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>

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>

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>