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Objective:To observe the effect of high expression of polypyrimidine tract-binding protein-associated splicing factor (PSF) on low concentration of 4-hydroxynonenal (4-HNE) induced human retinal microvascular endothelial cells (HRMECs), and explore the possible mechanism.Methods:The HRMECs cultured in vitro were divided into 4-HNE treated group, PSF overexpression group combined with 4-HNE group (PSF+4-HNE group), PSF overexpression+ML385 treatment combined with 4-HNE group (PSF+ML385+4-HNE group), and 4-HNE induced PSF overexpression group with LY294002 pretreatment (LY294002+4-HNE+PSF group). Cell culture medium containing 10 μmmol/L 4-HNE was added into 4-HNE treatment group, PSF+4-HNE group, PSF+ML385+4-HNE group for 12 hours to stimulate oxidative stress. 1.0 μg of pcDNA-PSF eukaryotic expression plasmid were transfected into PSF+4-HNE group and PSF+ML385+4-HNE group to achieve the overexpression of PSF. Also cells were pretreated with ML385 (5 μmol/L) for 48 hours in the PSF+ML385+4-HNE group, meanwhile within the LY294002+4-HNE+PSF group, after pretreatment with LY294002, cells were treated with plasmid transfection and 4-HNE induction. Transwell detects the migration ability of PSF to HRMECs. The effect of PSF on the lumen formation of HRMECs was detected by using Matrigel in vitro three-dimensional molding method. Flow cytometer was used to detect the effect of PSF overexpression on reactive oxygen (ROS) level in HRMECs. Protein immunoblotting was used to detect the relative expression of PSF, nuclear factor E2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1) protein, and phosphoserine threonine protein kinase (pAkt) protein. The comparison between the two groups was performed using a t-test. Results:The number of live cells, migrating cells, and intact lumen formation in the 4-HNE treatment group and the PSF+4-HNE group were 1.70±0.06, 0.80±0.13, 24.00±0.58, 10.00±0.67, and 725.00±5.77, 318.7±12.13, respectively. There were significant differences in the number of live cells, migrating cells, and intact lumen formation between the two groups ( t=12.311, 15.643, 17.346; P<0.001). The results of flow cytometry showed that the ROS levels in the 4-HNE treatment group, PSF+4-HNE group, and PSF+ML385+4-HNE group were 816.70±16.67, 416.70±15.44, and 783.30±17.41, respectively. There were statistically significant differences between the two groups ( t=16.311, 14.833, 18.442; P<0.001). Western blot analysis showed that the relative expression levels of pAkt, Nrf2, and HO-1 proteins in HRMECs in the 4-HNE treatment group, PSF+4-HNE group and LY294002+4-HNE+PSF group were 0.08±0.01, 0.57±0.04, 0.35±0.09, 0.17±0.03, 1.10±0.06, 0.08±0.11 and 0.80±0.14, 2.50±0.07, 0.50±0.05, respectively. Compared with the PSF+4-HNE group, the relative expression of pAkt, Nrf2, and HO-1 proteins in the LY294002+4-HNE+PSF group decreased significantly, with significant differences ( t=17.342, 16.813, 18.794; P<0.001). Conclusion:PSF upregulates the expression of HO-1 by activating the phosphatidylinositol 3 kinase/Akt pathway and inhibits cell proliferation, migration, and lumen formation induced by low concentrations of 4-HNE.
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OBJECTIVE： To determine and compare the contents of catalpol and aucubin in different parts （root， stem， leaf and flower） of wild Centranthera grandiflora， and to provide reference for the selection of medicinal parts and source development. METHODS： HPLC method was used to determine the contents of catalpol and aucubin in root， stem， leaf and flower of wild C. grandiflora， and the contents of different parts were analyzed comparatively. The determination of catalpol was performed on Agilent TC-C18 column with mobile phase consisted of methanol-0.1％ phosphoric acid （1 ∶ 99， V/V） at the flow rate of 1 mL/min； the detection wavelength was set at 210 nm， and sample size was 20 μL. The column temperature was 35 ℃； the determination of aucubin was performed on SPHERI-5RP-C18 column with mobile phase consisted of acetonitrile-water （3 ∶ 97， V/V） at the flow rate of 1 mL/min； the detection wavelength was set at 205 nm， and sample size was 20 μL； the column temperature was 25 ℃. RESULTS： The linear range of catalpol and aucubin were 0.061 5-3.321 and 0.000 36-0.216 mg/mL （all r＝0.999 9）. The limits of detection were 0.016 and 0.007 μg/mL. The limits of quantitation were 0.052 and 0.023 μg/mL. RSDs of precision， stability （24 h） and reproducibility tests were all lower than 2.00％ （n＝6）. The average recoveries were 99.34％ and 99.61％， and RSDs were 1.06％ and 1.12％， respectively （n＝6）. The average content of catalpol in root， stem， leaf and flower wild C. grandiflora were 1.609， 3.030， 11.095 and 1.921 mg/g， respectively. The contents of aucubin in different parts were 0.441， 0.020， 0.005 and 0.006 mg/g，respectively. CONCLUSIONS：The established HPLC method meets the requirements of quantitative analysis. Catalpol is mainly distributed in the leaves of wild C. grandiflora， and aucubin is mainly distributed in the roots of wild C. grandiflora. The experimental conclusion provides a reference for the reasonable selection of different medicinal parts as raw materials to develop medicine with different efficacy.
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Objective To prepare lansoprazole biphasic release pellet capsules. Methods The pellets carrying lansoprazole were directly prepared by centrifugal making-pill method,and the pellets of enteric coating and enteric pulsatile coating were adopted by fluidized bed coating method. Then the two kinds of pellets were filled by a fixed proportion to hollow capsules. In vitro dissolution method was used for the observation of the drug release behavior. Results The optimized formulation was as follows:the magnesium carbonate level was 15%,the L-HPC level was 20%in pellets carried drug,the isolation gown level was 9%-10%,the enteric coating level was above 41%in enteric coated pellets,the swelling agent level was 50-60%,the controlled layer level was 50%,the enteric coating level was above 41%in pulsatile enteric coated pellets,and the drying time was 4 h in the end. Conclusion The method is feasible for preparation of lansoprazole biphasic release pellet capsules by encapsulating enteric-coated pellets,and able to obtain good repeatability and stable quality.
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<p><b>OBJECTIVE</b>To investigate the post-transcriptional regulation of dual-specificity phosphatase-1 (DUSP1) by the RNA- binding protein HuR in heat shock.</p><p><b>METHODS</b>The recombinant plasmids carrying wild-type (WT) HuR or its mutants at threonine 118 were constructed and transiently transfected into NIH 3T3 cells via liposome, and the changes in the expressions of DUSP1 mRNA and protein were detected by quantitative real-time PCR and Western blotting, respectively.</p><p><b>RESULTS</b>Heat shock caused significantly enhanced phosphorylation of HuR at the residue T118. In 3T3 cells transfected with the plasmids carrying wild-type HuR for its over-expression showed significantly up-regulated DUSP1 mRNA and protein expressions at 24 h after transfection. Over-expression of HuR(T118A) down-regulated DUSP1 mRNA and protein expressions in cells challenged with heat shock, while HuR(T118E) over-expression significantly increased DISP1 expression at both mRNA and protein levels. After heat shock, HuR(WT) translocated from the cell nucleus to the cytoplasm to form particles. HuR(T118E) was diffusely distributed in the cytoplasm before heat shock and formed particles after heat shock. HuR(T118A) did not undergo such translocation in response to heat shock challenge.</p><p><b>CONCLUSION</b>HuR regulates DUSP1 mRNA and protein expression at the post-transcriptional level to increase its expression after heat shock by enhancing the phosphorylation HuR T118.</p>
Subject(s)
Animals , Mice , Cell Nucleus , Cytoplasm , Dual Specificity Phosphatase 1 , Genetics , Metabolism , ELAV Proteins , Metabolism , Gene Expression Regulation , Heat-Shock Response , Hot Temperature , NIH 3T3 Cells , Phosphorylation , RNA, Messenger , Real-Time Polymerase Chain Reaction , Transfection , Up-RegulationABSTRACT
In order to expand gene resources and improve Brassica napus cultivars, protoplasts isolated from hypocotyls of Brassica napus cv. Huayou No. 3 and Eruca sativa were fused by PEG-high Ca2+-high pH. Fusion frequency was up to 18.2% when fusion system contained 5 x 10(5) protoplasts/mL, and when PEG concentration of fusion agents were 35% and when fusion time was 25 min. Then the fused protoplasts were cultured by the method of thin liquid layer at the density of 1 x 10(5) protoplasts/mL in improved KM8p medium supplemented with 1.0 mg/L 2,4-D, 0.5 mg/L NAA, 0.5 mg/L 6-BA, 200 mg/L inositol, 300 mg/L protein hydrolysate, and the combinations of 0.1 mol/L sucrose and 0.2 mol/L glucose and 0.2 mol/L mannitol for osmotic regulator, the frequency of callus regeneration was up to 6.8%. When the micro-calli transferred to the proliferation medium that contained B5 salts, 0.087 mol/L sucrose, 0.2 mg/L 2,4-D, 0.5 mg/L NAA, 0.2 mg/L 6-BA and 0.5% Agar, pH 5.8, have grown up to 3-5 mm of diameter, the calli were transferred to the differentiation medium that contained MS salts, 0.087 mol/L sucrose, 0.1 mg/L IAA, 0.8 mg/L 6-BA, 0.8% Agar, pH5.8, the shoots were regenerated in 4 weeks and its frequency was up to 32.8%. Then 2-3 cm shoots were transferred to 1/2 MS medium with 0.5 mg/L IBA+0.2mg/L 6-BA, plantlets were obtained in 14 days and the plantlet frequency was up to 88%. When the protoplasts of Eruca sativa were treated with UV radiation for 2 minutes calli and plantlets have been regenerated, treated for 4 min only calli have been regenerated, and treated for more than 5 min calli have not been regenerated. The callus regeneration and callus proliferation and plant regeneration from symmetric fusion were more than from asymmetric fusion. 16 hybrid plantlets have been regenerated on 21 piece of hybrid calli identified by cytology method.