Effects of cysteinyl leukotriene receptors on phagocytosis of mouse microglial cells / 浙江大学学报·医学版

OBJECTIVE@#: To determine the effects of cysteinyl leukotriene receptors (CysLTR and CysLTR) on phagocytosis of mouse BV2 microglial cells.@*METHODS@#: BV2 cells were stimulated with microglial activators lipopolysaccharide (LPS) or CysLT receptor agonists LTD. The phagocytosis of BV2 cells was observed by immunofluorescence analysis and flow cytometry. The intracellular distributions of CysLTR and CysLTR in BV2 cells were examined with immunofluorescence staining.@*RESULTS@#: Both LPS and LTD could significantly enhance the phagocytosis of BV2 cells, and such effect could be inhibited by CysLTR selective antagonist Montelukast and CysLTR selective antagonist HAMI 3379. The activation of BV2 cells induced by LTD or LPS resulted in changes in intracellular distributions of CysLTR and CysLTR. CysLTR and CysLTR was co-localization with a similar distribution.@*CONCLUSIONS@#: CysLTR and CysLTR regulate the phagocytosis of mouse BV2 microglial cells with a synergistic effect.

Expression and distribution of cysteinyl leukotriene receptors CysLT1R and CysLT2R, and GPR17 in brain of Parkinson disease model mice / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To examine the spatiotemporal profiles and localization of CysLT1R, CysLT2R and GPR17 in mice with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced Parkinson disease (PD).</p><p><b>METHODS</b>PD model was induced by subcutaneous injection of MPTP (25 mg/kg) for 5 d in adult male C57BL/6 mice. At d10 after MPTP injection, the expression and cellular localization of CysLT1R, CysLT2R and GPR17 in the substantia nigra were detected by immunohistochemistry and immunofluorescence.</p><p><b>RESULTS</b>CysLT1R, CysLT22 and GPR17 were normally localized in TH-positive dopaminergic neurons and microglia, while CysLT2R was also expressed in astrocytes. In dopaminergic neurons, approximately 91% co-expressed GPR17, 77% co-expressed CysLT1R and 52% co-expressed CysLT2R. Compared with the control group, TH-positive cells in the substantia nigra were significantly reduced in PD mice. CysLT1R, CysLT2R and GPR17-positive cells were significantly reduced; and CysLT1R, CysLT2R, GPR17-positive dopaminergic neurons were also significantly reduced in the PD group. In the striatum, both CysLT1R and GPR17 were normally expressed in neurons; whereas CysLT2R was expressed in astrocytes. In PD striatum, CysLT1R and GPR17-positive cells were decreased, but CysLT2R expression was significantly increased which mainly expressed in the proliferating astrocytes.</p><p><b>CONCLUSION</b>CysLT1R, CysLT2R and GPR17 may be involved in the MPTP-induced PD damage in mice.</p>

Expression of 5-lipoxygenase in hippocampal CA1 neuronal damage following global cerebral ischemia in rats / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine 5-lipoxygenase (5-LOX) expression and the effect of zileuton, a selective 5-LOX inhibitor,on hippocampal neuron injury induced by global cerebral ischemia in rats.</p><p><b>METHODS</b>Global cerebral ischemia was induced by bilateral common carotid artery occlusion combined with hypotension in rats. 5-LOX expression was detected by Western blot analyses and 5-LOX localization was visualized by immunohistochemistry and double immunofluorescence methods. The 5-LOX inhibitor zileuton (10, 30, 50 mg/kg) was orally administered for 3 d after ischemia.</p><p><b>RESULTS</b>The 5-LOX expression was increased in the ischemic hippocampus on d1-7 (peaked at d3), and 5-LOX protein was primarily localized in neurons and translocated to the nuclei in the hippocampal CA1 region after ischemia. The 5-LOX inhibitor zileuton (30, 50 mg/kg) reduced ischemia-induced hippocampal neurons death 3d after ischemia.</p><p><b>CONCLUSION</b>5-LOX is involved in global cerebral ischemic damage in rats, and the 5-LOX inhibitor zileuton has a protective effect on neuronal damage in the rat hippocampus following global cerebral ischemia.</p>

Leukotriene D4 activates BV2 microglia in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of CysLT receptor agonist leukotriene D4(LTD4) and antagonists on activation of microglia BV2 cells.</p><p><b>METHODS</b>The expression of CysLT1 and CysLT2 protein was determined by Western blotting and immunostaining in microglia BV2 cells. BV2 cells were pretreated with or without CysLT1 receptor selective antagonist montelukast, CysLT2 receptor selective antagonist HAMI 3379, or CysLT1/CysLT2 receptor dual antagonist BAY u9773 for 30 min, then the cells were treated with LTD4 for 24 h. Cell viability was detected by MTT reduction assay. Phagocytosis and mRNA expression of IL-6 were determined by fluorescent bead tracking and RT-PCR, respectively.</p><p><b>RESULTS</b>In BV2 cells, LTD4 did not affect proliferation but significantly enhanced phagocytosis and increased IL-6 mRNA expression in a concentration-dependent manner. LTD4 at 100 nmol/L induced a 1.4-fold increase of phagocytic index and a 2-fold up-regulation of IL-6 mRNA expression (P<0.01). HAMI 3379 and BAY u9773 (100 nmol/L) further increased LTD4-induced phagocytosis; BAY u9773 and montelukast decreased LTD4-induced IL-6 mRNA expression, while HAMI 3379 had no effect on that.</p><p><b>CONCLUSION</b>LTD4 activates BV2 cells in vitro and enhances IL-6 mRNA expression mediated by CysLT1 receptor, LTD4 induces phagocytosis which might be negatively regulated by CysLT2 receptor in BV2 cells.</p>

Role of G protein-coupled receptor 17 in central nervous system injury / 浙江大学学报·医学版

G-protein-coupled receptor 17 (GPR17), an originally orphan receptor, was identified as a new uracil nucleotides/cysteinyl leukotriene receptor. However, whether GPR17 is really classified as a leukotriene receptor is a matter deserving further investigation. GPR17 is involved in many physiological and pathological processes including brain injury, spinal cord injury, and oligodendrocyte differentiation. GPR17 may become a new therapeutic target in these diseases. In this article, the research progress on the pharmacology and pathophysiological roles of GPR17 is reviewed.

Comparison of the effects of the locomotor activity between theophylline and caffeine in mice / 中华行为医学与脑科学杂志

Objective Using video tracking system to compare the effects of the locomotor activity between theophylline and caffeine in mice.Methods The KM mice were treated by theophylline and caffeine(both at 1,3,10,30,100 mg/kg)intraperitoneally respectively.After 10 min,the locomotor activity in the open field was recorded for 2 hours.The locomotor track,the total distance,the distances and distance ratio to total distance in central region were analyzed to evaluate the effects of these drugs on locomotor in mice.Results The mice administrated theophylline and caffeine both increased the total distances,and had similar bell-shaped dose-effect relationship.The distances reached the highest at 30 mg/kg theophylline((311±128)m)and 10 mg/kg caffeine ((279±89)m).The larger doses of caffeine inhibited the activity,and the total distance during 0～0.5 h was significantly decreased at the dose of 100 mg/kg(P＜0.05).Theophylline(30 and 100 mg/kg)and caffeine (30 mg/kg)significantly increased the distance ratio in central region(P＜0.01)and decreased the distance ratio in peripheral region(P＜0.01).Conclusion Theophylline and caffeine increase the total distance and the distance ratio in central region in mice,but have different valency and efficacy.

Effect and modulation of ICAM-1 and VCAM-1 in the inflammatory mechanisms following cerebral ischemia / 中国药理学通报

Intercellular adhesion molecules 1(ICAM-1) and vascular cell adhesion molecule 1(VCAM-1) play important roles in the inflammatory process of cerebral ischemic injury.The expression of ICAM-1 and VCAM-1 increases following cerebral ischemia;ICAM-1 and VCAM-1 promote ischemic inflammation through mediating leukocytes adhesion to the endothelial cells and eventually migration into brain tissue;the inhibition of the overexpression and effect of ICAM-1 and VCAM-1 can reduce cerebral ischemic injury.

Therapeutic effect of pranlukast, a cysteinyl leukotriene receptor antagoist, on focal cerebral ischemia in mice / 中国药理学通报

Aim To determine whether pranlukast (ONO-1078), a cysteinyl leukotriene receptor antagonist, possesses therapeutic effect when administered after focal cerebral ischemia in mice. Methods Persistent focal cerebral ischemia was induced by middle cerebral artery occlusion. Pranlukast and edaravone, a positive control drug, were ip injected 1, 6 and 24 h after ischemia. The neurological deficits were evaluated by neurological scores and inclined plane test 24 and 48 h after the surgery. Forty-eight h later, the brain slices were prepared for measurements of infarct volume and the ratio of ischemic/non-ischemic hemispheres. Brain sections were cut and examined for neuron densities in different regions of the brain. The effects of pranlukast and edaravone were evaluated by the changes of these variables. Results Pranlukast (0.1 and 0.2 mg?kg -1) and edaravone (3 and 10 mg?kg -1) significantly reduced the neurological deficits, infarct volume (maximally 82.3%), ratio of ischemic/non-ischemic hemispheres, and attenuated the reduction of neuron densities in hippocampal CA1 region, cortex and striatum. Conclusion Pranlukast possesses therapeutic effect on ischemic insults when administered after ischemia as effective as edravone, indicating a therapeutic potential in the treatment of ischemic stroke.

PROTECTIVE EFFECT OF ONO-1078, A LEUKOTRIENE ANTAGONIST, ON FOCAL CEREBRAL ISCHEMIA IN MICE / 药学学报

AIM　To determine whether ONO-1078 {pranlukast, 4-oxo-8-［p-(4-phenylbutyloxy) benzoyl-amino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate}, a potent leukotriene antagonist, has protective effect on focal cerebral ischemia in mice. METHODS　Focal cerebral ischemia was induced by permanent middle cerebral artery (MCA) occlusion in mice. ONO-1078 (0.01, 0.05, 0.10 mg*kg-1), dexamethasone (0.5 mg*kg-1), nimodipine (0.2 mg*kg-1) or saline (control) were injected ip once daily for 3 days, and 30 min before MCA occlusion. Twenty-four hours after cerebral ischemia, the neurological scores were evaluated, infarct volumes and areas of the right and left cerebral hemispheres were measured by computer imaging analysis. RESULTS　ONO-1078, dexamethasone and nimodipine reduced the neurological scores. ONO-1078 and dexamethasone reduced the ratio of right/left hemisphere area, indicating inhibition of brain edema, while nimodipine showed no effect. ONO-1078 dose-dependently reduced infarct size, and dexamethasone and nimodipine showed the same effect. CONCLUSION　ONO-1078 showed protective effect on focal cerebral ischemia. This may represent a novel approach to the treatment of acute cerebral ischemia.

Relationship between airway inflammation and bronchial hyperreactivity in rats and the effect of theophylline / 中国病理生理杂志

AIM: To determine the relationship between antigen-induced airway inflammation characterized by pulmonary eosinophilia and bronchial hyperreactivity in rats, and to evaluate the effect of theophylline at different doses. METHODS: In ovalbumin (OA)-sensitized rats, bronchiole wall area, eosinophils around bronchi, and the responses to methacholine (MCh) aerosol were measured after 1% OA aerosol challenge with computer-assisted techniques. RESULTS: OA challenge caused both inflammation and airway hyperreactivity, and there was a significantly positive correlation between them. Oral theophylline (1-12.5 mg/kg, bid for 7 days) attenuated antigen-induced inflammation (swelling of bronchiole walls and pulmonary eosinophilia) and bronchial hyperreactivity. CONCLUSION: These findings confirm that bronchial hyperreactivity positively correlates to airway inflammation in the rat, and suggest that theophylline at relatively lower doses has anti-inflammatory effect in airway allergic reaction.

Quantitative analysis of focal ischemic cerebral infarction in mice / 中国临床药理学与治疗学

AIM:To confirm the action of the light transmission method in evaluating focal ischemic cerebral infarction on persistent focal cerebral ischemia in mice. METHODS:Persistent focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Bederson's neurological scores, climbing board and hanging test were performed 24 h after ischemia, and infarct volume, brain hemisphere area, neuron density of cortex and subcortex were measured with computer-assisted imaging. Pranlukast ( 0.1 mg?kg -1) or nimodipine ( 0.4 mg?kg -1) were injected ip once daily for 3 days and to 1 h before MCAO assess the neuroprotective effect. RESULTS:The infarct volumes measured by light transmission closely correlated with that measured by TTC staining and neuron densities. The infarct volumes measured by light transmission well correlated with the neurological scores measured by integrated graded approach, too. Both pranlukast and nimodipine significantly attenuated infarct volumes and the ratio of ischemic/non-ischemic hemispheres, and reduced neurological deficits and neuron death. CONCLUSION:Light transmission and integrated graded approach can be used not only for qualitative analysis of focal cerebral ischemia, but also for evaluating the neuroprotective effect of drugs.

SURFACE EXPRESSION AND LOCALIZATION OF NMDARECEPTOR ON DENDRITIC TREE OF HIPPOCAMPAL NEURONS IN CULTURE / 解剖学报

Objective To investigate the developmental profiles on surface expression and distribution of NMDA receptor clusters especially on dendritic structures in cultured hippocampal neurons of rats. Methods A vector expressing green fluorescent protein N-terminally tagged NR1a subunit(GFP-NR1a) was generated and transfected into cultured hippocamplal neurons at 5 days in vitro (DIV 5).Surface expressed GFP-NR1a containing NMDA receptor clusters was then labeled in living neurons by using anti-GFP primary antibody followed by Cy3-conjugated secondary antibody.Furthermore,distribution of the surface clusters was observed on the detailed dendritic structures visualized with co-expression of cyan fluorescent protein(CFP). Results Punctate green flourescence clusters can be seen throughout the neurons transfected with GFP-NR1a and surface staining of living neurons showed that the most of these flourescence clusters on dendritic arbors were surface expressed in mature neurons.In parallel,no significant difference was found in terms of density of the surface NMDA clusters on dendritic trees in neurons at different developmental stage.Interestingly,at DIV 7,surface NMDA receptor clusters were mostly observed on the dendritic shafts,but rarely on filopodia.In contrast,two weeks later about 50% of clusters were found located at the dendritic spines.Conclusion This study shows that surface NMDA receptor clusters are distributed in a differentiated way related to dendritic structures during neural development.Especially at very early developmental stage,no clusters was found presented on filopodia,instead they were extensively clustered with a density estimated to be not significantly different from mature neurons.Therefore,our results suggested that NMDA receptors are likely to be incorporated or recruited to postsynaptic sites in the form of pre-expressed surface cluster on dendritic shafts during glutamatergic synapse formation.