脂氧素A4抑制内毒素诱导的人支气管上皮细胞环氧合酶2及前列腺素E2的表达

目的: 探讨脂氧素A₄对人支气管上皮细胞(HBECs)环氧合酶2(COX-2)表达的影响。方法: 应用不同浓度(0.1、1、10 mg/L)的内毒素(LPS)刺激HBECs 9 h,或者用1 mg/L LPS分别刺激HBECs不同时点(3 h、6 h、9 h)后,测定HBECs的COX-2 mRNA表达和细胞上清液前列腺素E₂(PGE₂)水平。应用不同浓度 (0、100、400 μmol/L) 的脂氧素A₄作用于经过LPS(1 mg/L)刺激培养9 h的HBECs,采用酶联免疫吸附法(ELISA)检测细胞上清液PGE₂的水平, 同时分别应用RT-PCR和Western blotting分别检测HBECs COX-2 mRNA及蛋白的表达。结果: LPS刺激培养条件下HBECs的COX-2 mRNA表达及其上清液PGE₂水平增加,并呈时间、剂量依赖性。脂氧素A₄能抑制LPS刺激培养HBECs COX-2蛋白和mRNA的表达及上清液PGE₂的水平,并呈剂量依赖性。结论: 脂氧素A₄能抑制LPS诱导的HBECs COX-2表达及上清液PGE₂的水平。     

Effect of PGE2 on the cell surface molecule expression in PMA treated thymocytes

PGE₂ is produced by cells of the thymic microenvironment. The effects of PGE₂ are mediated by cAMP through binding to its intracellular receptor protein kinase A (PKA). Phorbol 12-myristate 13-acetate (PMA) is known to modulate CD molecule expression on thymocytes, probably through activation of protein kinase C (PKC). We have hypothesized that cross-talk between these two signalling pathways may affect modulation of the CD molecules on the cell surface of thymocytes. For this purpose, we compare the effects of PMA alone or combined with PGE₂ on CD3, CD4 and CD8 expression on mouse thymocytes by flow-cytometric analysis. PMA treatment almost completely abolished CD4 expression and slightly decreased CD3 and CD8 expression. PGE₂ alone did not change the CD3, CD4 and CD8 molecule expression. Combined with PMA, PGE₂ can overcome the decrease induced by PMA of the CD3 expression and partially reduced the disappearance of the CD4 molecule. On the other hand PGE₂ accelerated the loss of CD8 molecule expression. These events occurred only in CD4+ CD8+ immature thymocytes. An analogue of cAMP (dibutryl cAMP) mimics the effect of PGE₂, but not Br-cGMP. This differential regulation by PGE₂ of the CD molecule expression on immature thymocytes may provide additional evidence on the role of PGE₂ during the process of thymic differentiation.     

The effects of vigabatrin on type II spike wave discharges in rats

The phospholipid mediator platelet-activating factor (PAF), and its non-hydrolyzable analog methylcarbamyl-PAF (mc-PAF) increase prostaglandin E₂ (PGE₂) release from astrocyte-enriched cortical cell cultures. Cyclooxygenase (COX) enzymes – of which there are two known isoforms – convert arachidonic acid to prostaglandin (PG) H₂ (PGH₂), which is further metabolized to various PGs, including PGE₂. COX-1 is generally considered to contribute to cell homeostasis, whereas COX-2 is thought to mediate inflammatory/immune PG formation. In this study we examined the involvement of the COX isoforms in PAF-induced PGE₂ release. Treatment of cells with the non-specific COX inhibitor indomethacin, or the specific COX-2 inhibitor NS-398, prior to mc-PAF stimulation completely blocked the PAF-induced release of PGE₂; treatment with more selective COX-1 inhibitors (i.e. piroxicam and SC-560) failed to significantly do so. These data suggest that COX-2 is responsible for PAF-mediated PGE₂ release in primary astrocytes.     

Selective inhibition of cyclooxygenase-2 exacerbates methamphetamine-induced dopamine depletion in the striatum in rats

Neuroinflammatory processes associated with induction of cyclooxygenase (COX) have been implicated in the deleterious events resulting in neurodegeneration. The present study was designed to investigate the impact of acute methamphetamine (MA) administration on COX expression and prostaglandin E2 (PGE₂) production, and to evaluate the effect of selective COX-2 inhibition using celecoxib in MA-induced degeneration of dopaminergic terminal and cell apoptosis in the striatum. Male Sprague–Dawley rats were treated with either a neurotoxic regimen of methamphetamine hydrochloride (5 mg/kg, i.p., every 2 h for four injections) with or without celecoxib (7.5 mg/kg) or vehicle. COX-1 expression was not affected by MA, while both COX-2 protein expression and number of COX-2 positive cells in striatum were significantly reduced 24 h after MA treatment. However, after 72 h, a significant upregulation of COX-2 protein was detected. PGE₂ production was correlated with altered COX-2 levels. NFkappa-B (NFκB), a key regulator of COX-2 expression, was activated 72 h after MA administration, and was accompanied by increased Ikappa B (IκB) phosphorylation. Animals receiving MA exhibited an increase in apoptotic cells and notable reductions in dopamine (DA) content (63.9%) in immunoreactivity of tyrosine hydroxylase (TH) and neuron specific microtubule-associated protein 2 (MAP2) in striatum. Administration of celecoxib exacerbated MA-induced DA depletion, and did not affect MA-induced MAP2 damage, apoptosis or proliferation of glial cells. Our findings suggest that COX-2 containing cells are targets of the damage during earlier stages of MA-related neurotoxicity, and that the selective inhibition of COX-2 enzyme is harmful rather than protective. The COX-2 induction appears during the recovery period, and NFκB activation may be an important mechanism.     

EP_2A体外可促进人CD34~+细胞归巢而不能促进其增殖

目的:探讨前列腺素E_2受体2激动剂(EP_2A)在体外对人CD34~+细胞的归巢与增殖作用。方法:收集健康供者经粒细胞集落刺激因子动员后的外周血,免疫磁珠法分选出人CD34~+细胞;同时收集健康供者动员前骨髓液,分离单个核细胞,并行骨髓间充质干细胞(BMMSC)培养。人CD34~+细胞和BMMSC经前列腺素E_2(阳性对照)、DMSO(阴性对照)、EP_2A和EP_2A+前列腺素E_2受体2拮抗剂(EP_2AA)处理后,对人CD34~+细胞用CCK-8法检测细胞活力,集落形成实验检测集落形成数目,流式细胞术检测G_2/M期细胞比例,Western blot检测细胞中survivin、β-catenin及CXC趋化因子受体4(CXCR4)的蛋白表达;ELISA法检测BMMSC中基质细胞衍生因子1α(SDF-1α)的含量。结果:EP_2A组与阴性对照组相比,人CD34~+细胞在细胞活力、集落形成数目、G_2/M期比例及survivin和β-catenin蛋白表达方面均无明显差别。但EP_2A组人CD34~+细胞CXCR4及BMMSC SDF-1α的表达均明显高于阴性对照组。结论:EP_2A体外可促进人CD34~+细胞归巢但不能促进其增殖。     

橄榄苦苷对IL-1β诱导的大鼠软骨细胞损伤的作用及机制研究

目的:研究橄榄苦苷对白细胞介素1β(IL-1β)诱导的SD大鼠关节软骨细胞的影响。方法:采用酶两步顺序消化法消化SD大鼠关节软骨分离细胞,体外培养软骨细胞,光学显微镜观察细胞形态,阿尔新蓝染色及Ⅱ型胶原免疫组化法对软骨细胞进行鉴定。橄榄苦苷对软骨细胞的细胞毒性采用CCK-8实验进行评估。取第3代软骨细胞,分别用浓度为10、50或100μmol/L的橄榄苦苷预先处理,随后用IL-1β刺激细胞24 h,所生成的NO和前列腺素E2(PGE2)的量分别用格里斯重氮化反应和酶联反应吸附实验进行评估。基质金属蛋白酶(MMP)-1和MMP-13 mRNA的表达利用实时荧光定量PCR进行定量检测。采用免疫印迹实验分析诱导型一氧化氮合酶(iNOS)、环氧化酶2(COX-2)和活化NF-κB的蛋白表达。结果:软骨细胞在不同浓度橄榄苦苷培养24 h后,其生存能力与对照组相比无明显差异。橄榄苦苷可以显著降低IL-1β刺激下软骨细胞MMP-1和MMP-13 mRNA的表达及NO、PGE2的生成。橄榄苦苷通过减少IκB蛋白的降解从而抑制IL-1β介导的NF-κB通路的活化。结论:橄榄苦苷可通过NF-κB信号转导通路调控炎症的发生发展,进一步明确了橄榄苦苷对关节炎防治作用的分子机制,为骨关节炎的免疫治疗提供了重要的基础理论依据。     

Rapamycin suppresses TLR4-triggered IL-6 and PGE(2) production of colon cancer cells by inhibiting TLR4 expression and NF-kappaB activation

Toll-like receptor 4 (TLR4) signaling in tumor cells can mediate tumor cell immune escape and tumor progression, being regarded as one of the mechanisms for chronic inflammation in tumorigenesis and progression. So, intervention of TLR4-mediated immune escape and metastasis has been proposed as one of the approaches to cancer prevention and treatment. Rapamycin, an immunosuppressant agent widely used for treatment of autoimmune diseases and transplantation rejection, is recently used for cancer therapy. However, the underlying mechanisms remain to be fully understood. In the present study, we demonstrate that rapamycin can significantly inhibit TLR4-triggered IL-6 and PGE₂ production and invasion of colon cancer cells. Suppression of TLR4-induced IL-6 and PGE₂ production is responsible for the rapamycin-mediated decrease of TLR4-evoked invasion of colon cancer cells. Furthermore, disruption of NF-κB pathway contributes to the inhibition of TLR4-induced IL-6, PGE₂ production and invasion by rapamycin in colon cancer cells. Rapamycin can also downregulate TLR4 expression. Therefore, we demonstrate that rapamycin may abrogate TLR4-triggered tumor cell immune escape and invasion by downregulating TLR4 expression and inhibiting TLR4-activated NF-κB pathway, thus providing new mechanistic explanation for the antitumor effect of rapamycin.     

Enhancement in Immunity of Tumor Bearing Mice by Immunization Against Prostaglandin E2

C57B1/6 mice hearing a palpable Lewis lung carcinoma (LLC) were immunized against prostaglandin E₂ (PGE₂) in order to prevent the immune suppression typical of tumor bearers. Proliferation in response to concanavalin A (Con A) by normal spleen cells cultured in the presence of PGE₂ or by spleen cells of LLC-bearing mice cultured with Con A alone was suppressed. However, the mitogenic response of spleen cells from PGE₂ immunized LLC-bearing mice was only partially suppressed. The random migration of normal macrophages cultured in the presence of PGE, or of macrophages from LLC-bearing mice cultured in medium alone was enhanced when compared to the random migration of normal macrophages cultured in the absence of PGE₂. In contrast, the random migration of macrophages obtained from PGE₂ immunized LLC-bearing mice was the same as that of normal macrophages cultured in the absence of PGE₂.     

The long-term exposure of rat cultured dorsal root ganglion cells to bradykinin induced the release of prostaglandin E2 by the activation of cyclooxygenase-2

The effects of long-term exposure of primary cultured rat dorsal root ganglion (DRG) cells to bradykinin (BK), compared to short-term exposure, were investigated to establish whether BK could induce prostaglandin E2 (PGE2) release from DRG cells. Short-term exposure (30 min) resulted in a small but significant amount of PGE2 release which was mainly inhibited by a selective COX-1 inhibitor, SC-560 but only partially by a selective COX-2 inhibitor, NS-398, and did not induce COX-2 protein as determined by Western blotting. In contrast, long-term exposure (3 h) induced a large amount of PGE2 release, which was completely abolished by indomethacin or NS-398. The level of COX-2 mRNA began to be detected by ribonuclease protection assay after 30 min of 100 nM BK exposure, maintained maximal expression for 1 h, and subsequently declined to the basal level. The level of COX-2 protein was expressed to follow the time course of COX-2 mRNA induction by BK in a delayed but similar kinetic manner. The expression of COX-2 induced by BK in DRG cells was inhibited by a BK B2 receptor antagonist, HOE140, but not a B1 receptor antagonist, Lys-des-Arg9, (Leu8)-BK. Thus, BK has been shown to induce COX-2 protein by B2 receptor, which may cause prostanoid generation in rat DRG cells, which may play an important role in the pathogenesis of inflammatory pain and hyperalgesia around the primary sensory neurons.     

Positive feedback effect of PGE2 on cyclooxygenase-2 expression is mediated by inhibition of Akt phosphorylation in human follicular dendritic cell-like cells

Prostaglandins (PGs) are bioactive lipid mediators generated from the phospholipids of cell membrane in response to various inflammatory signals. To understand the potential role of PGs in PG production itself during immune inflammatory responses, we examined the effect of PGE₂, PGF_2α, and beraprost on COX-2 expression using follicular dendritic cell (FDC)-like HK cells isolated from human tonsils. Those three PGs specifically augmented COX-2 protein expression in a dose-dependent manner after 4 or 8 h of treatment. The enhancing effect was also reflected in the actual production of PGs and the viable cell recovery of germinal center B-cells. To investigate the underlying molecular mechanism, we examined the impact of PI3K inhibitors on PG-induced COX-2 expression. Interestingly, COX-2 induction by PGE₂ and beraprost, but not PGF_2α, was enhanced by wortmannin and LY294002. In line with this result, Akt phosphorylation was inhibited by PGE₂ and beraprost but not by PGF_2α. The distinct effect of PGE₂ and beraprost from PGF_2α was reproduced in Akt-knockdowned HK cells. Our current findings imply that PGE₂ and PGI₂ stimulate COX-2 expression in FDC by inhibiting Akt phosphorylation. Additional studies are warranted to determine the potential role of Akt as a therapeutic target in patients with inflammatory disorders.     

The Effects of E Series Prostaglandins on Blastogenic Responses in Vitro and Graft Vs. Host Responses in Vivo

In this study the ability of prostaglandin E_l (PGE₁), Misoprostol (a stable analog of PGE₁), and 16,16-dimethyi PGE₂ (a stable analog of PGE₂) to suppress immune responses in vitro and in vivo was etermined. All of the compounds caused a titratable (10-6 to 10-9 M) suppression of Con A blastogenesis and the mixed lymphocyte response whereas there was only slight inhibition of the LPS response. When either 16,16-dimethyl PGE₂ (30 ug/mouse) or Misoprostol (60 ug/mouse) was administered daify in vivo, there was a significant suppression of splenomegaly in E₁ mice (C57B1/6 x CBA) which had been injected with parental (C57B1/6) spleen cells. We conclude that prostaglandins of the E series can function as immunosuppressive reagents both in vitro and in vivo. In the future they may serve to augment existing forms of immuno-suppressive therapy.     

Infection of human dendritic cells with herpes simplex virus type 1 dramatically diminishes the mRNA levels of the prostaglandin E2 receptors EP2 and EP4

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) of the immune system. Their migration to secondary lymphoid tissues is a crucial step for the priming of T cells and ultimately for the initiation of adaptive immune responses. Therefore, DCs are potential targets for immune evasion strategies of pathogens. The migration of DCs to the T cell areas of lymph nodes is guided by a gradient of chemokines, CCL19 and CCL21, which are constitutively expressed there. CCR7, the receptor for these chemokines, is expressed on activated DCs, enabling their homing to the lymph nodes. However, CCR7 expression alone is not sufficient for efficient migration. Prostaglandin E₂ (PGE₂) is a mandatory factor for CCR7-mediated migration of DCs and exerts its effects via prostaglandin E₂ receptor 2 (EP2) and prostaglandin E₂ receptor 4 (EP4). In this study, we investigated the effect of herpes simplex virus type 1 (HSV-1) infection of mature monocyte-derived dendritic cells (MODCs) on the EP2 and EP4 receptor expression. Affymetrix analyses and real-time polymerase chain reaction (PCR) demonstrated a dramatic down-regulation of the expression of those receptors. Additional real-time PCR and migration assays with a Δvhs mutant virus lacking the virion host shutoff (vhs) gene implicate a vhs independent mechanism. Therefore, our results suggest a novel immune evasion strategy for HSV-1.     

Expression of cyclooxygenase-1/-2, microsomal prostaglandin-E synthase-1 and E-prostanoid receptor 2 and regulation of inflammatory mediators by PGE2 in the amoeboid microglia in hypoxic postnatal rats and murine BV-2 cells

This study aimed to investigate the effect of hypoxia on the expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), microsomal prostaglandin-E synthase (mPGES-1), E-prostanoid receptor 2 (EP2) in microglia; and the roles of EP2-cyclic adenosine monophosphate (cAMP) signaling pathway in the prostaglandin E₂ (PGE₂) regulation of inflammatory mediators released by hypoxic BV-2 cells. Immunoexpression of COX-1, COX-2, mPGES-1 and EP2 was localized in the amoeboid microglial cells (AMC), a nascent brain macrophage in the developing brain, as confirmed by double labeling with OX-42 and lectin, specific markers of microglia. AMC emitted a more intense immunofluorescence in hypoxic rats when compared with the matching controls. In postnatal rats subjected to hypoxia, mRNA and protein expression levels of COX-1, COX-2 and mPGES-1 along with tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric-oxide synthase (iNOS) and PGE₂ product in the callosal tissue were significantly increased. The results were shared in the BV-2 cells except for COX-1 mRNA and protein whose levels remained unaltered. Interestingly, treatment with EP2 antagonist AH-6809 resulted in suppression of hypoxia induced EP2, IL-1β and iNOS mRNA and protein expression, TNF-α protein expression and intracellular cAMP level in BV-2 cells. It is suggested that PGE₂ may regulate above inflammatory mediators in the activated microglia via EP2-cAMP signaling pathway in hypoxic conditions.     

中枢前列腺素E_2在慢性心力衰竭交感兴奋中的作用

目的:观察中枢前列腺素E_2(PGE_2)在慢性心力衰竭(心衰)交感神经兴奋中的作用,并探讨其机制。方法:雄性SD大鼠冠脉结扎制备心衰模型,侧脑室渗透压泵持续给药,假手术组和心衰组给予人工脑脊液(0.25μL/h),心衰给药组给予塞来考昔(CLB;20 mg/h)。4周后,检测各组大鼠脑脊液内PGE_2浓度、交感神经兴奋性和心功能指标,同时测定下丘脑室旁核(PVN)内促肾上腺皮质激素释放激素(CRH)神经元激活指标和神经递质的变化。结果:与假手术组相比,心衰组大鼠脑脊液内PGE_2含量增加,肾交感神经放电活动增强,外周血去甲肾上腺素升高,左心室舒张末期压力、肺/体质量比和右心室/体质量比均增加,左室内压最大上升和下降速率均下降,PVN内CRH阳性神经元数目增多,外周血促肾上腺皮质激素浓度升高(P0.05);心衰大鼠侧脑室给予CLB后,脑脊液内PGE_2明显下降,PVN内CRH神经元激活减少,交感神经兴奋性减弱,心功能得到改善(P0.05)。与假手术组相比,心衰大鼠PVN内谷氨酸含量较高,γ-氨基丁酸含量和谷氨酸脱羧酶67阳性神经元数目较低(P0.05);侧脑室给予CLB后,以上各指标均被逆转(P0.05)。结论:慢性心衰时,中枢内升高的PGE_2可以激活下丘脑室旁核CRH神经元从而增强交感神经活动,而该作用可能是通过改变下丘脑室旁核神经递质系统来实现的。     

MODIFIED RESPONSES TO PGE2 IN POLYAMINE BIOSYNTHESIS BY T LYMPHOCYTES OF GASTRIC- AND CONJUNCTIVA BASAL CELL-CARCINOMA PATIENTS

The response to Prostaglandin (PG) E₂ of T cells from gastric carcinoma (GC)- and conjunctiva-basal cell carcinoma (conjunctiva-BCC)-bearing patients has been studied in relation to polyamine metabolism. Polyamines are crucial cofactors in cell growth as well as differentiation and many works report that lymphocyte spermine (SP), spermidine (SPD) and putrescine (PUT) levels may be related to tumor proliferation. The present work aims to detect the basal and PGE₂ induced concentrations of these polyamines and cAMP, ornithine decarboxylase (ODC), spermine N1-acetyltransferase (SAT) activities of T lymphocytes drawn from patients suffering from GC and conjunctiva-BCC since many carcinomas are characterized by high levels of PGE₂. Data obtained from lymphocytes of neoplastic subjects were compared with those derived from PGE₂-treated control lymphocytes. Results highlight a very significant increase of all the polyamine metabolites in PGE₂-treated T cells from neoplastic patients in respect to the untreated and PGE₂-treated control lymphocytes. Therefore, it is conceivable that the PGE₂ content increase, often occurring during the epithelial tumour development, may contribute, through enhancement of polyamine metabolism, to tumor progression.     

姜黄素对LPS刺激RAW264.7细胞PGE_2和IL-10影响

目的:观察姜黄素对脂多糖刺激小鼠RAW264.7细胞炎症因子的影响。方法:体外培养小鼠RAW264.7细胞,脂多糖刺激小鼠RAW264.7细胞分泌细胞因子,用不同浓度姜黄素进行干预,ELISA法检测培养上清液中抗炎细胞因子IL-10和炎症因子PGE2含量。结果:姜黄素可抑制脂多糖刺激小鼠RAW264.7细胞释放PGE2,促进抗炎因子IL-10表达,并呈一定量效关系,其影响细胞因子释放作用与细胞毒作用无关。结论:姜黄素呈浓度依赖性地抑制脂多糖刺激小鼠RAW264.7细胞释放PGE2,促进IL-10表达。     

Regulation of cyclooxygenase-2 by nitric oxide in activated hepatic macrophages during acute endotoxemia

Eicosanoids generated via cyclooxygenase-2 (COX-2) and nitric oxide produced from inducible nitric oxide synthase (NOSII) have been implicated in endotoxin-induced tissue injury. In the present studies, we characterized COX-2 and NOSII activity in rat hepatic macrophages and their interaction during acute endotoxemia. Kupffer cells from control animals were found to constitutively express COX-2 and NOSII mRNA and protein. Whereas treatment of the cells with lipopolysaccharide (LPS) and/or interferon-gamma (IFN-gamma) had no major effect on COX-2, NOSII expression increased. Induction of acute endotoxemia resulted in a rapid and transient increase in constitutive COX-2 expression and prostaglandin E2 (PGE2) production by liver macrophages as well as NOSII expression and nitric oxide release. Cells from endotoxin-treated rats were also sensitized to generate more nitric oxide and express increased NOSII in response to LPS and IFN-gamma. Inhibition of NOSII with aminoguanidine reduced COX-2 mRNA and protein expression as well as PGE2 production by activated macrophages from endotoxemic, but not control animals. In contrast, SC236, a specific COX-2 inhibitor, had no effect on NOSII mRNA or protein levels or on nitric oxide production by hepatic macrophages, even after endotoxin administration. These data suggest that activation of COX-2 may be important in the pathophysiological response of hepatic macrophages to endotoxin. Moreover, nitric oxide is involved in regulating COX-2 in activated liver macrophages during acute endotoxemia.