COX-2 is associated with cadmium-induced ICAM-1 expression in cerebrovascular endothelial cells

In order to get insight into the mechanism of cadmium (Cd)-induced brain injury, we investigated the effects of Cd on the induction of COX-2 and ICAM-1 in bEnd.3 mouse brain endothelial cells (EC). Cd stimulated PGE(2) release in a time and dose dependent manner, which was accompanied by increase of COX-2 expression. The thiol-reducing antioxidant N-acetylcyteine attenuated Cd-induced PGE(2) production and COX-2 expression. Cd increased phosphorylation of p38 MAPK, but not of JNK and ERK1/2. A blockade of p38 MAPK pathway abrogated Cd-induced COX-2 expression and PGE(2) production. Cd-induced ICAM-1 expression and leukocyte-EC adhesion were diminished by non-steroidal anti-inflammatory drugs such as indomethacin and NS-398, which was reversed by addition of PGE(2). Together, these data suggest that Cd induces COX-2 expression through the activation of p38 MAPK, an oxidative stress-sensitive cellular signaling molecule, and induction of COX-2 is associated with ICAM-1 expression in brain endothelial cells following Cd exposure.     

Urocortin induced expression of COX-2 and ICAM-1 via corticotrophin-releasing factor type 2 receptor in rat aortic endothelial cells

Background and purpose:

Our previous study showed that urocortin (Ucn1) exacerbates the hypercoagulable state and vasculitis in a rat model of sodium laurate-induced thromboangiitis obliterans. Furthermore, the inflammatory molecules COX-2 and ICAM-1 may participate in this effect. In the present study, the effects of Ucn1 on COX-2 and ICAM-1 expression in lipopolysaccharide (LPS)-induced rat aortic endothelial cells (RAECs) were investigated and the mechanisms involved explored.

Experimental approach:

RAECs were isolated from adult male Wistar rats, and identified at the first passage. Experiments were performed on cells, from primary culture, at passages 5–8. The expression of COX-2 and ICAM-1 at both mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blot analysis. Levels of PGE₂ and soluble ICAM-1 (sICAM-1) in culture medium were measured by enzyme-linked immunosorbent assay. Furthermore, the phosphorylation status of p38MAPK, ERK1/2, JNK, Akt and NF-κB was analysed by Western blot; nuclear translocation of NF-κB was observed by immunofluorescence.

Key results:

Ucn1 augmented LPS-induced expression of COX-2 and ICAM-1 in RAECs in a time- and concentration-dependent manner. Ucn1 increased PGE₂ and sICAM-1 levels. These effects were abolished by the CRF₂ receptor antagonist, antisauvagine-30, but not by the CRF₁ receptor antagonist, NBI-27914. Moreover, Ucn2 activated p38MAPK and augmented NF-κB nuclear translocation and phosphorylation, whereas ERK1/2, JNK and Akt pathways were not involved in this process.

Conclusions and implications:

These findings suggest that Ucn1 exerts pro-inflammatory effects by augmenting LPS-induced expression of COX-2 and ICAM-1 in RAECs via CRF₂ receptors and the activation of p38MAPK and NF-κB.     

siRNA沉默胃癌SGC-7901细胞COX-2基因对VEGF-C表达与细胞迁移的影响

目的：探讨RNA干扰技术（RNAi）沉默环氧化酶-2（COX-2）基因对血管内皮生长因子-C（VEGF-C）的表达及细胞迁移能力的影响。方法：利用siRNA干扰胃癌SGC-7901细胞COX-2基因,RT-PCR和免疫荧光检测干扰前后COX-2基因和VEGF-C基因在mRNA和蛋白水平表达差异;细胞划痕实验比较COX-2基因对细胞迁移能力变化的影响。结果：COX-2基因干扰后,胃癌SGC-7901细胞中COX-2基因和VEGF-C基因的mRNA和蛋白水平明显下调,组间差异具有统计学意义（P〈0.05）,且COX-2与VEGF-C的表达呈显著正相关性（P〈0.05）;同时胃癌细胞的迁移能力也明显降低。结论：siRNA能特异性地抑制胃癌SGC-7901细胞COX-2基因的表达,进而抑制VEGF-C的表达,降低胃癌细胞的迁移能力,减少胃癌细胞转移。     

Proinflammatory cytokines enhance COX-1 gene expression in cultured rat glomerular mesangial cells

Glomerular mesangial cells (GMC) exert an essential maintaining effect on hemodynamic integrity and immune competence of the kidney through arachidonate metabolism. To clarify this, cultured rat GMC were measured for the expression and production of cyclooxygenase (COX) and excretion of prostaglandin (PG). The rat GMC spontaneously expressed type 1 cyclooxygenase (COX-1), but not COX-2. The PGE2 and thromboxane B2 (TXB2) were spontaneously produced by the cells. Interleukin (IL)-1beta (25 ng/ml), IL-8 (25 ng/ml), growth-related oncogene-alpha (GRO, 50 ng/ml) and tumor necrosis factor-alpha (TNF-alpha, 25 ng/ml) stimulated the COX-1 protein production as demonstrated by Western blot and enhanced PGE2 synthesis in GMC, beginning on 2 h of incubation, and steadily enhanced TXB2 synthesis over a 24-h period. Lipopolysaccharide (LPS, 100 ng/ml) enhanced both PGE2 and TXB2 syntheses from 2 h to at least 24 h of incubation. Collectively, the proinflammatory cytokines could enhance COX-1 but not COX-2 expression in GMC leading to increased PGE2 and TXB2 production. These biochemical events may be implicated in normal renal physiology as well as in pathogenesis of glomerular diseases.     

The synthetic cannabinoid WIN 55,212-2 increases COX-2 expression and PGE2 release in murine brain-derived endothelial cells following Theiler's virus infection

Brain endothelial cells infection represents one of the first events in the pathogenesis of TMEV-induced demyelination disease (TMEV-IDD), a model of multiple sclerosis (MS). The fact that cyclooxygenase-2 (COX-2) expression in brain endothelium mediates a wide variety of actions during CNS inflammatory diseases such as MS, and that cannabinoids ameliorate the progression of TMEV-IDD, lead us to investigate the role of cannabinoids on COX-2 expression on murine brain endothelial cell cultures subjected or not to TMEV infection. Murine brain endothelial cells (b.end5) express both cannabinoid receptors CB1 and CB2. However, treatment of b.end5 with the cannabinoid agonist WIN 55,212-2 resulted in up-regulation COX-2 protein and PGE2 release by a mechanism independent on activation of these receptors. Other cannabinoids such as 2-arachidonoyl glycerol (2-AG) or the abnormal cannabidiol (Abn-CBD) failed to affect COX-2 in our conditions. TMEV infection of murine brain endothelial cell cultures induced a significant increase of COX-2 expression at 8h, which was maintained even increased, at 20 and 32h post-infection. The combination of TMEV infection and Win 55,212-2 treatment increased COX-2 expression to a greater amount than was seen with either treatment alone. 2-AG and Abn-CBD did not modify COX-2 expression after TMEV. COX-2 synthesis involved different signaling pathways when was induced by WIN 55,212-2 and/or by TMEV infection. WIN 55,212-2-induced COX-2 up-regulation involves the PI(3)K pathway, whereas COX-2 induction by TMEV needs p38 MAPK activation too. Overexpression of COX-2 and the subsequent increase of PGE2 could be affecting flow blood and/or immune reactivity.     

原花青素对脂多糖诱导RAW2647细胞COX-2酶活性、mRNA及蛋白表达的影响

目的探讨原花青素对脂多糖(LPS)诱导小鼠巨噬细胞株RAW264.7细胞COX-2酶活性及蛋白表达的影响。方法放射免疫法检测COX-2酶活性，RT-PCR检测COX-2 mRNA表达，Western blotting检测COX-2蛋白表达。结果原花青素(0.8，4和20 mg·L^-1)不影响LPS诱导RAW264.7细胞COX-2酶活性，可下调LPS诱导RAW264.7细胞COX-2 mRNA表达；原花青素(4和20 mg·L^-1)下调LPS诱导RAW264.7细胞COX-2蛋白表达。结论原花青素不影响LPS诱导RAW2647细胞COX-2酶活性，但对LPS诱导RAW264.7细胞COX-2 mRNA及蛋白表达抑制作用明显。     

CTGF inhibits cell motility and COX-2 expression in oral cancer cells

Oral squamous cell carcinoma (SCC) has a striking tendency to migrate and metastasize. Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin synthase, has been implicated in tumor metastasis. Connective tissue growth factor (CTGF), a secreted protein that binds to integrins, modulates the invasive behavior of certain human cancer cells. However, the effect of CTGF on migration activity and COX-2 expression in human oral cells is mostly unknown. Here we found that CTGF reduced the migration and expression of COX-2 in human oral cancer cells. αvβ5 monoclonal antibody (mAb), phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002 and wortmannin) and Akt inhibitor reversed the CTGF-inhibited the migration and COX-2 down-regulation of oral cancer cells. CTGF stimulation decreased the phosphorylation of focal adhesion kinase (FAK), PI3K and Akt. In addition, c-Jun siRNA also antagonized the CTGF-inhibited migration and COX-2 expression. Moreover, CTGF decreased the binding of c-Jun to the AP-1 element on the COX-2 promoter. Taken together, our results indicated that CTGF inhibits the migration of oral cancer cells by decreasing COX-2 expression through the αvβ5 integrin receptor, FAK, PI3K, Akt, c-Jun and AP-1 signal transduction pathways.     

Endogenous N-acyl-dopamines induce COX-2 expression in brain endothelial cells by stabilizing mRNA through a p38 dependent pathway

Cerebral microvascular endothelial cells play an active role in maintaining cerebral blood flow, microvascular tone and blood brain barrier (BBB) functions. Endogenous N-acyl-dopamines like N-arachidonoyl-dopamine (NADA) and N-oleoyl-dopamine (OLDA) have been recently identified as a new class of brain neurotransmitters sharing endocannabinoid and endovanilloid biological activities. Endocannabinoids are released in response to pathogenic insults and may play an important role in neuroprotection. In this study we demonstrate that NADA differentially regulates the release of PGE₂ and PGD₂ in the microvascular brain endothelial cell line, b.end5. We found that NADA activates a redox-sensitive p38 MAPK pathway that stabilizes COX-2 mRNA resulting in the accumulation of the COX-2 protein, which depends on the dopamine moiety of the molecule and that is independent of CB₁ and TRPV1 activation. In addition, NADA inhibits the expression of mPGES-1 and the release of PGE₂ and upregulates the expression of L-PGD synthase enhancing PGD₂ release. Hence, NADA and other molecules of the same family might be included in the group of lipid mediators that could prevent the BBB injury under inflammatory conditions and our findings provide new mechanistic insights into the anti-inflammatory activities of NADA in the central nervous system and its potential to design novel therapeutic strategies to manage neuroinflammatory diseases.     

环氧化酶-2和血管内皮细胞生长因子在妊娠滋养细胞肿瘤中的表达及意义

目的 探讨环氧化酶-2（COX-2）及血管内皮细胞生长因子（VEGF）对妊娠滋养细胞肿瘤发生发展及预后估计的作用.方法 采用免疫组化SABC法检测COX-2、VEGF在正常绒毛和妊娠滋养细胞疾病中的表达.结果 妊娠滋养细胞肿瘤组较正常绒毛及葡萄胎组COX-2、VEGF的表达率均明显增高（P＜0.01）;妊娠滋养细胞肿瘤中,COX-2与VEGF的表达呈正相关（r=0.795,P＜0.01）.结论 COX-2与VEGF的高表达协同积累可促使滋养细胞恶性转化,提示预后不良.     

Nitric oxide stimulates adrenomedullin secretion and gene expression in endothelial cells

Adrenomedullin, a peptide with vasorelaxant activity, stimulates nitric oxide (NO) synthesis. We tested whether or not NO regulates the function of the adrenomedullin system. Human umbilical vein endothelial cells (HUVEC) were incubated with the NO donors sodium nitroprusside (SNP), morpholinosydnonimine (SIN-1) and the phospodiesterase V inhibitor zaprinast. In HUVEC, adrenomedullin concentration in the supernatant was measured by radioimmunoassay and mRNA expression was studied by Northern blot and competitive quantitative PCR. SNP, SIN-1, and zaprinast (100 micromol/l each) significantly increased adrenomedullin concentration in the supernatant of HUVEC twofold. The same concentrations increased adrenomedullin mRNA expression four- to tenfold. Similar results were obtained by both quantitative PCR and Northern blot. Thus, NO donor exposure in vitro increases both adrenomedullin secretion and mRNA expression in a dose-dependent manner.     

环氧化酶-2在大鼠缺血再灌注脑损伤组织中的表达及与凋亡的关系

目的探讨环氧化酶-2（COX-2）在大鼠缺血再灌注脑（I/R）损伤组织中与凋亡的关系。方法大鼠I/R模型为研究对象,免疫组化和免疫印迹测定COX-2表达,TUNEL法测定神经元凋亡。结果 I/R后COX-2表达出现不同程度的增强,在24h达高峰,同假手术组比较差异有统计学意义（P〈0.05）;I/R后神经元凋亡明显,在24h达高峰,同假手术组比较差异有统计学意义（P〈0.05）;I/R前加入NS398,与I/R组比较,COX-2下降的同时（P〈0.05）,发现神经元凋亡有不同程度的降低（P〈0.05）。结论 COX-2在大鼠I/R脑损伤组织中参与神经元凋亡。     

小檗碱抑制结肠癌细胞中COX-2表达作用的研究

目的:研究小檗碱(Ber)抑制肿瘤细胞中环氧合酶-2(COX-2)表达与时间及浓度的关系,探讨Ber作为抗肿瘤药物的可能.方法:以结肠癌细胞系HT-29为研究对象,培养后加入不同浓度的Ber,用荧光定量PCR法观察其对COX-2的抑制作用,MTT法检测Ber对癌细胞的杀伤作用.结果:在浓度大于0.25μmol·L-1时Ber对COX-2有抑制作用,与时间、浓度正相关.在浓度大于3.0μmol·L-1时对癌细胞有杀伤作用.结论:Ber可抑制COX-2及癌细胞生长,推测Ber有一定的抑癌作用,可望成为一种新型的抗癌药.     

Tetramethylpyrazine downregulates angiotensin II-induced endothelin-1 gene expression in vascular endothelial cells

1. Tetramethylpyrazine (TMP) is one of the active ingredients of the Chinese herb Ligusticum wallichii Franchat. It is well documented that TMP exerts a cardiovascular protective effect. The aims of the present study were to examine whether TMP alters angiotenisn (Ang) II-induced endothelin (ET)-1 gene expression and to identify the putative underlying signalling pathways in vascular endothelial cells. 2. Cultured vascular endothelial cells were pre-incubated with TMP, stimulated with AngII and ET-1 gene expression was then examined. The effects of TMP pretreatment on AngII-induced extracellular signal-regulated kinase (ERK) phosphorylation were investigated to elucidate the intracellular mechanism responsible for the effects of TMP on ET-1 gene expression. 3. Tetramethylpyrazine inhibited AngII-induced ET-1 gene expression, as revealed by nothern blotting and a promoter activity assay. Tetramethylpyrazine also inhibited the AngII-induced increase in intracellular reactive oxygen species (ROS), as measured by the redox sensitive fluorescent dye 2' 7'-dichlorofluorescin diacetate and ERK phosphorylation. 4. In summary, we have demonstrated, for the first time, that TMP inhibits AngII-induced ROS generation, ERK phosphorylation and ET-1 gene expression in vascular endothelial cells. Thus, the present study delivers important new insights into the molecular pathways that may contribute to the proposed beneficial effects of TMP in the cardiovascular system.     

The flavouring phytochemical 2-pentanone reduces prostaglandin production and COX-2 expression in colon cancer cells

Many phytochemicals found in the diet may prevent colon carcinogenesis by affecting biochemical processes in the colonic mucosa. Inflammation and subsequent elevation of the enzyme cyclooxygenase-2 (COX-2) are two such factors involved in the development of colon cancer, and inhibition of these processes could be important targets for chemoprevention. We have previously shown COX-2 inhibitory activity locally in the colon; e.g. in human fecal water from a group of vegetarians. In this study we focus on 2-pentanone, a frequently occurring compound in common foods such as banana and carrot. The aim was to study the inhibitory effects on prostaglandin production and COX-2 protein expression in tumour necrosis factor-alpha stimulated colon cancer cells (HT29) by radioimmunoassay and Western blotting. 2-Pentanone inhibited both prostaglandin production and COX-2 protein expression in human colon cancer cells. A concentration of 400 mumol/l 2-pentanone inhibited the prostaglandin production by 56.9+/-12.9% which is in the same range as the reference compound NS398 (59.8+/-7.6%). The two highest concentrations of 2-pentanone were further analyzed by Western blot, and 400 micromol/l and 200 micromol/l 2-pentanone resulted in a 53.3+/-9.6% and +/-27.1% reduction of the COX-2 protein levels respectively. Further studies on flavouring compounds, for example 2-pentanone, as colon cancer chemopreventives would be very valuable, and such results may contribute to future dietary recommendations.