Relationships among gender,age, time,and temperature in methamphetamine-induced striatal dopaminergic neurotoxicity

A neurotoxic regimen of methamphetamine (MA—40 mg/kg ip) administered at 0 (control—MA vehicle), 0.5 and 72 h prior to determinations of striatal dopamine (DA) and DOPAC (3,4-dihydroxyphenylacetic acid)/DA ratios were compared among juvenile and adult female and male mice. Adult females and males showed similar depletions in striatal DA at 0.5 h post-MA, but males showed greater DA depletions and DOPAC/DA ratios at 72 h post-MA. Juvenile mice showed neither sex differences, nor any MA neurotoxicity upon striatal DA or DOPAC/DA ratios. Following MA, body temperatures increased in all mice, but increases in adult males were greater than adult females; juveniles showed no sex differences and body temperature increases were similar to that of adult males. MA-evoked DA output was greater in adult compared to juvenile males and a biologically effective regimen of testosterone to juvenile males neither increased MA-evoked DA output nor decreased MA-induced striatal DA like that observed in adult males. These results demonstrate: (1) Unlike adults, juvenile mice show neither a sex difference for MA-induced neurotoxicity or body temperature increases, nor MA neurotoxicity, (2) Initial effects of MA (0.5 h) in adult females and males are similar, but at 72 h post-MA females show no further striatal DA depletion, (3) Increased striatal DA depletion within adult versus juvenile males may be related to initially higher MA-evoked DA responses, and (4) Testosterone fails to convert juvenile males into adults with regard to MA effects.     

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。     

甲基苯丙胺对相关脑区的神经毒性作用

目的:研究甲基苯丙胺(MA)对大鼠纹状体、海马、额叶皮质等脑区神经细胞的毒性作用以及对大鼠行为的影响。方法:H-E染色、Glees银浸染观察神经元和轴突的变化;高效液相色谱检测上述脑区多巴胺(DA)及其代谢产物含量;免疫组化胶质纤维酸性蛋白(GFAP)检测胶质细胞增生情况。结果:MA对上述脑区神经细胞和轴突有损伤作用,表现为神经细胞变圆,极性消失;胶质细胞增生,噬神经细胞现象、胶质小结形成;神经轴索扭曲,节段性增粗,轴索间隙增宽;GFAP阳性星形细胞增多;纹状体DA及其代谢产物含量显著降低,海马、额叶皮质DA含量明显降低;大鼠行为改变明显。结论:MA对大鼠中枢神经系统多脑区神经元有明显的毒性作用,可导致上述脑区DA含量下降和大鼠行为改变。     

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.     

Testosterone enhances dopamine depletion by methamphetamine in male, but not female, mice

We examined the effects of varying concentrations of testosterone propionate (T) treatment within intact and gonadectomized male and female mice with regard to its capacity to alter striatal dopamine (DA) depletion in response to a neurotoxic regimen of methamphetamine (MA). Administration of T at 24h prior to MA significantly increased striatal DA depletion in intact and gonadectomized male mice. Similar treatments administered to intact and gonadectomized female mice failed to alter striatal DA concentrations in response to MA. These results demonstrate that T can enhance MA-induced neurotoxicity in male, but not in female, mice. Such findings have important implications with regard to sex differences in nigrostriatal dopaminergic function, in general, and, in specific, to sex differences related to nigrostriatal dopaminergic neurotoxicity and neurodegeneration like that in response to MA and in Parkinson's disease, where a greater incidence is typically reported for males.     

Methamphetamine enhances 1-methyl-4-phenylpyridinium ion-induced hydroxyl radical generation in the rat striatum

We determined the methamphetamine (MA), a potent dopamine (DA) releaser, enhances 1-methyl-4-phenylpyridinium ion (MPP(+))-induced hydroxyl radical (&z.rad;OH) generation in the rat striatum. Rats were anesthetized, and sodium salicylate in Ringer's solution (0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of .OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. After administration of MA (5 mg/kg i.v., every 2 h, four times), MA drastically increased DA release and the &z.rad;OH formation. When iron (II) was administered to the MA-treated animals, a marked elevation of DHBA was observed, compared with MPP(+)-only treated animals, that showed a positive linear correlation between DA and .OH formation trapped as DHBA (R(2)=0.985) in the dialysate. These results suggest that MA enhances the &z.rad;OH products of efflux/oxidation due to MPP(+).     

Selective COX-2 inhibition prevents progressive dopamine neuron degeneration in a rat model of Parkinson's disease

Several lines of evidence point to a significant role of neuroinflammation in Parkinson's disease (PD) and other neurodegenerative disorders. In the present study we examined the protective effect of celecoxib, a selective inhibitor of the inducible form of cyclooxygenase (COX-2), on dopamine (DA) cell loss in a rat model of PD. We used the intrastriatal administration of 6-hydroxydopamine (6-OHDA) that induces a retrograde neuronal damage and death, which progresses over weeks. Animals were randomized to receive celecoxib (20 mg/kg/day) or vehicle starting 1 hour before the intrastriatal administration of 6-OHDA. Evaluation was performed in vivo using micro PET and selective radiotracers for DA terminals and microglia. Post mortem analysis included stereological quantification of tyrosine hydroxylase, astrocytes and microglia. 12 days after the 6-OHDA lesion there were no differences in DA cell or fiber loss between groups, although the microglial cell density and activation was markedly reduced in animals receiving celecoxib (p < 0.01). COX-2 inhibition did not reduce the typical astroglial response in the striatum at any stage. Between 12 and 21 days, there was a significant progression of DA cell loss in the vehicle group (from 40 to 65%) that was prevented by celecoxib. Therefore, inhibition of COX-2 by celecoxib appears to be able, either directly or through inhibition of microglia activation to prevent or slow down DA cell degeneration.     

Protection against titanium particle-induced osteoclastogenesis by cyclooxygenase-2 selective inhibitor

Wear particle-induced osteoclastogenesis is the most common cause of aseptic loosening in total joint arthroplasty. Although cyclooxygenase (COX)-2, an inducible regulator of prostaglandin E2 (PGE2) synthesis, is known to be involved in osteoclast differentiation, its effect on osteoclastogenesis in response to wear particles remains unclear. In this study, we investigated the role of COX-2 in the regulation of osteoclast differentiation in the osteoclast precursor cell line RAW264.7 stimulated with titanium (Ti) particles. The results showed COX-2 expression in the early stages of RAW264.7 differentiation when stimulated with receptor activator of nuclear factor kappa B ligand (RANKL) and Ti particles. Blockade of COX-2 by celecoxib, a COX-2 selective inhibitor, effectively reduced the expression of PGE2 and inhibited differentiation of RAW264.7 cells into tartrate-resistant acid phosphatase-positive (TRAP+) osteoclastic cells. Quantitative real-time polymerase chain reaction revealed that celecoxib inhibited mRNA expression of RANK, cathepsin K (CPK), TRAP, and the nuclear factor of activated T cells c1 (NFATc1) in RAW264.7 cells stimulated by Ti particles and RANKL. Moreover, exogenous PGE2 reversed the inhibitory effects of celecoxib. These results provide direct evidence that COX-2 dependent PGE2 induced by RANKL and Ti particles is required for osteoclastogenesis and suggests that reduced production of PGE2 by inactivation of COX-2 would provide a promising therapeutic target for the treatment of osteoclastogenesis induced by wear particles.     

Morphological changes in rat striatal boutons after chronic methamphetamine and haloperidol treatment

Dopaminergic (DA) synaptic boutons were identified in rat striatum with an electron microscopic histochemical method. In rats which developed behavioral hypersensitivity after treatment with methamphetamine (MAP) for about 2 weeks, significantly fewer DA boutons were found. This effect was specific to DA boutons without mitochondria and was not seen in boutons with mitochondria. The density of granular synaptic vesicles in DA boutons, however, did not change significantly. These morphological changes would represent a high capability of nerve cells to reorganize synaptic connections under altered chemical environments, but they could not be related uniquely to behavioral hypersensitivity, as similar effects were observed in rats treated with haloperidol which did not develop behavioral hypersensitivity. Peculiarly, the effects of MAP treatment on both behavior and DA boutons were prevented by combined administration of haloperidol.     

Morphoproteomics identifies constitutive activation of the mTORC2/Akt and NF-κB pathways and expressions of IGF-1R,Sirt1, COX-2, and FASN in peripheral T-cell lymphomas: pathogenetic implications and therapeutic options

Background: Gaining a better understanding of the molecular circuitries and pathways implicated in the malignant growth and biological behavior of T cell lymphomas may identify potential cellular targets with clinical therapeutic potential. The immunohistochemical characterization of key cellular proteins participating in these pathways can provide surrogate markers of biological activity. The mammalian target of rapamycin complex (mTORC) signaling pathway has been implicated in T-cell lymphopoiesis. The mTORC2 pathway involves downstream activation of nuclear factor (NF)-κB and p-Akt (Ser 473). Fatty acid synthase (FASN) and insulin-like growth factor-1 receptor (IGF-1R) are expressed upstream of the mTORC and NF-κB signaling pathways. Cyclooxygenase (COX)-2 products influence these pathways. Our goal was to use morphoproteomics to characterize the expression patterns of the proteins in various peripheral T-cell lymphomas. Design: Ten cases of peripheral T-cell lymphoma (PTCL) were examined for expression of proteins along the mTORC, Akt and NF-κB pathways and affiliated tumorigenic molecules. These included two angioimmunoblastic PTCL, one natural killer/PTCL, one anaplastic large PTCL, and six PTCL not otherwise specified. Immunostaining for phosphorylated (p) mTOR (Ser 2448), p-Akt (Ser 473), p-NF-κBp65 (Ser 536), IGF-1R (Tyr1165/1166), silent mating type information regulation 2 homolog 1 (Sirt1), COX-2 and FASN was performed on paraffin-embedded tissue for each case. Percent expression was scored using bright-field microscopy with high expression designated as more than 50% of the cells with positive stain in the appropriate subcellular compartment. Results: All ten cases demonstrated nuclear staining for p-mTOR (Ser 2448) corresponding to mTORC 2, and all cases showed strong, diffuse nuclear staining for p-NF-κBp65 (Ser 536). All ten also showed nuclear and cytoplasmic staining for p-Akt (Ser 473) and cytoplasmic staining for IGF-1R. High expressions for nuclear Sirt1, and cytoplasmic COX-2 and FASN were detected in 7, 9, and 8 out of 10 cases, respectively. Six out of 10 cases demonstrated high expression of all the mentioned markers. Conclusion: The constitutive activation of mTORC2, NF-κB, p-Akt and the concomitant expression of IGF-1R suggests convergence of these molecular pathways in T-cell lymphoma. The results of this study also suggest that mTORC2 may be a common denominator among this heterogeneous group of lymphomas. Interference of key nodes of this pathway may carry a clinical therapeutic benefit. Agents that may be considered based on existing data include: bortezomib to inhibit NF-κB pathway activation; metformin to inhibit both NF-κB and mTORC2 and histone deacteylase inhibitors to inhibit mTORC2 pathway signaling. Furthermore, panobinostat inhibits Sirt1 pathway when present, and celecoxib inhibits NF-κB pathway activation independent of COX2.     

选择性环氧合酶2抑制剂塞来昔布对人子宫内膜癌细胞的凋亡诱导

目的 研究环氧合酶2(COX-2)选择性抑制剂塞来昔布诱导人子宫内膜癌细胞凋亡作用.方法 体外培养的人子宫内膜癌细胞HEC-1-B经塞来昔布处理后,采用Giemsa染色、流式细胞术和DNA Ladder观察HEC-1-B细胞凋亡变化;通过半定量RT-PCR方法检测HEC-1-B细胞COX-2及凋亡相关基因Fas、survivin的表达.结果 20 μmol/L塞来昔布处理HEC-1-B细胞后,Giemsa染色即可见细胞核固缩、碎裂,出现凋亡小体等细胞凋亡形态变化;流式细胞学检测示50 μmol/L塞来昔布可使HEC-1-B细胞凋亡率达46.9%,PI荧光直方图出现凋亡细胞峰,DNA电泳可见典型的细胞凋亡梯形条带.塞来昔布处理HEC-1-B细胞后,COX-2 mRNA表达下降,细胞凋亡相关基因Fas表达增加,细胞凋亡抑制基因surviyin表达下降,50 μmol/L塞来昔布可使survivin表达难以检出.结论 塞来昔布对HEC-1-B细胞有明显的凋亡诱导作用,通过抑制COX-2表达进而使Fas表达上调,survivin表达下调可能是其诱导细胞凋亡机制之一.     

Immunocytochemical localization of cyclo-oxygenase isoforms in cultured human airway structural cells

BACKGROUND: Cyclo-oxygenase (COX) exists as two isoforms, COX-1, the constitutive isoform, and COX-2, which is inducible by cytokines or inflammatory stimuli and may participate in airway inflammation. OBJECTIVE: To determine the basal distribution of COX isoforms, and their regulation by interleukin-1 beta (IL-1beta), bradykinin (BK) and dexamethasone (Dex) in cultured airway structural cells. METHODS: We measured COX-1 and COX-2 in cultured human airway smooth muscle (HASM) cells, MRC5 fibroblasts and normal human epithelial cells (NHBE) using immunocytochemical analysis. RESULTS: The majority of all types of untreated cultured cells expressed COX-1 (75% of HASM, 75% of MRC5 fibroblasts and 72% of NHBE cells). Fibroblasts and smooth muscle cells showed low constitutive COX-2 expression (2 and 8%, respectively) but this was higher in NHBE cells (28%). IL-1beta (24 h incubation) or BK (4 h incubation) had no effect on COX-1 expression in any of the cells studied. In contrast, there was a two- and 1.5-fold rise in the percentage of NHBE cells expressing COX-2; a 7.5- and sixfold rise in the percentage of HASM cells expressing COX-2 and a 33.5- and 20.5-fold increase in the percentage of fibroblasts expressing COX-2 after IL-1beta or BK treatment, respectively. Pretreatment with dexamethasone abolished IL-1beta- and BK-stimulated COX-2 induction in all cells studied. CONCLUSION: COX-1 is expressed constitutively in human airway fibroblasts, smooth muscle and epithelial cells but epithelial cells also show constitutive expression of COX-2. Both IL-1beta and BK induced COX-2 expression in all cells studied and this induction was blocked by dexamethasone. Immunocytochemical techniques can be successfully used to detect the distribution of COX isoforms in cell cultures.     

Reduced apomorphine sensitivity of dopamine metabolism in rat striatum after repeated administration of methamphetamine

Rats received daily injections of saline or methamphetamine (MAP; 4 mg/kg/day) for 14 days. Seven days after the completion of this regime, dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels were measured in the striatum following intraperitoneal injections of either saline plus gamma-butyrolactone (GBL; 750 mg/kg) or apomorphine (2 mg/kg) plus GBL. The saline plus GBL challenges produced no difference in the DA or DOPAC levels between saline- and MAP-treated rats. By contrast, the apomorphine plus GBL challenges produced higher DOPAC levels in MAP-treated rats than saline-treated rats, although they produced no difference in the DA levels between the two groups. These results indicate that apomorphine depresses the striatal DA metabolism less in MAP-treated rats than in saline-treated control rats. Repeated MAP administration might produce this effect through apomorphine subsensitivity of presynaptic DA autoreceptors.     

Anti-tumor effect and mechanism of cyclooxygenase-2 inhibitor through matrix metalloproteinase 14 pathway in PANC-1 cells

Objective: To investigate whether celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, can attenuate proliferation, migration, invasion and MMP-14 expression in pancreatic cancer cells PANC-1 and the possible anti-tumor mechanism of celecoxib. Methods: Human pancreatic cancer cell line PANC-1 cells were treated with diverse concentrations of celecoxib (20, 60, 100 μmol/L). Cell proliferation, invasion and migration capabilities were measured by MTT colorimetry, transwell invasion assay, and scratch assay separately. At the same time, the protein expression of COX-2 and MMP-14 was assessed by ELISA. Results: The capabilities of proliferation, invasion and migration in PANC-1 cells were attenuated in a concentration-dependent manner after treated with celecoxib, followed by the down-regulation of the protein expression of COX-2 and MMP-14. In addition, MMP-14 expression was significantly positively correlated with COX-2 expression. Conclusions: COX-2 inhibitor celecoxib can inhibit the proliferation, invasion and migration of PANC-1 cells via down-regulating the expression of MMP-14 in a concentration-dependent manner, thus contributing to its anti-tumor effect in pancreatic cancer.     

细胞外信号调节激酶/miR-133b在甲基苯丙胺致PC12细胞神经毒性的作用及机制

目的 探讨甲基苯丙胺(MA)致神经细胞毒性过程中细胞外信号调节激酶(ERK)和miR-133b的表达变化及调控机制。方法 用MA建立PC12神经细胞损伤模型,采用四甲基偶氮唑盐(MTT)检测细胞活性及镜下形态观察确定MA最佳损伤浓度;应用流式细胞术检测细胞内活性氧（ROS）水平;通过 Western blotting技术测定总ERK1/2和磷酸化ERK1/2 (p-ERK1/2)的表达变化;并应用实时定量聚合酶链反应（Real-time PCR）测定miR-133b的表达变化。为进一步分析ERK/miR133b分子通路的作用关系,经U0126特异阻断ERK通路,检测miR-133b的表达变化。 结果 给予不同浓度的MA,均可导致PC12细胞损伤,其中800μmol/L MA处理后,大部分胞体变圆,神经突起退缩,神经网络消失。MTT结果显示细胞活性明显下降。进一步的细胞毒性机制分析显示,MA处理后,细胞内ROS水平升高,p-ERK表达增高,miR-133b表达降低;并且给予ERK通路抑制剂U0126（10μmol/L）后,miR-133b表达升高,细胞活性增强,胞内ROS水平降低,镜下细胞损伤改善。 结论 MA可通过上调ERK磷酸化抑制miR-133b表达,介导神经元毒性损伤。     

COX2 expression in neuroblastoma increases tumorigenicity but does not affect cell death in response to the COX2 inhibitor celecoxib

COX2 is an inducible cyclooxygenase implicated in the metastasis and migration of tumour cells. In neuroblastoma, COX2 expression has been detected in both cell lines and tumours. The treatment of neuroblastoma cells in vitro with celecoxib, a COX2 inhibitor, induces apoptosis. The aim of this study was to investigate the role of COX2 in neuroblastoma tumour biology by creating a cell line in which COX2 could be conditionally expressed. Xenograft studies showed that the conditional expression of COX2 enhanced tumour growth and malignancy. Elevated COX2 expression enhanced the proliferation and migration of neuroblastoma cells in vitro. However, elevated COX2 expression or variation between cell lines did not affect sensitivity to the COX2 inhibitor celecoxib, indicating that celecoxib does not promote cell death through COX2 inhibition. These data show that increased COX2 expression alone can enhance the tumorigenic properties of neuroblastoma cells; however, high levels of COX2 may not be a valid biomarker of sensitivity to non-steroidal anti-inflammatory drugs such as celecoxib.     

Protease Allergens Induce the Expression of IL-25 via Erk and p38 MAPK Pathway

Allergic diseases, including asthma, are characterized by T helper type 2 (Th2) cell-mediated inflammations, coupled with tissue infiltration by eosinophils. In this study, we demonstrate that multiple protease allergens, including papain and DerP1, efficiently induce interleukin (IL)-25 and thymic stromal lymphopoietin (TSLP) gene expression, and this phenomenon is dependent on the protease activities of these allergens. The IL-25 cytokine level in bronchial alveolar lavage (BAL) was also profoundly and significantly increased after treatment with papain. Additionally, the levels of Th2 cytokines were significantly increased, as compared to those in the OVA-only treatment group. The various protease allergens triggered the expression of IL-25 and TSLP mRNA in mouse lung epithelial cells (MLE12) and primary mouse lung epithelial cells; these effects were inhibited by the deactivation of the protease activity of papain. The allergen papain activates the ErK and p38 MAP pathways; the inhibition of these pathways, but not the NFκB or PI-3 kinase pathways, impairs the induction of IL-25 and TSLP expression by proteases. In this study, we demonstrate that the protease allergens induce IL-25 and TSLP via the MAP kinase signal pathways, and their protease activities are essential to this pathway.