Research progress on epithelial-mesenchymal transition in cancer recurrence and metastasis / 浙江大学学报·医学版

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-β, 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.

Research advances on the roles of nicotinamide phosphoribosyltransferase in inflammation / 浙江大学学报·医学版

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.

Antioxidative effects of cysteinyl leukotriene receptor antagonists montelukast and HAMI 3379 on ischemic injury in rat cortical neurons in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>In ischemic neuronal injury, montelukast and HAMI 3379 exert a moderate antioxidative effect, and this effect may be receptor-independent.</p>

Effect of histone deacetylase inhibitor NL101 on rat neurons / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Effects of the water channel aquaporin 4 deficiency on bleomycin-induced lung fibrosis in mice / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To evaluate the effect of water channel aquaporin 4 (AQP4) on bleomycin-induced lung fibrosis in mice.</p><p><b>METHODS</b>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-β1 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.</p><p><b>RESULTS</b>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-β1 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.</p><p><b>CONCLUSION</b>AQP4 might not be involved in bleomycin-induced lung fibrosis in mice.</p>

Effects of leukotriene D4 on proliferation and migration of lung epithelial A549 cells in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effects of cysteinyl leukotriene (CysLT) receptor agonist leukotriene D4 (LTD4) on proliferation and migration in lung epithelial A549 cells.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSION</b>The proliferation and migration of A549 cells are not changed when treated with 0.01-100 nmol LTD4 for up to 72h.</p>

Cysteinyl leukotriene receptor 1 is involved in rotenone-induced injury of PC12 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate whether cysteinyl leukotriene receptor 1 (CysLT₁ receptor) is involved in rotenone-induced injury of PC12 cells.</p><p><b>METHODS</b>After 24 h treatment with rotenone or with rotenone and the CysLT₁ receptor antagonist montelukast, PC12 cell viability was determined by the colorimetric MTT reduction assay. After PC12 cells were treated with various concentrations of rotenone for 24 h or with 3 μmol/L rotenone for various durations, the expression of CysLT(1) receptor was determined by Western blotting, and its intracellular distribution was detected by immunocytochemistry.</p><p><b>RESULTS</b>Rotenone (0.3-30 μmol/L) induced PC12 cell injury; this injury was significantly attenuated by montelukast at 1 and 5 μmol/L.The expression of CysLT(1) receptor increased after rotenone treatment at 1-10 μmol/L, or at 3 μmol/L for 3 and 24 h. Rotenone caused concentration-and time-dependent translocation of CysLT₁ receptor from the nucleus to the cytosol.</p><p><b>CONCLUSION</b>Cysteinyl leukotriene receptor 1 is involved in rotenone-induced injury of PC12 cells.</p>

Effect of montelukast on morphological changes in neurons after ischemic injury / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine the effect of montelukast, a cysteinyl leukotriene receptor 1 antagonist, on morphological changes in rat neurons after ischemic injury.</p><p><b>METHODS</b>The in vivo ischemia injury was induced by oxygen-glucose deprivation (OGD) for 2 h and reperfusion (R) for 24 h (OGD/R) in rat neurons primary culture and mixed cortex culture. In the presence or absence of various concentrations of montelukast, neuron number, area of neuron, number of neuritis per neuron, branch number of primary neuritis and primary neurite length were determined for evaluating morphological changes in neurons.</p><p><b>RESULTS</b>OGD/R significantly reduced neuron number, and altered neuron morphology. In cortical neuron cultures, montelukast (0.0001-1 μmol/L) attenuated OGD/R-induced reduction in neuron number, and inhibited OGD/R-induced increase in branch number of primary neuritis. In the mixed cultures, montelukast (0.0001-0.1 μmol/L) increased the primary neurite length, and reduced number of neuritis and branch number of primary neurite after OGD/R.</p><p><b>CONCLUSION</b>Montelukast has a protective effect on ischemic injury in neurons.</p>

Construction of HEK293 cell lines expressing hCysLT₂ receptor and its application in screening of antagonists / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct HEK293 cell lines stably expressing hCysLT(2) receptor, and to evaluate its application in screening of synthetic compounds with antagonist activity.</p><p><b>METHODS</b>The recombinant plasmid pcDNA3.1(+)-hCysLT(2) was transfected into HEK293 cells using Lipofectamin 2000. The transfected HEK293 cells were selected in 96 well plates by limiting dilution with 600 μg/ml C418 for 8 weeks. The expression of human CysLT(2) receptor was detected by RT-PCR and immunofluorescence staining. In HEK293 cells stably transfected with hCysLT(2), the agonist LTD(4)-induced elevation of intracellular calcium concentration ([Ca2(+)]i) was measured as the index for screening compounds with antagonist activity.</p><p><b>RESULT</b>After selection in 96 well plates by limiting dilution, 12 monoclones were obtained and 11 of them highly expressed hCysLT(2) receptor. The positive control ATP at 50 μmol/L and LTD(4) at 100 nmol/L elevated [Ca2(+)]i in hCysLT(2)-HEK293 cells. AP-2100984 inhibited LTD(4)-induced [Ca2(+)]i elevation, but selective CysLT(1) receptor antagonists did not exert such an effect. The newly synthesized compounds DXW2, DXW3, DXW4, DXW5, DXW9, DXW25, DXW26, DXW29 and DXW35 at 1 μmol/L significantly inhibited LTD(4)-induced [Ca2(+)]i elevation. The IC(50) values of DXW4 and DXW5 were 0.25 μmol/L and 7.5 μmol/L.</p><p><b>CONCLUSION</b>HEK293 cell lines stably expressing hCysLT(2) receptor have been successfully constructed, and can be used to screen compounds with CysLT(2) receptor antagonist activity.</p>

Rotenone-induced changes of cysteinyl leukotriene receptor 1 expression in BV2 microglial cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare and identify a polyclonal antibody (pAb) against (mouse) cysteinyl leukotriene receptor 1 (CysLT(1)) and to investigate the changes of CysLT(1) receptor expression in BV2 microglial cells after rotenone treatment.</p><p><b>METHODS</b>Rabbits were immunized with KLH-coupled CysLT(1) peptide to prepare the pAb. The titer of the pAb in rabbit plasma was detected by ELISA method, and the specificity of the pAb was tested by antigen blockade. After BV2 cells were treated with rotenone (0.01-1 μmol/L) for 24 h, the expression of CysLT(1) was determined by immunostaining, Western blotting and RT-PCR.</p><p><b>RESULT</b>The pAb showed a titer of 1/32728, and was not cross-reacted with antigens of CysLT(2) receptor and GPR17. Immunostaining, Western blotting and RT-PCR analysis showed the expression of CysLT(1) receptor in BV2 microglia. Rotenone at 1μmol/L significantly induced an increased expression of CysLT(1) receptor.</p><p><b>CONCLUSION</b>The prepared CysLT(1) receptor polyclonal antibody has a high titer and high specificity to meet testing requirements of Western blotting and immunostaining; CysLT(1) is associated with rotenone-induced injury of BV2 microglial cells.</p>

Effects of agonist and antagonist of cysteinyl leukotriene receptors on differentiation of rat glioma C6 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the role of cysteinyl leukotriene (CysLT) receptors in the differentiation of rat glioma C6 cells.</p><p><b>METHODS</b>Rat glioma C6 cells were treated with the agonist LTD(4), the CysLT(1) receptor antagonist montelukast and the differentiation inducer forskolin. Cell morphology and GFAP protein expression were determined after treatments.</p><p><b>RESULT</b>Forskolin (10 μmol/L) induced morphological changes and GFAP protein expression (cell differentiation) in C6 cells, but LTD(4) (0.1-100 nmol/L) did not induce these changes. Montelukast (1 μmol/L) alone did not affect C6 cell differentiation, while it induced the differentiation when combined with the LTD(4) (100 nmol/L).</p><p><b>CONCLUSION</b>The CysLT(2) receptor may modulate the differentiation of rat glioma C6 cells.</p>

Deficiency of water channel AQP4 aggravates NMDA-induced brain injury in mice / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To evaluate the role of water channel AQP4 in NMDA-induced brain injury in mice.</p><p><b>METHODS</b>In AQP4 gene knockout (AQP4(-/-)) mice, brain injury was induced by microinjection of NMDA into the cortex. The injured area was determined by toluidine blue staining, degenerated neurons were detected by Fluro-Jade B staining, and increased blood-brain barrier (BBB) permeability was evaluated by IgG immunostaining.</p><p><b>RESULT</b>Compared with wild-type mice, AQP4(-/-) mice exhibited increased cortical lesion area, aggravated neuron degeneration, and increased BBB disruption after NMDA microinjection.</p><p><b>CONCLUSION</b>AQP4 may play a protective role in NMDA-induced brain injury in mice.</p>

Enzymatic activities of recombinant human NAMPT and NAMPT (H247A) proteins / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare and purify recombinant human NAMPT and NAMPT (H247A) proteins and to detect their enzymatic activity.</p><p><b>METHODS</b>Using pcDNA3.1-hnampt as template, full-length hnampt was sub-cloned into pET-11a(+) plasmid. The hnampt (H247A) mutant was obtained by site-directed mutagenesis. The plasmids were introduced in Escherichia coli BL21star for protein expression. The recombined NAMPT and NAMPT (H247A) proteins were purified by flowing through nickel column and size-exclusion column. The target proteins were confirmed by SDS-PAGE and mass spectrometry detection. The enzymatic activities of recombinant proteins were assessed by solution NMR.</p><p><b>RESULT</b>The DNA sequences showed that hnampt (wild type) and hnampt (H247A) (mutation) were cloned into pET-11a(+). The recombinant proteins were expressed in Escherichia coli BL21star in soluble form. The purified protein was confirmed to be NAMPT with a molecular weight of 56 KD. The enzyme activity of NAMPT (H247A) was dramatically decreased compared to wild-type NAMPT.</p><p><b>CONCLUSION</b>The recombinant hNAMPT and hNAMPT (H247A) proteins have been successful prepared and purified. The H247A mutation dramatically decreases the enzymatic activity of NAMPT.</p>

Construction and identification of eukaryotic expression vector of rat GPR17 gene / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct the eukaryotic expression vector of rat GPR17 (rGPR17) cDNA,and to identify its function in HEK293 cells.</p><p><b>METHODS</b>Total RNA was extracted from rat brain tissue; full-length GPR17 cDNA was prepared by RT-PCR, and cloned into pcDNA3.1(+) plasmid. The recombinant plasmid was converted into E.coli DH5alpha and confirmed by PCR, double enzyme digestion analysis and DNA sequencing. The recombinant plasmid pcDNA3.1(+)-rGPR17 was transiently transfected into HEK293 cells using Lipofectamin 2000. Expression of rGPR17 gene was confirmed by RT-PCR and immunofluorescence staining. The exogenous LTD(4) enhanced intracellular calcium was measured using Fluo-4.</p><p><b>RESULT</b>RT-PCR, double enzyme digestion analysis and sequencing showed that the rGPR17 gene was cloned into recombinant vector, and the recombinant rGPR17 was expressed after transfection in HKE293 cells. LTD(4) increased intracellular calcium release in the transfected HEK293 cells.</p><p><b>CONCLUSION</b>The eukaryotic expression vector of rGPR17 cDNA has been constructed; it is functionally expressed in HEK293 cells. This work provides a basis for further research of the GPR17 receptor and its antagonists.</p>

Preparation and identification of polyclonal antibody against cysteinyl leukotriene receptor 2 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare and identify a polyclonal antibody against cysteinyl leukotriene receptor (CysLT(2)receptor).</p><p><b>METHODS</b>Rabbits were immunized with KLH-coupled CysLT(2) receptor peptide to prepare the polyclonal antibody (pAb). The titer of the pAb in rabbit plasma was detected by indirect ELISA, and the specificity of the pAb was tested by antigen blockade. The tissue distribution of CysLT(2) receptor was detected by Western blot and immunohistochemistry with the prepared pAb.</p><p><b>RESULT</b>The pAb showed a titer higher than 1/1047296, and was specific to CysLT(2) receptor, without cross-reaction with the antigens of CysLT(1) receptor and GPR17. A higher expression of CysLT(2) receptor in kidney, brain and lung of rats and mice was detected by Western blot analysis using the prepared pAb. The molecular weight of CysLT(2) receptor protein was about 40 kD. Immunohistochemical examination showed that CysLT(2) receptor was expressed mainly in the neuron, and partly in astrocytes in rat brain.</p><p><b>CONCLUSION</b>The prepared CysLT(2) receptor pAb has high sensitivity and specificity, and can be used in Western blot and immunohistochemistry.</p>

Method for screening cysteinyl leukotriene receptor 2 antagonists and preliminary screening of compounds / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a method for screening cysteinyl leukotriene receptor 2 (CysLT(2)) antagonists and to preliminarily screen a series of synthetic compounds.</p><p><b>METHODS</b>Rat glioma cell line (C6 cells) highly expressing CysLT(2) receptor was used. Intracellular calcium concentration was measured after stimulation with the agonist LTD(4),which was used to screen compounds with antagonist activity for CysLT(2) receptor. Bay u9773, a CysLT1/CysLT(2) receptor non-selective antagonist, and AP-100984, a CysLT(2) receptor antagonist, were used as control.</p><p><b>RESULT</b>PT-PCR showed a higher expression of CysLT(2) receptor in C6 cells. LTD(4) at 1 mumol/L significantly increased intracellular calcium in C6 cells; the maximal effect was about 37.5% of ATP, a positive stimulus.LTD(4)-induced increase of intracellular calcium was blocked by CysLT(2) receptor antagonists, but not by CysLT(1) receptor antagonists. Among the synthetic compounds, D(XW-)1,2,13,23,29 and 30 inhibited LTD(4)-induced increase of intracellular calcium.</p><p><b>CONCLUSION</b>LTD(4)-induced change in intracellular calcium in C6 cells can be used as a screening method for CysLT(2) receptor antagonists. The compounds, D(XW-)1,2,13,23,29 and 30, possess antagonist activity for CysLT(2) receptor.</p>

Preparation and identification of a polyclonal antibody against novel cysteinyl leukotriene receptor GPR17 / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To prepare and identify a polyclonal antibody (pAb) against GPR17, a novel cysteinyl leukotriene receptor.</p><p><b>METHODS</b>Rabbits were immunized with KLH-coupled GPR17 peptide to prepare the pAb. The titer of the pAb in rabbit plasma was detected by indirect ELISA, and the specificity of the pAb was tested by antigen blockade. GPR17 tissue distribution was detected by Western blot with the pAb.</p><p><b>RESULTS</b>The pAb showed a titer as high as 1:16 364,and was not cross-reacted with the antigens of CysLT(1) and CysLT(2) receptors. A higher expression of GPR17 in the rat brain and heart was detected using the newly prepared pAb. The molecular weigh of GPR17 protein was about 43 kD.</p><p><b>CONCLUSION</b>The prepared GPR17 pAb has high sensitivity and specificity,and can be used in Western blot for detecting GPR17.</p>

5-Lipoxygenase/cysteinyl leukotriene pathway is not involved in injury of rat C6 glioma cells induced by oxygen-glucose deprivation / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine whether oxygen-glucose deprivation (OGD) induces C6 cell injury, and whether 5-lipoxygenase (5-LOX)/cysteinyl leukotriene (CysLT) pathway is involved in OGD-induced injury.</p><p><b>METHODS</b>After OGD treatment and recovery for various durations, the viability of C6 cells was determined, and the effects of 5-LOX inhibitors and CysLT receptor antagonists were investigated. Intracellular distribution of 5-LOX protein was detected by immunocytochemistry, and the mRNA expressions of CysLT1 and CysLT2 receptors were detected by RT-PCR. The effect of leukotriene D(4) (LTD(4)) on C6 cells was also investigated.</p><p><b>RESULT</b>OGD for 4-8 h followed by recovery for 24-72 h significantly induced C6 cell injury. Neither 5-LOX inhibitors nor CysLT receptor antagonists inhibited OGD-induced injury. OGD did not induce 5-LOX translocation into the nuclear membrane. C6 cells highly expressed CysLT(2) receptor, but the expression of CysLT1receptor was much weaker; the expression was not affected by OGD. In addition, LTD(4) did not affect C6 cells significantly.</p><p><b>CONCLUSION</b>OGD can induce C6 cell injury, but 5-LOX/CysLT pathway might be not involved in OGD-induced injury.</p>

Cysteinyl leukotriene receptor 1 antagonist pranlukast modulates differentiation of SK-N-SH cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine whether cysteinyl leukotriene receptor agonist LTD(4) and cysteinyl leukotriene receptor 1 (CysLT(1)) antagonist pranlukast affect the differentiation of human neuroblastoma SK-N-SH cells.</p><p><b>METHODS</b>SK-N-SH cell morphological changes induced by LTD(4), pranlukast and LTD(4) + pranlukast were observed with retinoid acid (RA) as the positive control. The expressions of CysLT(1) and CysLT(2) receptors were detected by immunoblotting analysis, and the expression of microtubule-associated protein-2 (MAP-2), a neuron marker, was detected by fluorescent immunostaining.</p><p><b>RESULT</b>The immunoblotting results showed that SK-N-SH cells expressed CysLT(1) receptor moderately, and CysLT(2) receptor highly. The morphological results showed that RA, pranlukast and LTD(4) + pranlukast induced the compaction of the cell bodies and the outgrowth of neurites, while LTD(4) had no significant effect. The immunostaining results showed that MAP-2 was distributed in the cell bodies in control or pranlukast-treated cells; it was distributed in cell bodies and neuritis in RA-treated cells. Pranlukast increased the numbers of MAP-2-positive cells.</p><p><b>CONCLUSION</b>The CysLT(1)receptor antagonist pranlukast modulates the differentiation of SK-N-SH cells.</p>

Homeostatic conditions affect the protective effect of edaravone on ischemic injury in neurons / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine whether homeostatic conditions (pH, glycine or ion concentration) affect the protective effects of edaravone on ischemic injury in rat cortical neurons.</p><p><b>METHODS</b>In cultured rat cortical neurons, the compositions in the experimental solutions were changed to mimic the disturbance of homeostasis after cerebral ischemia. In vitro ischemic injury was induced by oxygen-glucose deprivation (OGD) for 3 h and reperfusion for 12 h, and the neuron injury was evaluated by 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) reduction assay and lactate dehydrogenase (LDH) release. Effect of edaravone on OGD injury was observed in different experimental solutions.</p><p><b>RESULT</b>In weak alkalified solution (pH 7.8) or the solution containing glycine (10 micromol/L), OGD injury became more serious; but in weak acidic (pH 6.5) or higher Mg(2+) (1.8 mmol/L) solutions, OGD injury was attenuated. Edaravone (1 micromol/L) reversed the injury in the solutions with pH 6.1,7.4 and 7.8 or the solution containing glycine, but did not show protective effect in the solution with pH 6.5 and the higher Mg(2+) or lower Ca(2+) solution.</p><p><b>CONCLUSION</b>The changes of homeostatic conditions affect the severity of ischemic injury of neurons and the protective effect of edaravone.</p>