Design, synthesis and anticancer activity studies of oleanolic acidderivatives targeting mTOR / 药学学报

Ten novel oleanolic acid (OA) derivatives containing urea or thiourea group were designed and synthesized, the chemical structures were confirmed by 1H NMR, 13C NMR and HR-MS. All of these compounds were evaluated for the inhibitory activity against growth of HepG2 and SGC7901 cells. The results showed that compounds I3 and II3 exhibited significant antitumor activities with IC50 of 9.4 and 5.5 μmol·L-1, respectively. Molecular docking studies showed that all these compounds exhibit inhibitory ability against mTOR kinase. Compounds I3 and II3 were further evaluated for the inhibitory activity against mTOR kinase. The results showed that I3 and II3 exhibited strong inhibitory effect on mTOR kinase with IC50 values of 0.83 and 0.26 μmol·L-1.

Design, synthesis and anti-tumor activity studies of oleanolic acid derivatives using VEGFR as target / 药学学报

In this study, twenty containing ethylenediamine groups derivatives of oleanolic acid (OA) were synthesized, their structures were determined by 1H NMR, 13C NMR and HR-MS. The anti-tumor activities in HepG2 and SGC7901 cells were evaluated by MTT assay. The results showed that all compounds exhibited anti-tumor activity, compounds I6, I8 and I9 exhibited significant anti-tumor activities with IC50 values of 16.7, 9.8 and 6.3 μmol·L-1, respectively. Molecular docking studies showed that compounds I6-I9 produce higher combining ability with VEGFR. Compound I6-I9 were further evaluated for the inhibitory activity against VEGFR-2, the result showed I9 had a strong inhibitory effect on VEGFR with IC50 values of 0.56 μmol·L-1.

Protective Effects of Silibinin and Its Possible Mechanism of Action in Mice Exposed to Chronic Unpredictable Mild Stress

Silibinin, a natural flavonoid antioxidant isolated from extracts of the milk thistle herb, has recently been identified as having anti-hepatotoxic and anticancer properties. In this paper, we investigated the effects of silibinin on behavior and neuroplasticity in mice subjected to chronic unpredictable mild stress (CUMS). After 5 consecutive weeks of CUMS, the mice were treated with silibinin (100 mg/kg, 200 mg/kg and 400 mg/kg by oral gavage) for 3 consecutive weeks. The results showed that silibinin administration significantly alleviated the CUMS-induced depressive-like behavior, including the total number of squares crossed and the frequency of rearing in the open field test, the immobility time in the tail suspension test and the forced swimming test. Furthermore, silibinin treatment increased the levels of brain-derived neurotrophic factor (BDNF), serotonin (5-HT) and norepinephrine (NE) in the prefrontal cortex and hippocampus. Our study provides new insight into the protective effects of silibinin on the depressive status of CUMS mice, specifically by improving neuroplasticity and neurotransmission.

Culture and identification of neural stem cells from mouse embryos / 中国当代儿科杂志

<p><b>OBJECTIVE</b>The purpose of this study was to culture and identify neural stem cells from mouse embryos in vitro using a modified method and provide a basis for further study of the biology of neural stem cells under hypoxia.</p><p><b>METHODS</b>The cells were isolated mechanically from the front cortex of fetal Institute of Cancer Research (ICR) mice on embryonic day 14. They were passaged by mechanical dissociation and enzymatic digestion. The neurospheres were identified by immunofluorescent staining of nestin. Cell differentiation was induced by 1% fetal bovine serum and then the cells were identified by immunohistochemistry of β-tubulin III and GFAP.</p><p><b>RESULTS</b>The cells obtained from the front cortex of fetal ICR mice had the capacity of forming neurospheres which showed nestin immunoreactive positivity. After being induced by 1% fetal bovine serum, the cells were differentiated into β-tubulin III-positive cells and GFAP-positive cells.</p><p><b>CONCLUSIONS</b>Using mechanical dissociation of primary cells and mechanical dissociation with enzymatic digestion of primary cells, the NSCs from the front cortex of mouse embryos can be obtained.</p>

Effect of prenatal heroin exposure on p-ERK1/2 expression in the prefrontal lobe cortex, hippocampus and nucleus accumbens in mice / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the expression of phosphorylated-ERK1/2(p-ERK1/2)MAPK in the prefrontal lobe cortex (PFC), hippocampus (HP) and nucleus accumbens (Acb) in mice exposed to heroin in the uterus, and elucidate whether ERK MAPK signal transduction pathway participates in neurobehavioral teratogenicity induced by maternal heroin abuse.</p><p><b>METHODS</b>Animal model was established by subcutaneous administration of diacetylmorphine (10 mg/kg.d) to pregnant BALB/c mice on embryonic days 9-18, and their offspring were assigned to heroin and normal saline groups according to the maternal treatment. P-ERK1/2 expression in the PFC, HP and Acb were detected by RT-PCR and Western blot.</p><p><b>RESULTS</b>The heroin group had body weights similar to the normal saline group after birth. There were no significant differences in the p-ERK1/2 expression in the PFC, HP and Acb between the two groups.</p><p><b>CONCLUSIONS</b>Prenatal exposure to 10 mg/kg heroin altered neither the body weight nor the general development in mice. The ERK1/2 MAPK signal pathway might not be involved in the neurobehavioral teratogenicity induced by prenatal heroin exposure.</p>

Proliferation of neural progenitor cells in the subventricular zone and around the hematoma after intracerebral hemorrhage in adult rat / 南方医科大学学报

<p><b>OBJECTIVE</b>To examine the proliferation of the neural progenitor cells in the subventricular zone (SVZ) and around the hematoma after intracerebral hemorrhage (ICH) in adult rats.</p><p><b>METHODS</b>ICH was induced by stereotactic injection of type VII collagenase into the corpus striatum of adult rats, followed by pulse or continuous intrapenitoneal injection of bromodeoxyuridine (Brdu) to label the proliferating cells. The rats were sacrificed on days 2, 7, 14 and 28 following the ICH for immunohistochemistry of the tissues in the SVZ and around the hemotoma to determine the number of Brdu- immunoreactive cells.</p><p><b>RESULTS</b>With pulse Brdu labeling, a significant increase in the number of Brdu-immunoreactive cells in the ipsilateral and contralateral tissues in the SVZ and around the hematoma was observed 2-14 days, and the cell number reached the maximum on day 7 after ICH as compared with that of the sham-operated group. With continuous Brdu injection, the increase was observed on day 14 after ICH, and till day 28, the Brdu-immunoreactive cells in the SVZ decreased to the control level, but some positive cells still persisted in the tissues around the hematoma.</p><p><b>CONCLUSION</b>ICH induces transient and regional increase in the cell proliferation in the ipilateral and contraletral SVZ and tissues around the hematoma, and the proliferating cells in the SVZ may migrate towards the hematoma area.</p>

Effect of ligustrazine on cell proliferation in subventricular zone in rat brain with focal cerebral ischemia-reperfusion injury / 中南大学学报(医学版)

OBJECTIVE@#To observe the effect of ligustrazine on cell proliferation in subventricular zone (SVZ) in rat brain with focal cerebral ischemia reperfusion injury.@*METHODS@#Male SD rats were randomly divided into a normal group,a sham operation group,a ligustrazine treatment group, and a control group. The ligustrazine treatment group and the control group were further divided into 5 subgroups: 1d, 3d, 7d, 14d, and 21d reperfusion after 2h middle cerebral artery occlusion (MCAO). The focal cerebral ischemia-reperfusion model was made by MCAO. S phase cells were labelled with BrdU. Immunohistochemistry method was conducted to detect the BrdU positive cells. The total number of BrdU positive cells in the SVZ was measured. The expression of neuro nitric oxide synthase (nNOS) was detected with Western blot method.@*RESULTS@#There was a significant increase of BrdU positive cells in SVZ of ligustrazine treatment in the 1d and 3d group compared with that of the control group (P<0.01). The total number of BrdU positive cells reached a peak in 7d group and declined afterwards. Cells proliferated also in SVZ on the contralateral side, and peaked at 7d. The nNOS expression of ligustrazine administration after the focal cerebral ischemia-reperfusion decreased at 1d and 3d after the reperfusion compared with that of the control group (P<0.05), and increased at 7d, but with no significant difference compared with that of the control group.@*CONCLUSION@#Ligustrazine may promote the cell proliferation in SVZ of adult rats with ischemia-reperfusion injury by decreasing the nNOS expression.

Effect of ligustrazine on nNOS expression and neuranagenesis in adult rats after cerebral ischemia-reperfusion injury / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of ligustrazine on cell proliferation in the subventricular zone (SVZ) and dentate gyrus (DG) and nNOS expression in rat brain after cerebral ischemia-reperfusion injury.</p><p><b>METHODS</b>Male SD rats were randomly divided into normal control group, sham operation group, model group and ligustrazine treatment group. The latter two groups were further divided into 5 subgroups for observation at 1, 3, 7, 14 and 21 days after reperfusion following a 2-hour middle cerebral artery occlusion (MCAO). The cells in S phase were labeled with BrdU, and immunohistochemistry was employed to detect BrdU- and nNOS-positive cells. The numbers of BrdU-positive cells in the SVZ and DG were measured. The expression of nNOS was detected by Western blotting.</p><p><b>RESULTS</b>nNOS expression increased significantly in the model group as compared to the sham operation group (P<0.05), and ligustrazine treatment significantly lowered the expression level in comparison with the model group (P<0.05). Compared with the model group, a significant increase in BrdU-positive cells occurred in the SVZ of rats 1 and 3 days after igustrazine treatment (P<0.05), along with an increase of DG BrdU-positive cells.</p><p><b>CONCLUSION</b>Ligustrazine significantly restrains ischemia-reperfusion injury-induced nNOS activity enhancement and promotes cell proliferation in the SVZ and DG of adult rats after ischemia-reperfusion injury.</p>

Construction and identification of a plasmid with EGFP reporter gene for enhancer function analysis / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a plasmid vector with EGFP reporter gene for functional analysis of enhancers.</p><p><b>METHODS</b>EGFP DNA was amplified by PCR from plasmid pEGFP-N1 DNA and subcloned into plasmid PGL3-promoter backbone without luc(+) gene to construct the enhancer-identifying vector pEGFP-enhancer. Different copies of hypoxia response element (HRE) sequence were synthetized and subcloned into the multiple cloning site of the plasmid pEGFP-enhancer. Using Lipofectamine 2000, the recombined pEGFP-HRE and pEGFP-5HRE plasmids were transfected into the Hela cells respectively. After hypoxic or normoxic cell culture, EGFP expression in the cells was detected by flow cytometry and fluorescence microscopy.</p><p><b>RESULTS</b>After hypoxic exposure, the fluorescence intensity of EGFP in the Hela cells transfected with the plasmid increased with the enhancer HRE copies, while the fluorescence intensity underwent no significant changes after normoxic cell culture.</p><p><b>CONCLUSION</b>we have successfully constructed the enhancer expression vector plasmid pEGFP-enhancer, which can identify the activity of the enhancers through EGFP expression.</p>

Effect of citicoline on spatial learning and memory of rats after focal cerebral ischemia / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of citicoline on spatial learning and memory of rats after focal cerebral ischemia.</p><p><b>METHODS</b>The rats were randomly divided into sham-operation group, ischemia control group and citicoline group. In the later two groups, focal cerebral ischemia model was established by introducing an intraluminal filament into the left middle cerebral artery, and citicoline (500 mg/kg) or 0.9% NaCl was administered intraperitoneally once a day for 2 weeks after the operation. The rats in the sham-operation group were not subjected to middle cerebral artery occlusion (MCAO) with intraluminal filament. The spatial learning and memory functions of the rats were evaluated by Morris water maze test 15 days after MCAO for 5 days.</p><p><b>RESULTS</b>The rats in ischemia control group exhibited serious spatial learning and memory deficits in both place navigation test and spatial probe test. In the former test, the mean escape latency of citicoline-treated rats were significantly shorter than that of ischemia control rats (P<0.01), and in the latter test significant diffidence was noted between citicoline and ischemia control groups in the percentage time spent in the former platform quadrant and frequency of crossing the former platform (P<0.05).</p><p><b>CONCLUSION</b>Citicoline can improve the spatial learning and memory function of rats after focal cerebral ischemia.</p>

Cloning, expression, and antibody preparation of nestin with immunohistochemical analysis / 南方医科大学学报

<p><b>OBJECTIVE</b>To obtain recombinant nestin and prepare anti-nestin polyclonal antibody (mAb) to explore the biological roles of nestin in the central nervous system development.</p><p><b>METHODS</b>The nestin cDNA was cloned from human neural stem cells by RT-PCR and ligated to prokaryotic expression plasmid pQE30 for construction of the recombinant vector pQE30-nestin. After sequencing, the recombinant vector was transformed into E.coli M15 and His-tagged nestin was induced by IPTG. The nestin was purified by Ni-NTA affinity chromatography column and characterized by SDS-PAGE and Western blotting. BALB/c mice were immunized with the purified recombinant protein to prepare the antiserum, which was analyzed by Western blotting, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry.</p><p><b>RESULTS</b>The nestin gene was successfully cloned from human neural stem cells, which was identical to that reported in GenBank. After IPTG induction, the E.coli transformed with pQE30-nestin plasmid expressed a 25,000 His-tagged protein, which was successfully purified and identified as nestin by Western blotting. Western blotting, ELISA and immunohistochemistry demonstrated that the antiserum could specifically bind to the recombinant nestin as well as to nestin in fetal human and rat brains.</p><p><b>CONCLUSION</b>We successfully cloned the nestin gene and expressed the nestin, and nestin mAb prepared can specifically recognize not only the recombinant nestin, but also nestin from human and rats brain tissues.</p>

Effects of ketamine-midazolam anesthesia on the expression of NMDA and AMPA receptor subunit in the peri-infarction of rat brain / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Activation of N-methyl-D-aspartate (NMDA) receptors and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors play an important role in the neurons death induced by ischemia. The mitigating effect of intravenous anesthetics on ischemic neuron injury is related to their influence on NMDA receptors. This study was performed to investigate the effect of ketamine-midazolam anesthesia on the NMDA and AMPA receptor subunits expression in the peri-infarction of ischemic rat brain and explore its potential mechanism of neuroprotection.</p><p><b>METHODS</b>Thirty Sprague Dawley (SD) rats were subjected to permanent middle cerebral artery occlusion under ketamine/atropine (100/0.05 mg/kg) or ketamine-midazolam/atropine (60/50/0.05 mg/kg) intraperitoneal anesthesia (n=15 each). Twenty-four hours after ischemia, five rats in each group were killed by injecting the above dosage of ketamine or ketamine-midazolam intraperitoneally and infarct size was measured. Twenty-four and 72 hours after ischemia, four rats in each group were killed by injecting the above dosage of ketamine or ketamine-midazolam intraperitoneally. After staining the brain tissue slices with toluidine blue, the survived neurons in the peri-infarction were observed. Also, the expression level of NMDA receptors 1 (NR1), NMDA receptors 2A (NR2A), NMDA receptors 2B (NR2B) and AMPA (GluR1 subunit) were determined by grayscale analysis in immunohistochemical stained slices.</p><p><b>RESULTS</b>Compared with ketamine anesthesia, ketamine-midazolam anesthesia produced not only smaller infarct size [(24.1+/-4.6)% vs (38.4+/-4.2)%, P<0.05], but also higher neuron density (24 hours: 846+/-16 vs 756+/-24, P<0.05; 72 hours: 882+/-22 vs 785+/-18, P<0.05) and lower NR2A (24 hours: 123.0+/-4.9 vs 95.0+/-2.5, P<0.05; 72 hours: 77.8+/-4.1 vs 54.2+/-3.9, P<0.05) and NR2B (24 hours: 98.5+/-2.7 vs 76.3+/-2.4, P<0.05; 72 hours: 67.2 +/-7.5 vs 22.2+/-2.6, P<0.05) expression level in the peri-infarction following ischemia.</p><p><b>CONCLUSION</b>The protective effects of ketamine-midazolam anesthesia on ischemic brain injury may related to decreasing NR2A and NR2B expression.</p>

Effect of histamine on intracortical blood vessels of rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate histamine-induced changes of the intracortical vessels in the cortical slice of rat brain.</p><p><b>METHODS</b>Immunohistochemistry was employed to detect the expression of H1 and H2 receptors in the intracortical blood vessels of rats. Histamine-induced constriction of the intracortical blood vessels of the brain slices was observed with differential interference contrast microscope. Measurements of the luminal diameter were made on-line during the course of the experiment and confirmed off-line from the stored images. In order to observe whether histamine H1 and H2 receptors affected histamine-induced constriction, the intracortical blood vessels in the brain slices were pre-treated with H1 receptor antagonist diphenhydramine and H2 receptor antagonist cimetidine.</p><p><b>RESULTS</b>Expression of H1 and H2 receptors was detected in the intracortical blood vessels of the rat brain. Histamine (1-100 micromol/L) induced a concentration-dependent constriction from (1.48-/+0.67)% to (32.91-/+7.91)%. The reactions to each histamine concentration were significantly (P<0.01) different from each other, with the exception of the highest histamine concentrations (30 and 100 micromol/L) when maximal constriction due to histamine were observed (P>0.05). With pre-treatment of the slice with 10 micromol/L diphenhydramine, application of histamine did not elicit constriction. Pre-treatment of the slice with 10 micromol/L cimetidine did not completely inhibit but somehow significantly weakened vascular constriction in response to histamine treatment at 10 and 30 micromol/L (P<0.05).</p><p><b>CONCLUSION</b>Histamine can induce constriction of the intracortical blood vessels, which is mediated by H1 receptor.</p>

Effects of ligustrazine on hippocampal dentate gyrus cell proliferation after focal cerebral ischemia in adult rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the effects of ligustrazine on cell proliferation in hippocampal dentate gyrus subgranular zone (SGZ) after focal cerebral ischemia in adult rats.</p><p><b>METHODS</b>Middle cerebral artery occlusion (MCAO) model was established in adult rats by placement of an intraluminal filament at the origin of the MCA. Ligustrazine was administered intraperitoneally at a daily dose of 80 mg/kg starting at 2 h after MCAO, and BrdU (50 mg/kg daily) was also injected intraperitoneally starting at 4 h after MCAO. BrdU-positive cells in the SGZ were counted 7, 14 and 24 days after MCAO, respectively.</p><p><b>RESULTS</b>Compared with sham operation group, ischemic ipsilateral BrdU-positive cells in the ischemic model group increased 7 days after MCAO, reaching the peak on day 14, and decreased on day 21 (P<0.01). The number of ischemic ipsilateral BrdU-positive cells in ligustrazine group was significantly greater than that in the ischemic model group on days 7, 14 and 21 (P<0.01), and maintained the high level on day 21.</p><p><b>CONCLUSION</b>Ligustrazine possesses long lasting effect of promoting cell proliferation in the SGZ after focal cerebral ischemia in adult rats.</p>