 9 vs. 236. 4±11.8, 1 hour: 524. 7±17. 5 vs. 211. 6±9. 8, 2 hours: 584. 9±15. 6 vs. 222. 4±12. 3, all P＜0. 01), whereas the p65 protein level was similar between the two groups. BLP stimulation also induced the nuclear translocation of p50 and p65 in control group (1-hour p50: 344. 2±13. 6 vs. 79. 0±5. 2, p65:78. 4 ±4.5 vs. 0, both P＜0. 05), but not in tolerance group. In addition, the phosphorylation of p65 at serine 536 was induced after BLP stimulation in control THP-1 cells (0. 5 hour: 0. 67±0. 08 vs. 0. 04±0. 01,1 hour: 0.71±0.11 vs. 0.04±0.01, both P＜0.05), but this change was not detected in BLP-tolerant cells. Conclusion It was found that in BLP-tolerant cells, the expression of inhibitory subunit p50 was increased and the nuclear translocation and phosphorylation of p65 with trans-activation ability was inhibited.These changes are likely responsible for the reduced gene expression of NF-ΚB dependent genes in BLP-tolerant cells.     

CD44单克隆抗体诱导THP-1细胞分化和凋亡作用及其机制探讨

目的 观察CD44单克隆抗体A3D8对急性单核细胞白血病细胞系THP-1细胞的增殖抑制作用,并探讨其作用途径.方法 采用MTT法检测A3D8对THP-1细胞的增殖抑制效应;流式细胞术分析THP-1细胞CD33、CD15、CD11b、CD14、Annexin-V、caspase-3、细胞周期;Western blot法分析p-Akt、磷酸化细胞外调节蛋白激酶(P-ERK)、bcl-2和p27kip1蛋白的表达.结果 A3D8能显著抑制THP-1 细胞增殖,呈剂量和时间的量效关系.2.0 μg/ml A3D8作用THP-1细胞1～6 d后细胞明显分化.A3D8处理4 d,THP-1细胞CD33、CD15、CD11b、CD14表达水平与对照组比较[平均荧光强度(MFI)]分别为68.9±2.0对39.3±1.5、61.7±5.5对12.9±2.6、67.3±3.8对14.0±2.0、83.0±5.7对8.0±1.0(P均<0.01).细胞周期阻滞在G0/G1期.处理4 d实验组和对照组p-Akt、p-ERK、bcl-2蛋白分别为0.24±0.06对1.20±0.15、0.32±0,05对1.24±0.09、0.11±0.05对0.65 ±0.07,实验组较对照组显著减少(P均<0.01);处理4 d实验组和对照组p27kipl.蛋白分别为1.08±0.09对0.10±0.02,实验组较对照组显著增加(P<0.05).A3D8处理5 d THP-1细胞Annexin-V、caspase-3阳性率与对照组比较,分别为(32.5±2.5)%对(2.4±0.3)%、(33.3±2.5)%对(3.6±0.3)%(P均<0.01).结论 CD44单克隆抗体A3D8可能通过抑制PI3K/Akt和ERK1/2信号通路诱导THP-1细胞分化和凋亡.

Abstract：

Objective To investigate the effects of anti-CD44 mAb A3D8 on the cell proliferation of human acute monocytic leukemia cell line THP-1 and its mechanism. Methods Cell proliferation was assayed with MTT method, the expression of CD33, CD15, CD11b, CD14, Annexin-V, caspase-3 and cell cycle with flow cytometry, and the expression of p-Akt, p-ERK, bcl-2 and p27kipl with Western blot. Results A3D8 could remarkably inhibit the proliferation capacity of the THP-1 cells in a dosage- and time-dependent manner. THP-1 differentiation was observed when treated with A3D8 (2.0 μg/ml) for one to six days. Expression of CD33 (68. 9 ±2.0 vs 39.3 ± 1.5), CD15(61. 7 ±5.5 vs 12.9 ±2.6), CD11b (67.3 ± 3.8vs 14.0±2.0) and CD14 (83.0 ±5.7 vs 8.0 ± 1.0) was significantly increased at day 4 compared with the control group ( all P < 0.01). Cell cycle of the THP-1 cells was arrested in G1/G1. Expression of the Annexin-V [(32.5±2.5)% vs (2.4±0.3)%] and caspase-3 [(33.3 ±2.5)% vs (3.6±0.3)%] was much higher than that in normal controls (all P <0. 01) , and apoptosis was observed in THP-1 cells at day 5. Expression of p-Akt (0.24 ±0.06 vs 1.20 ±0.15), p-ERK (0. 32 ±0.05 vs 1. 24 ±0. 09), and bcl-2 (0. 11 ±0.05 vs 0. 65 ± 0. 07) was much lower than that of the controls ( all P < 0. 01), while p27kipl (1.08 ± 0.09 vs 0. 10 ± 0.02) was significantly increased at day 4 (P < 0.05). Conclusion Anti-CD44 antibody can induce the differentiation and apoptosis of THP-1 cell through inhibiting PI3K/Akt and ERK1/2 signaling pathway.     

糖尿病大鼠肺组织中核转录因子-κB及其抑制因子信号通路的表达变化

目的 探讨核转录因子-κB(NF-κB)信号通路激活对糖尿病肺损伤的影响.方法 通过高糖、高脂饮食加腹腔注射小剂量链尿佐菌素(STZ)的方法,建立2型糖尿病大鼠模型,采用光镜动态观察12、24周时,对照组(20只)、糖尿病组(30只)大鼠肺组织的形态学改变情况,用Masson三色染色观察肺组织胶原沉积情况,应用免疫组织化学方法检测2组肺组织NF-κB p65、IκBα、蛋白激酶C的变化情况.结果 光镜下糖尿病大鼠肺组织结构紊乱,肺泡间隔增厚,周围细胞外基质增多,局部纤维化,随病情进展,表达愈加明显.12、24周糖尿病大鼠肺组织NF-κB p65的吸光度值为0.20±0.0l、0.35±0.06,高于同期对照组的0.12±0.02、0.17±0.03,差异有统计学意义(P均＜0.01),12、24周糖尿病肺组织IκB的吸光度为0.29±0.02、0.36±0.03,分别高于同期对照组的0.08±0.02、0.22±0.08,差异有统计学意义(P均＜0.01).结论 NF-κB及其IκB信号通路的激活参与糖尿病肺组织病变的发生发展,可能是肺损伤的机制之一.

Abstract：

Objective To investigate the effect of activation of NF-κB signaling pathway on pathogenesis of lung in diabetes mellitus(DM) rat. Methods The experimental type 2 diabetic rats were built by injecting streptozotocin (STZ) and feeding with high fat and glucose food. At the 12nd and 24th week, we observed the alteration of morphology in the lung of rats in the control group(20 rats) ,the DM group(30 rats)using spectroscopic analysis. The collagen accumulation of lung was observed by masson trihrome staining, and alteration of NF-κB P65, IκBα, and PKC in lung was observed by immunohistochemistry. Results The tissue structure of lung in the DM rats distributed deranged in the light microscope, alveolar wall were thicken, extracellular matrixes increased and pulmonary fibrosis appeared. With the development of pathogenic condition, the expression increased obviously. The staining optical density value of NF-κB P65 in tissue of lung in the 12 w and 24 w DM group were 0. 20 ± 0. 01 and 0. 35 ± 0. 06 respectively, which was significantly higher than those of the control group at the corresponding time point ( 0. 12 ± 0. 02 and 0. 17 ± 0. 03, respectively, Ps ＜ 0. 0l ). The staining optical density value of IκB in tissue of lung in the 12 w and 24 w DM group were 0. 29 ±0. 02 and 0. 36 ± 0. 03, respectively, which were significantly higher than those of the control at the corresponding time point (0. 08 ± 0. 02 and 0. 22 ± 0. 08, respectively, Ps ＜ 0. 01 ). Conclusion The signaling pathway of NF-κB/IκB participate in the occurrence and development in the pathogenesis of lung in DM, and may be one of the mechanisms of lung injury.     

血管内皮生长因子和核转录因子p65在多形性腺瘤中的表达及其意义

目的 探讨血管内皮生长因子(VEGF)和核转录因子(NF-κB)p65在不同分型的多形性腺瘤中的表达及意义.方法 涎腺多形性腺瘤患者60例,其中细胞丰富型31例,基质丰富型29例,取瘤旁正常涎腺组织30份,应用免疫组织化学技术检测组织中VEGF、NF-κBp65的表达.结果 (1)VEGF表达主要以肿瘤性上皮组织较多,多为实性上皮条索、腺管样结构区域,染色强度不尽相同.(2)多形性腺瘤组织中NF-κB p65阳性表达主要见于腺泡上皮和导管上皮,呈强阳性染色;间质成分中血管上皮呈弱阳性表达、纤维结缔组织呈弱阳性表达;黏液样组织和软骨样组织未见明显阳性表达.(3)VEGF在细胞丰富型腺瘤中平均吸光度(MA)值为955.67±305.79,基质丰富型MA值为149.13±60.85,正常对照组MA值为53.46±9.66,两两比较差异均有统计学意义(P均＜0.05).(4)NF-κB p65在细胞丰富型腺瘤MA值为529.80±164.81,基质丰富型为43.40±5.46,正常对照组为6.84±1.91,两两比较差异均有统计学意义(P均＜0.05).<5)涎腺多形性腺瘤中NF-κB p65与VEGF表达呈正相关(r=0.854,P＜0.05).结论 (1)涎腺多形性腺瘤新生血管形成能力及细胞增殖活性随着瘤体中细胞成分的增多而逐渐升高,细胞丰富型多形性腺瘤较基质丰富型多形性腺瘤具有更易恶变的倾向.(2)NF-κB p65可能是通过作用于VEGF来实现对新生血管的调控作用.

Abstract：

Objective To investigate the expression and significance of VEGF and NF-κB/p65 in the two subtypes of pleomorphic adenoma. Methods Immunohistochemistry was applied to detect the expression of VEGF and NF-κB/p65 in pleomorphic adenoma of 60 patients including 31 cases of cell-rich and 29 cases of stroma-rich as well as normal salivary gland tissues of 30 cases from adjacent tumor. Results VEGF positive staining was mainly found in tumor epithelia,while NF-κB/p65 positive was detected in gland alveolus cell and ductal epithelia. The Mean optical density( MA ) values of VEGF were 955.67 ± 305.79,149. 13 ± 60. 85 and 53.46 ± 9. 66, respectively, in cell-rich adenoma, stroma-rich adenoma and normal control. The difference in VEGF expression between the groups was significant (Ps ＜ 0. 05 ) . The MA values of NF-κB/p65 were 529. 80 ± 164. 81,43.40 ±5.46 and 6. 84 ± 1.91 ,respectively,in three groups mentioned above. The difference in NF-κB/p65 expression between the groups was significant ( Ps ＜ 0. 05 ). In pleomorphic adenoma, the expression level of NF-κB/p65 was positively correlated with VEGF. Conclusion ( 1 ) The angiogenesis and proliferation potential of carcinomas increased with the cell component in pleomorphic adenoma. Stroma-poor adenoma is more frequently subjected to malignant transformation than stroma-rich adenoma. (2) NF-κB/p65 may have effects on angiogenesis by activating VEGF. Detecting the expression of VEGF and NF-κB/p65 may be helpful to predict the biological behavior of pleomorphic adenoma and prognosis of the patients, which couldprovide useful information for future targeted therapy of pleomorphic adenoma.     

T淋巴细胞趋化因子受体5表达上调介导了肠毒素超抗原对肺动脉内皮的损伤

目的 观察革兰阳性菌肠毒素活化的T淋巴细胞(T细胞)对人肺动脉内皮细胞(HPAEC)的损伤作用,初步探讨其损伤机制.方法 将葡萄球菌肠毒素B(SEB)活化后的T细胞上清加入HPAEC,检测内皮细胞趋化因子的分泌情况;用Transwell小室观察内皮细胞分泌的趋化因子对T细胞趋化的影响.用10 ng/ml SEB刺激的T细胞与HPAEC共培养,检测T细胞与内皮细胞的黏附率;用原位末端缺刻标记法(TUNEL)检测内皮细胞凋亡情况.结果 不同浓度T细胞培养上清刺激的HPAEC都表现出趋化因子随刺激时间延长而释放增加的趋势,72 h后1×10-2、1×10-1、1×100T细胞中单核细胞趋化蛋白-1(MCP-1,ng/ml)分别为1.240±0.103、4.200±0.305、6.500±0.500,巨噬细胞炎性蛋白-1α(MIP-1α,ng/ml)分别为0.210±0.015、0.287±0.012、0.531±0.037,正常T细胞表达和分泌因子(Rantes,ng/ml)分别为1.420±0.074、7.634±0.630、15.700±1.300,其中Rantes表现出早期(6 h)快速释放和后期(12、24、48、72 h)延迟释放的特点.与未经SEB作用的对照组相比,超抗原组的T细胞从Transwell小室上室趋化移动至聚碳酸酯膜的数量(个)显著增多(86.38±14.50比16.50±2.50,P＜0.01＝;与T细胞组比较,超抗原组24 h T细胞黏附率显著增加[(15.50±1.08)%比(1.60±0.22)%,P＜0.01＝,加入1 μg/ml甲基化Rantes组T细胞黏附率[(4.39±0.66)%]较超抗原组显著下降(P＜0.01＝;黏附到下层HPAEC的T细胞趋化因子受体5(CCR5)/CD4较100 ng/ml SEB诱导3 d的单个核细胞(PBMC)增加了约2.5倍,CCR5/CD8增加了约2.8倍;与正常培养的HPAEC组比较,超抗原组HPAEC细胞凋亡指数增加[(32.50±4.50)%比(3.50±0.50)%,P＜0.01＝.结论 SEB活化的T细胞增加了对HPAEC的趋化和黏附,进一步导致HPAEC的损伤;这种趋化作用的增强与SEB活化的T细胞表面CCR5表达上调有关.

Abstract：

Objective To observe the injurious effect of T cell activated by Staphylococcus enterotoxin B (SEB) on human pulmonary artery endothelial cell (HPAEC) and explore its possible mechanism. Methods HPAEC was cocultured with SEB-activated T cells supernatant, and the secretion of chemotactic factors from HPAEC was examined. The Transwell inserts was used in chemoattraction assays. After HPAECs were cocultured with T cells and 10 ng/ml SEB for 3 days, HPAEC damage was monitored by microscopy and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Results Three kinds of tested chemokines showed a time-dependent increase in all supernatant of HPAEC incubated with different concentrations of T cells. After 72 hours, the monocyte chemoattractant protein-1 (MCP-1,ng/ml) in 1 × 10-2, 1 × 10-1 , 1× 100 T cell supernatant groups was 1. 240± 0. 103, 4. 200± 0. 305, 6. 500±0. 500, respectively, macrophage inflammatory protein-1a (MIP-1α, ng/ml) was 0. 210 ± 0. 015, 0. 287 ±0. 012, 0. 531 ± 0. 037, respectively, and Rantes (ng/ml) was 1. 420 ± 0. 074, 7. 634 ± 0. 630, 15. 700 ±1. 300, respectively. Rantes presented a two-phase secretion mode : in early 6 hours it increased swiftly, but relatively slow at 12, 24, 48, 72 hours. T cell adherent to polycarbonate membrane increased after SEB stimulation in superantigen group compared with control group without SEB stimulation (86. 38± 14. 50 vs.16. 50± 2. 50, P＜ 0. 01 ). When 10 ng/ml SEB activated T cell was cocultured with HPAEC, more of originally suspended cultured T cells adhered to HPAEC monolayer [(15. 50±1.08)% vs. (1.60±0. 22)%,P＜0.01], whereas the cell adhesion ratio decreased markedly in 1 μg/ml Met-Rantes group [(4. 39±0. 66)%, P ＜ 0. 01 ). FACs test of HPAEC-adherent T cell showed lymphocyte chemokine receptor 5 (CCR5)/CD4 and CCR5/CD8 increased over 2. 5 folds and 2.8 folds compared with 100 ng/ml SEB activated T cell. Cell death rate of HPAEC was increased when cocultured with SEB-activated T cell in superantigen group compared with HPAEC normal incubation group [(32. 50±4. 50)% vs. (3. 50±0. 50)%, P＜0. 01].Conclusion Increased chemoattraction and adherence of SEB-activated T cells to HPAEC could damage HPAEC; this effect was possibly due to up-regulation of CCR5 on T cell.     

抑制胞浆型磷脂酶A2活性对内毒素诱导人脐静脉内皮细胞分泌瘦素的影响

目的 通过改变胞浆型磷脂酶A2(cPLA2)的活性,检测细菌脂多糖(LPS)及Ca2+载体A23187诱导的人脐静脉内皮细胞株(ECV-304)上清液中瘦素(Leptin)水平的变化,探讨在体外炎症状态下cPLA2活性与细胞分泌Leptin的关系.方法 体外培养ECV-304细胞.实验1:将细胞分为空白对照组,LPS 3个浓度5、10、20 μg/ml刺激组,Ca2+载体A23187 3个浓度0.1、 1.0、10.0 μmol/L刺激组共7个组,分别作用6、12、24 h后收集上清液.实验2:根据实验1结果将细胞分为空白对照组,LPS 20 μg/ml刺激组,cPLA2特异性抑制剂AACOCF3 3个浓度0.1、1.0、10.0 μmol/L与LPS合用刺激组,丝裂素活化蛋白激酶上游激酶1/2(MEK1/2)抑制剂 U0126 3个浓度0.1、1.0、5.0 μmol/L与LPS合用刺激组共8个组,在LPS刺激前1 h加入AACOCF3或U0126,LPS刺激24 h后收集上清液.采用放射免疫分析法检测Leptin水平.结果 实验1:随LPS刺激浓度增加和时间延长,细胞释放Leptin浓度逐渐减少,LPS 20 μg/ml组作用24 h后Leptin浓度(ng/ml)较空白对照组显著下降(0.540±0.109比0.823±0.048,P<0.05).但A23187对细胞分泌Leptin并无显著影响.实验2:LPS刺激能使细胞分泌Leptin浓度(ng/ml)明显下降(0.558±0.069比0.825±0.067,P<0.05);而用不同浓度AACOCF3或U0126干预后,细胞分泌Leptin的浓度(ng/ml)有所回升,且呈浓度依赖性(AACOCF3 0.1、1.0、10.0 μmol/L组分别为0.673±0.135、 0.723±0.055、 0.797±0.062;U0126 0.1、 1.0、5.0 μmol/L组分别为0.698±0.112、 0.862±0.184、0.935±0.145),AACOCF3 1.0 μmol/L、10.0 μmol/L组和U0126 1.0 μmol/L、5.0 μmol/L组Leptin浓度均显著高于LPS 20 μg/ml刺激组(均P<0.05).结论 在由LPS诱导的体外炎症状态下,Leptin的分泌与cPLA2的活性具有一定的关系.

Abstract：

Objective To determine Leptin levels in supernatant fluid of culture of human umbilical vein endothelial cells (ECV-304) after being challenged by lipopolysaccharide (LPS) and calcium ion vector A23187, and to explore the possible relation between Leptin release and cytosolic phospholipase A2 (cPLA2) activity in an inflammatory cell model. Methods ECV-304 cells were cultured in vitro. Experiment 1: the cells were divided into seven groups: blank control group, LPS 5, 10, 20 μg/ml stimulation groups, A23187 0.1, 1.0, 10.0 μmol/L stimulation groups. The supernatants were collected at 6, 12 and 24 hours. Experiment 2: according to the results of experiment 1, the cells were divided into eight groups: blank control group, LPS 20 μg/ml stimulation group, the inhibitor of cPLA2 AACOCF3 0.1, 1.0, 10.0 μmol/L plus LPS stimulation groups, the inhibitor of mitogen-activated protein/extracellular signal-regulated protein kinase kinase 1/2 (MEK1/2) U0126 0.1, 1.0, 5.0 μmol/L plus LPS stimulation groups, with AACOCF3 or U0126 added 1 hour before the addition of LPS, and the supernatants were collected 24 hours after the addition of LPS. Leptin level was determined by radioimmunoassay. Results Experiment 1: with increase in LPS concentration and prolongation of time, Leptin release was decreased gradually. After 24 hours of interaction the concentration of Leptin (ng/ml) in LPS 20 μg/ml group was decreased significantly compared with the blank control group (0.540±0.109 vs. 0.823±0.048, P<0.05). However, A23187 had no significant effect on Leptin release. Experiment 2: LPS rendered cells to release less Leptin (ng/ml: 0.558±0.069 vs. 0.825±0.067, P<0.05); by adding AACOCF3 or U0126 in different concentration before adding LPS rendered the cells to release more Leptin (ng/ml), and it showed concentration-dependent (the AACOCF3 0.1, 1.0, 10.0 μmol/L groups were 0.673±0.135, 0.723±0.055, 0.797±0.062, respectively; the U0126 0.1, 1.0, 5.0 μmol/L groups were 0.698±0.112, 0.862±0.184, 0.935±0.145, respectively). The release of Leptin in AACOCF3 1.0 μmol/L, 10.0 μmol/L and U0126 1.0 μmol/L, 5.0 μmol/L groups was significantly higher than LPS 20 μg/ml stimulation group (all P<0.05). Conclusion There is a possible relation between Leptin release and cPLA2 activity in inflammatory cells induced by LPS.     

地塞米松对脑损伤大鼠核转录因子-κB水平的影响

Objective To explore the effects of dexamethasone on nuclear factor-kB (NF-κB) expression in brain tissue after traumatic brain injury (TBI). Methods Forty rats were randomly divided into two groups: dexamethasone treatment and no treatment, and severe brain injury was produced by gas percussion in both groups. At 0, 6, 24, 72 and 120 hours after injury, 5 rats of each group were executed and the histopathological changes in brain tissue in rats were observed by hematoxylin-eosin (HE) stain. The expression of NF-κB in brain tissue of rats was detected by immunohistochemical method. Results NF-κB expression was significantly up-regulated at 6 hours in brain tissue of rats after TBI (P<0.05), reaching the highest level at 24 hours (P<0. 01). It showed a tendency to lower, but was still high at 120 hours after TBI (P<0. 05 or P<0. 01). After treatment with dexamethasone, NF-κB level was lowered at 6, 24 and 72 hours (all P<0. 01). Conclusion NF-κB expression is up-regulated in brain tissue in early period after TBI, and keeps on a high level, thus inducing inflammatory response to produce secondary injury to brain tissue. Dexamethasone shows protective effects by regulating the levels of NF-κB and prevents secondary injury which is caused by the inflammatory cytokines in rat brain tissue after TBI.     

地塞米松对脑损伤大鼠核转录因子-κB水平的影响

Objective To explore the effects of dexamethasone on nuclear factor-kB (NF-κB) expression in brain tissue after traumatic brain injury (TBI). Methods Forty rats were randomly divided into two groups: dexamethasone treatment and no treatment, and severe brain injury was produced by gas percussion in both groups. At 0, 6, 24, 72 and 120 hours after injury, 5 rats of each group were executed and the histopathological changes in brain tissue in rats were observed by hematoxylin-eosin (HE) stain. The expression of NF-κB in brain tissue of rats was detected by immunohistochemical method. Results NF-κB expression was significantly up-regulated at 6 hours in brain tissue of rats after TBI (P<0.05), reaching the highest level at 24 hours (P<0. 01). It showed a tendency to lower, but was still high at 120 hours after TBI (P<0. 05 or P<0. 01). After treatment with dexamethasone, NF-κB level was lowered at 6, 24 and 72 hours (all P<0. 01). Conclusion NF-κB expression is up-regulated in brain tissue in early period after TBI, and keeps on a high level, thus inducing inflammatory response to produce secondary injury to brain tissue. Dexamethasone shows protective effects by regulating the levels of NF-κB and prevents secondary injury which is caused by the inflammatory cytokines in rat brain tissue after TBI.     

地塞米松对脑损伤大鼠核转录因子-κB水平的影响

Objective To explore the effects of dexamethasone on nuclear factor-kB (NF-κB) expression in brain tissue after traumatic brain injury (TBI). Methods Forty rats were randomly divided into two groups: dexamethasone treatment and no treatment, and severe brain injury was produced by gas percussion in both groups. At 0, 6, 24, 72 and 120 hours after injury, 5 rats of each group were executed and the histopathological changes in brain tissue in rats were observed by hematoxylin-eosin (HE) stain. The expression of NF-κB in brain tissue of rats was detected by immunohistochemical method. Results NF-κB expression was significantly up-regulated at 6 hours in brain tissue of rats after TBI (P<0.05), reaching the highest level at 24 hours (P<0. 01). It showed a tendency to lower, but was still high at 120 hours after TBI (P<0. 05 or P<0. 01). After treatment with dexamethasone, NF-κB level was lowered at 6, 24 and 72 hours (all P<0. 01). Conclusion NF-κB expression is up-regulated in brain tissue in early period after TBI, and keeps on a high level, thus inducing inflammatory response to produce secondary injury to brain tissue. Dexamethasone shows protective effects by regulating the levels of NF-κB and prevents secondary injury which is caused by the inflammatory cytokines in rat brain tissue after TBI.     

地塞米松对脑损伤大鼠核转录因子-κB水平的影响

Objective To explore the effects of dexamethasone on nuclear factor-kB (NF-κB) expression in brain tissue after traumatic brain injury (TBI). Methods Forty rats were randomly divided into two groups: dexamethasone treatment and no treatment, and severe brain injury was produced by gas percussion in both groups. At 0, 6, 24, 72 and 120 hours after injury, 5 rats of each group were executed and the histopathological changes in brain tissue in rats were observed by hematoxylin-eosin (HE) stain. The expression of NF-κB in brain tissue of rats was detected by immunohistochemical method. Results NF-κB expression was significantly up-regulated at 6 hours in brain tissue of rats after TBI (P<0.05), reaching the highest level at 24 hours (P<0. 01). It showed a tendency to lower, but was still high at 120 hours after TBI (P<0. 05 or P<0. 01). After treatment with dexamethasone, NF-κB level was lowered at 6, 24 and 72 hours (all P<0. 01). Conclusion NF-κB expression is up-regulated in brain tissue in early period after TBI, and keeps on a high level, thus inducing inflammatory response to produce secondary injury to brain tissue. Dexamethasone shows protective effects by regulating the levels of NF-κB and prevents secondary injury which is caused by the inflammatory cytokines in rat brain tissue after TBI.     

地塞米松对脑损伤大鼠核转录因子-κB水平的影响

Objective To explore the effects of dexamethasone on nuclear factor-kB (NF-κB) expression in brain tissue after traumatic brain injury (TBI). Methods Forty rats were randomly divided into two groups: dexamethasone treatment and no treatment, and severe brain injury was produced by gas percussion in both groups. At 0, 6, 24, 72 and 120 hours after injury, 5 rats of each group were executed and the histopathological changes in brain tissue in rats were observed by hematoxylin-eosin (HE) stain. The expression of NF-κB in brain tissue of rats was detected by immunohistochemical method. Results NF-κB expression was significantly up-regulated at 6 hours in brain tissue of rats after TBI (P<0.05), reaching the highest level at 24 hours (P<0. 01). It showed a tendency to lower, but was still high at 120 hours after TBI (P<0. 05 or P<0. 01). After treatment with dexamethasone, NF-κB level was lowered at 6, 24 and 72 hours (all P<0. 01). Conclusion NF-κB expression is up-regulated in brain tissue in early period after TBI, and keeps on a high level, thus inducing inflammatory response to produce secondary injury to brain tissue. Dexamethasone shows protective effects by regulating the levels of NF-κB and prevents secondary injury which is caused by the inflammatory cytokines in rat brain tissue after TBI.     

地塞米松对脑损伤大鼠核转录因子-κB水平的影响

Objective To explore the effects of dexamethasone on nuclear factor-kB (NF-κB) expression in brain tissue after traumatic brain injury (TBI). Methods Forty rats were randomly divided into two groups: dexamethasone treatment and no treatment, and severe brain injury was produced by gas percussion in both groups. At 0, 6, 24, 72 and 120 hours after injury, 5 rats of each group were executed and the histopathological changes in brain tissue in rats were observed by hematoxylin-eosin (HE) stain. The expression of NF-κB in brain tissue of rats was detected by immunohistochemical method. Results NF-κB expression was significantly up-regulated at 6 hours in brain tissue of rats after TBI (P<0.05), reaching the highest level at 24 hours (P<0. 01). It showed a tendency to lower, but was still high at 120 hours after TBI (P<0. 05 or P<0. 01). After treatment with dexamethasone, NF-κB level was lowered at 6, 24 and 72 hours (all P<0. 01). Conclusion NF-κB expression is up-regulated in brain tissue in early period after TBI, and keeps on a high level, thus inducing inflammatory response to produce secondary injury to brain tissue. Dexamethasone shows protective effects by regulating the levels of NF-κB and prevents secondary injury which is caused by the inflammatory cytokines in rat brain tissue after TBI.     

地塞米松对脑损伤大鼠核转录因子-κB水平的影响

Objective To explore the effects of dexamethasone on nuclear factor-kB (NF-κB) expression in brain tissue after traumatic brain injury (TBI). Methods Forty rats were randomly divided into two groups: dexamethasone treatment and no treatment, and severe brain injury was produced by gas percussion in both groups. At 0, 6, 24, 72 and 120 hours after injury, 5 rats of each group were executed and the histopathological changes in brain tissue in rats were observed by hematoxylin-eosin (HE) stain. The expression of NF-κB in brain tissue of rats was detected by immunohistochemical method. Results NF-κB expression was significantly up-regulated at 6 hours in brain tissue of rats after TBI (P<0.05), reaching the highest level at 24 hours (P<0. 01). It showed a tendency to lower, but was still high at 120 hours after TBI (P<0. 05 or P<0. 01). After treatment with dexamethasone, NF-κB level was lowered at 6, 24 and 72 hours (all P<0. 01). Conclusion NF-κB expression is up-regulated in brain tissue in early period after TBI, and keeps on a high level, thus inducing inflammatory response to produce secondary injury to brain tissue. Dexamethasone shows protective effects by regulating the levels of NF-κB and prevents secondary injury which is caused by the inflammatory cytokines in rat brain tissue after TBI.     

子宫颈癌化疗前后核转录因子-κB和P-糖蛋白表达的比较

目的 探讨子宫颈癌患者新辅助化疗(NACT)前后癌组织中核转录因子-κB(NF-κB)、P-糖蛋白(P-gp)的表达及相互关系.方法 采用real time RT-PCR和免疫组织化学法检测40例子宫颈癌患者NACT前后癌组织中NF-κB、P-gp的表达.结果 化疗前后子宫颈癌组织中NF-κB mRNA的表达量分别为50.340±0.780、67.595±0.914,差异有统计学意义(t=-73.384,P＜0.01);子宫颈癌组织中MDR1 mRNA的表达量分别为45.800±0.636、62.381±0.743,差异有统计学意义(t=-66.582,P＜0.01);子宫颈癌组织中NF-κB蛋白的阳性表达率分别为50%(20/40)、80%(32/40),差异有统计学意义(χ2=7.912,P=0.005);子宫颈癌组织中P-gp的阳性表达率分别为40%(16/40)、70%(28/40),差异有统计学意义(χ2=7.273,P=0.007);新辅助化疗后NF-κB、MDR1 mRNA及其编码蛋白NF-κB、P-gp表达水平较化疗前明显升高,2组比较差异有统计学意义(P＜0.01).化疗前后NF-κB与P-gp表达均成正相关(r前=0.408,P＜0.05;r后=0.327,P＜0.05).结论 NF-κB与P-gp可能参与了子宫颈癌和多药耐药的发生,本组化疗药物可能诱导了NF-κB的活化与多药耐药,NF-κB的活化与P-gp介导的子宫颈癌多药耐药有关.

Abstract：

Objective To investigate the relationship between the expression of NF-κB and Pglycoprotein(P-gp) in cervical cancer tissue before and after neoadjuvant chemotherapy(NACT). Methods The mRNA and the protein expression levels of NF-κB and P-gp were determined by Real time-PCR and immunohistochemical assay respectively in cancer specimens from 40 cevical cancer patients, which were collected before and after NACT. Results The mRNA expression levels of NF-κB were 50. 340 ± 0. 780 and 67. 595 ± 0. 914 before and after NACT respectively, which showed significant difference statistically ( t = -73. 384 ,P ＜ 0. 01 ). We also found significant difference in the comparison between MDR1 mRNA expression before and after NACT(45. 800 ±0. 636 vs 62. 381 ±0. 743 ,t = -66. 582,P ＜0. 01 ). The positive rate of NF-κB protein expression before and after NACT were 50% ( 20/40 ), 80% ( 32/40 ) respectively, which showed significant difference statistically( χ2 = 7. 912, P = 0. 005 ). The positive rate of P-gp protein expression after NACT was significantly higher than that before NACT [ 40% ( 16/40 ) vs 70% ( 28/40 ), χ2 = 7. 273, P = 0. 007 ]The mRNA and protein expressions of NF-κB were positively correlated with those of P-gp before and after NACT( r = 0. 408,r = 0. 327 respectively, Ps ＜ 0. 05 ). Conclusion NF-κB and P-gp may play an important role in multi-drug resistance of cervical carcinoma. Chemotherapy drugs in our NACT strategy may induce the activation of NF-κB and multi-drug resistance. Activation of NF-κB may correlate with multi-drug resistance mediated by P-gp.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.     

Effects of thalidomide on Annexin Ⅱ gene regulation

Objective To investigate the effect of thalidomide on Annexin Ⅱ (AnxA2) gene regula-tion in multiple myeloma cell line RPMI8226 and human microvascular endothelial cell line HMEC-1 cells in vitro, and explore the potential mechanism of thrombosis induced by thalidomide. Methods RPMI8226 and HMEC-I cells were cultivated in vitro. Real time quantitative PCR (RQ-PCR) was used to detect the influ-ence of thalidomide at different concentration on the expression of AnxA2 mRNA, flow cytometry(FCM) and confocal microscopy were used to detect the cell surface protein level after the samples were stimulated with different concentrations of thalidomide. Results AnxA2 mRNA level in RPMI8226 cells treated with thalido-mide at 12.5 μg/ml, 25.0 μg/ml and 50.0 μg/ml was decreased compared with the control group (0.60 ±0. 15, 0.33 ± 0. 14, 0.42 ±0. 16, vs 1.07 ±0. 16, respectively, P <0.05)and did so in HMEC-1 cells (0.21 ±0.20, 0.08 ±0.08, 0.17 ±0. 16 vs 1.16 ±0.24, respectively, P <0.05). The AnxA2 protein lev-el in RPMI8226 cells treated with above mentioned concentrations of thalidomide was also decreased compared with the control (3.39 ± 0.32, 2.82 ± 0.28, 3.21 ± 0.23 vs 5.53 ± 0.32, repectively, P < 0. 05) and that did so in HMEC-1 cells (0.72±0. 11, 0.64 ±0.08, 0.67 ±0.08 vs 1.40 ±0. 15, respectively, P<0.05). Conclusions Thalidomide can inhibit the expression of AnxA2 mRNA and protein in RPMI8226 and HMEC-1 cells, which may be one of the mechanisms for the development of thrombosis induced by thalido-mide in multiple myeloma patients.