GPR17 mediates ischemia-like neuronal injury via microglial activation.

GPR17 is a G (i)-coupled dual receptor, linked to P2Y and CysLT receptors stimulated by uracil nucleotides and cysteinyl leukotrienes, respectively. Recent evidence has demonstrated that GPR17 inhibition ameliorates the progression of cerebral ischemic injury by regulating neuronal death and microglial activation. The present study aimed to assess the detailed regulatory roles of this receptor in oxygen­glucose deprivation/recovery (OGD/R)­induced ischemia­like injury in vitro and explore the underlying mechanism. The results demonstrated that OGD/R induced ischemic neuronal injury and microglial activation, including enhanced phagocytosis and increased inflammatory cytokine release in neuron­glial mixed cultures of cortical cells. GPR17 upregulation during OGD/R was spatially and temporally correlated with neuronal injury and microglial activation. In addition, GPR17 knockdown inhibited OGD/R­induced responses in neuron­glial mixed cultures. GPR17 knockdown also attenuated cell injury induced by the agonist leukotriene D4 (LTD4) or uridine 5'­diphosphate (UDP) in neuron­glial mixed cultures. However, GPR17 knockdown did not affect OGD/R­induced ischemic neuronal injury in primary cultures of neurons. In primary astrocyte cultures, neither GPR17 nor OGD/R induced injury. By contrast, GPR17 knockdown ameliorated OGD/R­induced microglial activation, boosting phagocytosis and inflammatory cytokine release in primary microglia cultures. Finally, the results demonstrated that the conditioned medium of microglia pretreated with OGD/R induced neuronal death, and the neuronal injury was significantly inhibited by GPR17 knockdown. These findings suggested that GPR17 may mediate ischemia­like neuronal injury and microglial activation in vitro; however, the protective effects on ischemic neuronal injury might depend upon microglial activation. Whether GPR17 regulates neuronal injury mediated by oligodendrocyte linkage remains to be investigated.

NAMPT inhibitor protects ischemic neuronal injury in rat brain via anti-neuroinflammation.

Nicotinamide phosphoribosyltransferase (NAMPT) is an important neuroprotective factor in cerebral ischemia, and it has been reported that NAMPT inhibitors can aggravate neuronal injury in the acute phase. However, because it is a cytokine, NAMPT participates in many inflammatory diseases in the peripheral system, and its inhibitors have therapeutic effects. Following cerebral ischemia, the peripheral and resident inflammatory and immune cells produce many pro-inflammatory mediators in the ischemic area, which induce neuroinflammation and impair the brain. However, the effects of NAMPT inhibitors in the neuroinflammation after ischemic brain injury remain unknown. Here, we found that FK866, a potent NAMPT inhibitor, decreased the level of TNF-α, NAMPT and IL-6 in the ischemic brain tissue one day after middle-cerebral-artery occlusion and reperfusion (MCAO/R), improved neurological dysfunction, decreased infarct volume and neuronal loss, and inhibited microgliosis and astrogliosis 14days after MCAO/R. The expression of NAMPT protein was induced in Iba1-positive microglia/macrophages in the ischemia core 14days after MCAO/R. In vitro studies show that oxygen-glucose deprivation and recovery (OGD/R) activate microglia. Activated microglia increased the activity of NF-κB, increased the mRNA synthesis of TNF-α, NAMPT and IL-6, and increased the secretion of TNF-α, NAMPT and IL-6. On the other hand, NAMPT can act synergistically with other cytokines and activate microglia. FK866 strongly inhibited these changes and alleviated OGD/R-induced activation of microglia. As such, NAMPT is a crucial determinant of cellular inflammation after cerebral ischemia. NAMPT inhibitors are novel compounds to protect neuronal injury from ischemia via anti-inflammatory effects.

Specific cell surface labeling of GPCRs using split GFP.

Specific cell surface labeling is essential for visualizing the internalization processes of G-protein coupled receptors (GPCRs) and for gaining mechanistic insight of GPCR functions. Here we present a rapid, specific, and versatile labeling scheme for GPCRs at living-cell membrane with the use of a split green fluorescent protein (GFP). Demonstrated with two GPCRs, GPR17 and CysLT2R, we show that two ß-stands (ß-stands 10 and 11) derived from a superfolder GFP (sfGFP) can be engineered to one of the three extracellular loop of a GPCR. The complementary fragment of sfGFP has nine ß-strands (ß-stands 1-9) that carries the mature fluorophore, and can be proteolytically derived from the full-length sfGFP. Separately the GFP fragments are non-fluorescent, but become fluorescent upon assembly, thus allowing specific labeling of the target proteins. The two GFP fragments rapidly assemble and the resulting complex is extremely tight under non-denaturing conditions, which allows real-time and quantitative assessment of the internalized GPCRs. We envision that this labeling scheme will be of great use for labeling other membrane proteins in various biological and pharmacological applications.

Nicotinamide phosphoribosyltransferase inhibitor APO866 induces C6 glioblastoma cell death via autophagy.

APO866 is a potent inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), and inhibits nicotinamide adenine dinucleotide (NAD) synthesis. Our previous study showed that APO866 inhibits the proliferation of C6 glioblastoma cells, but failed to induce apoptosis. Since APO866 inhibits cellular metabolism and such metabolic stress is closely related with autophagy, thus we determined whether APO866 can induce autophagy in C6 glioblastoma cells and whether the autophagy induced by APO866 is pro-death or pro-survival. Using LC3 immunofluorescence imaging and transmission electron microscopy detection, we found that APO866 at 1-100 nM induced autophagy in C6 glioblastoma cells. APO866 at 1 nM mainly induced initial autophagic vacuoles. Whereas APO866 at 100 nM induced degrading autophagic vacuoles, as well as induced nuclei malformation and mitochondria swelling. In addition, APO866 concentration-dependently decreased the cell viability of C6 glioblastoma cells, and this effect was attenuated by autophagy inhibitors, including 3-methyladenine and LY294002. APO866 concentration-dependently decreased intracellular NAD level. Interestingly, APO866 at 1 nM slightly decreased intracellular NAD level, but dramatically increased autophagy-positive cells. The dramatical cell viability decreasing required the decreasing of intracellular NAD level to a very low threshold. Thus, our results indicated that APO866 induced pro-death autophagy in C6 glioblastoma cells by decreasing intracellular NAD, and low concentration of APO866 can be used as an autophagy inducer in autophagic-death sensitive glioblastoma.

CysLT2 receptor mediates lipopolysaccharide-induced microglial inflammation and consequent neurotoxicity in vitro.

Neuroinflammation induced by microglial activation plays a critical role in many neurodegenerative diseases, including Parkinson's disease (PD). Recent studies have indicated that cysteinyl leukotriene receptor 2 (CysLT2R) is involved in inflammation and brain injury after cerebral ischemia. However, the role of CysLT2R in microglial responses associated with PD remains unclear. In the present study, we determined the regulatory roles of CysLT2R in microglial inflammation and subsequent neurotoxicity in an in vitro brain inflammation model induced by the microglial activator lipopolysaccharide (LPS). We found that LPS induced phagocytosis of a murine microglial cell line (BV-2 cells) and increased production of the proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß). The expression of CysLT2R protein was up-regulated and the nuclear translocation of CysLT2R was induced in LPS-activated BV-2 cells. CysLT2R selective antagonist HAMI 3379 significantly inhibited LPS-induced phagocytosis and overproduction of the cytokines in BV-2 cells. Similarly, the CysLT2R silencing by specific short hairpin RNA (shRNA) had the same effects as those of HAMI 3379, suggesting that the effect might be CysLT2R-dependent. Furthermore, the conditioned medium (CM) derived from LPS-treated BV-2 cells induced the cell death of a rat adrenal pheochromocytoma cell line (PC12). HAMI 3379 and CysLT2R shRNA attenuated neuronal death by suppressing the production of neurotoxic cytokines released from LPS-activated microglia. Collectively, these results suggest that CysLT2R mediates LPS-induced microglial inflammation and consequent neurotoxicity. CysLT2R may be a promising molecular target that modulates microglia-related neuroinflammation in neurodegenerative disorders, such as PD.

Autophagy in synaptic development, function, and pathology.

In the nervous system, neurons contact each other to form neuronal circuits and drive behavior, relying heavily on synaptic connections. The proper development and growth of synapses allows functional transmission of electrical information between neurons or between neurons and muscle fibers. Defects in synapse-formation or development lead to many diseases. Autophagy, a major determinant of protein turnover, is an essential process that takes place in developing synapses. During the induction of autophagy, proteins and cytoplasmic components are encapsulated in autophagosomes, which fuse with lysosomes to form autolysosomes. The cargoes are subsequently degraded and recycled. However, aberrant autophagic activity may lead to synaptic dysfunction, which is a common pathological characteristic in several disorders. Here, we review the current understanding of autophagy in regulating synaptic development and function. In addition, autophagy-related synaptic dysfunction in human diseases is also summarized.

[Research progress on epithelial-mesenchymal transition in cancer recurrence and metastasis].

Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose their morphology and function and gradually transformed into mesenchymal-like cells. It is considered that EMT is the main cause for tumor recurrence and metastasis. Many factors are involved in the regulation of EMT, such as E-cadherin, transforming growth factor-ß, Wnt signaling pathway, microRNA and EMT-related transcription factors. This article reviews the research progress on EMT and the involved mechanisms, and thus to provide a new perspective on cancer therapy in the future.

[Protective effect of grape seed proanthocyanidin on cultured RGC-5 cells against CoCl2-induced hypoxic injury].

OBJECTIVE: To investigate the protective effects of grape seed proanthocyanidin extracts (GSPE) against CoCl2-induced hypoxic injury in cultured RGC-5 cells. METHODS: CoCl2(400 µmol/L) was used to induce hypoxic injury in cultured RGC-5 cells; the cells were pretreated with 0,100,200,400 and 800µmol/L GSPE for 24h. The cell viability was assayed by MTT; the apoptosis was detected by Hoechst 33342 staining; the intracellular reactive oxygen species (ROS) was measured by H2DCFDA oxidative reaction. The mRNA expression of Bcl-2, caspase 9 and caspase 3 was determined by real-time PCR. RESULTS: Compared to hypoxic control group, pretreatment with GSPE significantly increased viability of RGC-5 cells (P<0.001), reduced cell apoptosis (P<0 .001) and intracellular ROS(P <0 .001). In addition, GSPE significantly increased the mRNA expression of Bcl-2(P<0 .001) and decreased mRNA expression of caspase 9(P<0 .001) and caspase 3(P<0 .001) compared to hypoxic control group. CONCLUSION: GSPE may have a protective effect against CoCl2-induced hypoxic injury in cultured RGC-5 cells. The decrease of intercellular ROS, up-regulation of Bcl-2 and down-regulation of caspase 9 and caspase 3 may be involved in the mechanism of the protective effect of GSPE.

[Antioxidative effects of cysteinyl leukotriene receptor antagonists montelukast and HAMI 3379 on ischemic injury in rat cortical neurons in vitro].

OBJECTIVE: To investigate the antioxidative effects of two cysteinyl leukotriene receptors antagonists (CysLT1R and CysLT2R) montelukast and HAMI 3379 on ischemic injury of rat cortical neurons in vitro. METHODS: Cultured rat cortical neurons were pretreated with CysLT1R antagonist montelukast and CysLT2R antagonist HAMI 3379, and then exposed to oxygen-glucose deprivation/recovery (OGD/R）or H2O2. Reactive oxygen species (ROS) mitochondrial membrane potential (MMP) depolarization, neuronal viability and lactate dehydrogenase (LDH) release were determined. Meanwhile, RNA interference was used to inhibit the expression of CysLT1R and CysLT2R，and the effects were observed. RESULTS: ROS production in neurons was significantly increased after 1 h OGD, which reached the peak at 30 min and lasted for 1.5 h after recovery. Montelukast and HAMI 3379 at 0.01-1µmol/L moderately decreased OGD/R-induced ROS production (P<0.05). Montelukast mildly attenuated OGD/R-induced MMP depolarization (P<0.05)，but HAMI 3379 had no effect. H2O2 reduced neuronal viability and increased LDH release, namely inducing neuronal injury. Montelukast and HAMI 3379 at 0.1-1µmol/L moderately attenuated H2O2-induced neuronal injury (P<0.05). However, both CysLT1R siRNA and CysLT2R shRNA did not significantly affect the responses mentioned above. CONCLUSION: In ischemic neuronal injury, montelukast and HAMI 3379 exert a moderate antioxidative effect, and this effect may be receptor-independent.

[Effect of histone deacetylase inhibitor NL101 on rat neurons].

OBJECTIVE: To investigate the protective effect of histone deacetylase inhibitor NL101 on L-homocysteine (HCA)-induced toxicity in rat neurons, and the toxic effect on normal rat neurons. METHODS: In the presence of NL101 at various concentrations, HCA (5 mmol/L)-induced changes in cell density, necrosis, and viability were determined in the mixed cultures of rat cortical cells and the primary cultures of rat neurons. The direct effect of NL101 on primary neurons was also observed in the absence of HCA. Histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) was used as the control. After the treatments, cell viability, the density, and morphology of neurons and glial cells, and cell necrosis were determined. RESULTS: In the mixed cultures of cortical cells, NL101 had no effect on HCA (5 mmol/L)-induced cell number reduction at 0.001-10µmol/L; however, it significantly attenuated necrosis at 1-10 µmol/L, and increased neuronal number at 1 µmol/L. NL101 had no effect on the mixed cortical cells in the absence of HCA. In the primary neurons, NL101 reduced neuronal viability and mildly increased necrosis at 1-10 µmol/L in the absence of HCA, while it significantly attenuated HCA-induced neuronal viability reduction at 0.01-10 µmol/L and reduced neuronal necrosis at 1-10 µmol/L. The effects of NL101 were apparently similar to those of SAHA. CONCLUSION: NL101 has protective effect on HCA-induced neuronal injury but it is neurotoxic at high concentrations, which is similar to the typical histone deacetylase inhibitor SAHA.

[Zileuton, a 5-lipoxygenase inhibitor, attenuates mouse microglial cell-mediated rotenone toxicity in PC12 cells].

OBJECTIVE: To examine the effect of a selective inhibitor of 5-lipoxygenase (5-LOX) zileuton on microglia-mediated rotenone neurotoxicity. METHODS: The supernatant from different concentrations of rotenone-stimulated mouse microglia BV2 cells was used as the conditioned media (CM) for PC12 cells. The viability of PC12 cells was determined by MTT assay and lactate dehydrogenase (LDH) release. Cell death was observed by LDH release and double fluorescence staining with Hoechst/propidiumiodide (PI). The effect of zileuton on microglia-mediated rotenone toxicity was evaluated by the above methods. RESULTS: Rotenone at 1-10 nmol/L was nontoxic to PC12 cells directly. However, the CM from BV2 cells that were treated with rotenone (1-10 nmol/L) resulted in toxicity of PC12 cells. The BV2 CM which stimulated with rotenone (1-10 nmol/L) induced morphological changes, reduced cell viability, and increased LDH release and cell necrosis in PC12 cells. Pretreatment of BV2 cells with the 5-LOX inhibitor zileuton (0.01-1 µmol/L) protected PC12 cells from the microglia-mediated rotenone toxicity. CONCLUSION: The 5-LOX inhibitor zileuton effectively attenuates microglia-mediated rotenone toxicity in PC12 cells. These results suggest that 5-LOX pathway may be involved in neuronal death induced by microglial inflammation.

[Effects of the water channel aquaporin 4 deficiency on bleomycin-induced lung fibrosis in mice].

OBJECTIVE: To evaluate the effect of water channel aquaporin 4 (AQP4) on bleomycin-induced lung fibrosis in mice. METHODS: In wild type and AQP4 gene knockout (AQP4-/-) mice, lung fibrosis was induced by injection of bleomycin (3 mg/kg) into the trachea and saline injection was used as a control. At d3, 7, 14, 28 after bleomycin-treatment, mice were randomly sacrificed in batch and the lung coefficient was determined. Serum levels of TGF-ß1 and TNF-α were measured by ELISA and hydroxyproline contents in lung tissue were determined by Alkaline hydrolysis method. H-E staining and Masson's staining were performed to examine the pathological changes of lung tissues after bleomycin-treatment. RESULTS: On d14 after bleomycin-treatment, the lung coefficients in wild type mice and AQP4-/- mice were 1.9-fold (12.69 ± 6.05 vs 6.80 ± 0.82, q=4.204, P<0.05) and 2.3-fold (14.05 ± 5.82 vs 6.05± 0.58, q=5.172, P<0.01) of that in control, respectively, but no significant difference was found between wild type and AQP4-/- mice in the lung coefficient value (P>0.05). The hydroxyproline contents in the lung increased after bleomycin-treatment; on d28, the lung hydroxyproline contents in wild type and in AQP4-/- mice were 1.55-fold (0.85 ± 0.22 g/mg vs 0.55 ± 0.14 µg/mg, q=4.313, P<0.05) and 1.4-fold (0.84 ± 0.13 µg/mg vs 0.60 ± 0.14µg/mg, q=4.595，P<0.05) of that in control, respectively, but no significant difference was noticed between wild type and AQP4-/- mice in lung hydroxyproline contents. There was a tendency that serum TGF-ß1 and TNF-α levels increased in bleomycin-treated mice, but no significant difference was found between wild type and AQP4-/- mice. AQP4-knockout showed no effects on pathological changes of lung tissues with H-E staining and Masson's staining in mice with bleomycin-induced lung fibrosis. CONCLUSION: AQP4 might not be involved in bleomycin-induced lung fibrosis in mice.

[Effects of leukotriene D4 on proliferation and migration of lung epithelial A549 cells in vitro].

OBJECTIVE: To investigate the effects of cysteinyl leukotriene (CysLT) receptor agonist leukotriene D4 (LTD4) on proliferation and migration in lung epithelial A549 cells. METHODS: The expression of CysLT1 receptor and CysLT2 receptor was determined by immunofluoresence staining in A549 cells. A549 cells were treated with LTD4 (0.01-100 nmol/L) for 24-72 h. Cell viability was detected by MTT reduction assay. Cell migration was determined by modified scratch and healing model. RESULTS: In A549 cells, CysLT1 receptor and CysLT2 receptor were mainly expressed in the cytoplasm, membrane and few in the nuclei. The treatment of LTD4 (0.01-100 nmol/L) for 24-72 h caused no effect on cell viability (Ps>0.05); when A549 cells were treated with 100 nmol/L LTD4 for 24, 48 and 72 h the cell viability was (103.00±4.46)%，(107.00±9.45)% and (105.00±9.02)% of control, respectively (Ps>0.05). The migration rate of A549 cells after scratching during the first 24 h was markedly greater than that during the second and third 24 h in the same concentration groups; however, no significant difference in migration rate was noticed when the cells were treated with different concentrations of LTD4 (0.01-100 nmol/L)(Ps>0.05). The migration of A549 cells was 1.15-fold, 1.21-fold and 1.06-fold of that of control when the cells were treated with 100 nmol/L LTD4 for 24, 48 and 72 h, respectively (Ps>0.05). CONCLUSION: The proliferation and migration of A549 cells are not changed when treated with 0.01-100 nmol LTD4 for up to 72h.

[Application of locomotor activity test to evaluate functional injury after global cerebral ischemia in C57BL/6 mice].

OBJECTIVE: To evaluate the application of locomotor activity test in functional injury after global cerebral ischemia (GCI) in C57BL/6 mice. METHODS: GCI was induced by bilateral carotid arteries occlusion for 30 min in C57BL/6 mice. Mice were divided into sham group, GCI group and minocycline group. Saline or minocycline (45 mg/kg) was i.p. injected once daily for 6 d after ischemia. At Day 6 after ischemia, locomotor activity was recorded for 1 h in open field test. Total distance, central distance, central distance ratio, periphery distance, periphery distance ratio, central time and periphery time were used to evaluate the behavior characteristics of locomotor activity in C57BL/6 mice after ischemia. The survival neuron density was detected by Nissl staining in hippocampus, cortex and striatum. RESULTS: Compared with sham group, total distance, central distance and central time increased and periphery time decreased in C57BL/6 mice after GCI (Ps<0.05). However, minocycline significantly reduced the central distance and central time and increased the periphery time (Ps<0.05). Neurons were damaged in hippocampus, cortex and striatum after GCI, which manifested by decreased neurons and the most serious damage in hippocampal CA1 region. Minocycline significantly improved the neuron appearance and increased the neuron number in hippocampus and striatum (P<0.001 or P<0.05). CONCLUSION: Locomotor activity in open field test can objectively evaluate the behavior injury after GCI in mice. Central distance and central time can be used as indexes of quantitative assessment.

[Influence of object material and inter-trial interval on novel object recognition test in mice].

OBJECTIVE: To investigate the efficacy of novel object recognition (NOR) test in assessment of learning and memory ability in ICR mice in different experimental conditions. METHODS: One hundred and thirty male ICR mice were randomly divided into 10 groups: 4 groups for different inter-trial intervals (ITI: 10 min, 90 min, 4 h, 24 h), 4 groups for different object materials (wood-wood, plastic-plastic, plastic-wood, wood-plastic) and 2 groups for repeated test (measured once a day or every 3 days, totally three times in each group). The locomotor tracks in the open field were recorded. The amount of time spent exploring the novel and familiar objects, the discrimination ratio (DR) and the discrimination index (DI) were analyzed. RESULTS: Compared with familiar object, DR and DI of novel object were both increased at ITI of 10 min and 90 min (P<0.01). Exploring time, DR and DI were greatly influenced by different object materials. DR and DI remained stable by using identical object material. NOR test could be done repeatedly in the same batch of mice. CONCLUSION: NOR test can be used to assess the learning and memory ability in mice at shorter ITI and with identical material. It can be done repeatedly.

Regulation of rotenone-induced microglial activation by 5-lipoxygenase and cysteinyl leukotriene receptor 1.

The 5-lipoxygenase (5-LOX) products cysteinyl leukotrienes (CysLTs) are potent pro-inflammatory mediators. CysLTs mediate their biological actions through activating CysLT receptors (CysLT(1)R and CysLT(2)R). We have recently reported that 5-LOX and CysLT(1)R mediated PC12 cell injury induced by high concentrations of rotenone (0.3-10 µM), which was reduced by the selective 5-LOX inhibitor zileuton and CysLT(1)R antagonist montelukast. The purpose of this study was to examine the regulatory roles of the 5-LOX/CysLT(1)R pathway in microglial activation induced by low concentration rotenone. After mouse microglial BV2 cells were stimulated with rotenone (0.3-3 nM), phagocytosis and release of pro-inflammatory cytokine were assayed as indicators of microglial activation. We found that rotenone (1 and 3 nM) increased BV2 microglial phagocytosis and the release of the pro-inflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). Zileuton and montelukast prevented rotenone (3 nM)-induced phagocytosis and cytokine release. Furthermore, rotenone significantly up-regulated 5-LOX expression, induced 5-LOX translocation to the nuclear envelope, and increased the production of CysLTs. These responses were inhibited by zileuton. Rotenone also increased CysLT(1)R expression and induced nuclear translocation of CysLT(1)R. In primary rat microglia, rotenone (10 nM) increased release of IL-1ß and TNF-α, whereas zileuton (0.1 µΜ) and montelukast (0.01 µΜ) significantly inhibited this response. These results indicated that 5-LOX and CysLT(1)R might be key regulators of microglial activation induced by low concentration of rotenone. Interference of 5-LOX/CysLT(1)R pathway may be an effective therapeutic strategy for microglial inflammation.

[Research advances on the roles of nicotinamide phosphoribosyltransferase in inflammation].

Nicotinamide phosphoribosyltransferase (Nampt) is also called visfatin or pre-B-cell colony-enhancing factor. The functions of Nampt have been reported as a cytokine, an adipokine and the rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis. As a pleiotropic multifunctional protein, Nampt is involved in a variety of physiological and pathological conditions including innate immunity, metabolic disorders, and stress; and Nampt also participates in inflammatory disorders such as acute lung injury, atherosclerosis, myocardial infarct, obesity, type 2 diabetes, and rheumatoid arthritis. The studies indicate that Nampt might be a potential target for pharmacological intervention against inflammatory diseases. We review research advances on the roles of Nampt in inflammation.

Cysteinyl leukotriene receptor 1 mediates LTD4-induced activation of mouse microglial cells in vitro.

AIM: To investigate the roles of cysteinyl leukotriene receptors CysLT1R and CysLT2R in leukotriene D4 (LTD4)-induced activation of microglial cells in vitro. METHODS: Mouse microglial cell line BV2 was transfected with pcDNA3.1(+)-hCysLT1R or pcDNA3.1(+)-hCysLT2R. The expression of relevant mRNAs and proteins in the cells was detected using RT-PCR and Western blotting, respectively. Phagocytosis was determined with flow cytometry analysis. The release of interleukin-1ß (IL-1ß) from the cells was measured using an ELISA assay. RESULTS: The expression of CysLT1R or CysLT2R was considerably increased in the transfected BV2 cells, and the receptors were mainly distributed in the plasma membrane and cytosol. Treatment of the cells expressing CysLT1R or CysLT2R with CysLT receptor agonist LTD4 (0.1-100 nmol/L) concentration-dependently enhanced the phagocytosis, and increased mRNA expression and release of IL-1ß. Moreover, the responses of hCysLT1R-BV2 cells to LTD4 were significantly larger than those of hCysLT2R-BV2 or WT-BV2 cells. Pretreatment of hCysLT1R-BV2 cells with the selective CysLT1R antagonist montelukast (1 µmol/L) significantly blocked LTD4-induced phagocytosis as well as the mRNA expression and release of IL-1ß, whereas the selective CysLT2R antagonist HAMI 3379 (1 µmol/L) had no such effects. CONCLUSION: CysLT1R mediates LTD4-induced activation of BV2 cells, suggesting that CysLT1R antagonists may exert anti-inflammatory activity in brain diseases.

HAMI 3379, a CysLT2 receptor antagonist, attenuates ischemia-like neuronal injury by inhibiting microglial activation.

The cysteinyl leukotrienes (CysLTs) are inflammatory mediators closely associated with neuronal injury after brain ischemia through the activation of their receptors, CysLT1R and CysLT2R. Here we investigated the involvement of both receptors in oxygen-glucose deprivation/recovery (OGD/R)-induced ischemic neuronal injury and the effect of the novel CysLT2R antagonist HAMI 3379 [3-({[(1S,3S)-3- carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy)benzoic acid] in comparison with the CysLT1R antagonist montelukast. In primary neurons, neither the nonselective agonist leukotriene D4 (LTD4) nor the CysLT2R agonist N-methyl-leukotriene C4 (NMLTC4) induced neuronal injury, and HAMI 3379 did not affect OGD/R-induced neuronal injury. However, in addition to OGD/R, LTD4 and NMLTC4 induced cell injury and neuronal loss in mixed cultures of cortical cells, and neuronal loss and necrosis in neuron-microglial cocultures. Moreover, they induced phagocytosis and cytokine release (interleukin-1ß and tumor necrosis factor-α) from primary microglia, and conditioned medium from the treated microglia induced neuronal necrosis. HAMI 3379 inhibited all of these responses, and its effects were the same as those of CysLT2R interference by CysLT2R short hairpin RNA, indicating CysLT2R dependence. In comparison, montelukast moderately inhibited OGD/R-induced primary neuronal injury and most OGD/R- and LTD4-induced (but not NMLTC4-induced) responses in mixed cultures, cocultures, and microglia. The effects of montelukast were both dependent and independent of CysLT1Rs because interference by CysLT1R small interfering RNA had limited effects on neuronal injury in neuron-microglial cocultures and on cytokine release from microglia. Our findings indicated that HAMI 3379 effectively blocked CysLT2R-mediated microglial activation, thereby indirectly attenuating ischemic neuronal injury. Therefore, CysLT2R antagonists may represent a new type of therapeutic agent in the treatment of ischemic stroke.

Biomechanical properties of retinal glial cells: comparative and developmental data.

The biomechanical properties of Müller glial cells may have importance in understanding the retinal tissue alterations after retinal surgery with removal of the inner limiting membrane and during the ontogenetic development, respectively. Here, we compared the viscoelastic properties of Müller cells from man and monkey as well as from different postnatal developmental stages of the rat. We determined the complex Young's modulus E = E' + iE″ in a defined range of deforming frequencies (30, 100, and 200 Hz) using a scanning force microscope, where the real part E' reflects the elastic property (energy storage or elastic stiffness) and the imaginary part E″ reflects the viscous property (energy dissipation) of the cells. The viscoelastic properties were similar in Müller cells from man, monkey, and rat. In general, the elastic behavior dominated over the viscous behavior (E' > E″). The inner process of the Müller cell was the softest region, the soma the stiffest (Einnerprocess(')Eglia(')). These relations were also observed during the postnatal development of the rat. It is concluded that, generally, retinal cells display mechanics of elastic solids. In addition, the data indicate that the rodent retina is a reliable model to investigate retinal mechanics and tissue alterations after retinal surgery. During retinal development, neuronal branching and synaptogenesis might be particularly stimulated by the viscoelastic properties of Müller cell processes in the inner plexiform layer.