1.Effects of deltamethrin on cell survival rate and intracellular free Ca2+ concentration in primary cultured astrocytes of rat.
You-tong WU ; Nian SHI ; Su-qing WANG ; Tao LI ; Jiang-hai CHEN ; Li ZHOU ; Liang CHEN ; Jie DONG
Chinese Journal of Industrial Hygiene and Occupational Diseases 2003;21(3):194-196
OBJECTIVETo study the effects of deltamethrin (DM) on cell survival rate and intracellular Ca(2+) ([Ca(2+)]i) concentration in primary cultured astrocytes of rat.
METHODSThe cell survival rate was measured by Typan Blue assay; the intracellular [Ca(2+)]i concentration was determined by the fluorescent Ca(2+) indicator Fura-2/AM.
RESULTSThe survival rate of astrocytes was decreased to 91.9% after astrocytes were incubated with 1 x 10(-5) mol/L DM for 72 h (P < 0.05). The cell survival rates were 89.0%, 84.8%, 81.2% and 79.2% respectively when astrocytes were administered with 1 x 10(-4) mol/L DM for 4, 12, 24 and 72 h, which were remarkably lower than control groups (P < 0.01). Comparing with controls and before DM treatment, sharp increases in [Ca(2+)]i concentration [(451.4 +/- 42.3), (536.9 +/- 47.5) and (870.9 +/- 100.5) nmol/L respectively] were observed when astrocytes were incubated with 1 x 10(-7), 1 x 10(-6) and 1 x 10(-5) mol/L DM for 5 minutes (P < 0.01). After astrocytes were treated with 1 x 10(-8), 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) mol/L DM for 15 minutes, the [Ca(2+)]i concentrations were decreased to (124.3 +/- 6.0), (131.3 +/- 19.1), (118.9 +/- 1.4), (136.6 +/- 3.8) nmol/L respectively, which were significantly different from those of controls and before treatment. And this situation was almost keeping stable to 30 min.
CONCLUSIONThe cell survival rate was decreased and the [Ca(2+)]i concentration was temporarily increased when astrocytes were treated with DM.
Animals ; Astrocytes ; cytology ; drug effects ; metabolism ; Calcium ; metabolism ; Cell Survival ; drug effects ; Cells, Cultured ; Insecticides ; toxicity ; Nitriles ; Pyrethrins ; toxicity ; Rats
2.Effect of propofol on thrombospondin-1 expression in cultured newborn rat cortical astrocytes.
Yu ZHANG ; Yu LI ; Weiguang LI ; Chenggang ZHANG ; Tiehu YE
Journal of Southern Medical University 2013;33(9):1316-1320
OBJECTIVETo investigate the effect of propofol on the expression of thrombospondin-1 (THBS-1) mRNA and protein in purified newborn rat cortical astrocytes in vitro.
METHODSAstrocytes were isolated from newborn rat cortex and grown in culture before exposure to propofol at 3, 10, 30, 100 or 300 µmol/L for 6 h, 12, or 24 h. The mRNA level of THBS-1 was detected by RT-PCR, and the protein level of THBS-1 was detected by immunofluorescence cytochemistry and Western blotting.
RESULTSPropofol exposure caused significantly upregulated THBS-1 level in cultured astrocytes (P<0.05) to a level about 1.3 times higher than that in control cells. The mRNA and protein levels of THBS-1 in cultured rat cortical astrocytes were upregulated by exposures to 10, 30 and 100 µmol/L propofol (P<0.01). High expression of THBS-1 mRNA and protein was detected in the cells with exposures for different durations (P<0.05), especially in the 12 h group (P<0.01).
CONCLUSIONPropofol at clinically relevant concentrations can modulate the level of THBS-1 secreted by astrocytes of rat cerebral cortex in vitro.
Animals ; Astrocytes ; drug effects ; metabolism ; Cells, Cultured ; Cerebral Cortex ; cytology ; Propofol ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Thrombospondin 1 ; metabolism
3.Okadaic acid induces the expression of glutamate transporter EAAT1 in the neurons of rat brain.
Jian-She WEI ; Ling-Mei ZHANG ; Ya-Lin HUANG ; Cui-Qing ZHU ; Feng-Yan SUN
Acta Physiologica Sinica 2002;54(4):287-293
To study the relationship between tau hyperphosphorylation and the function of glutamate transporter okadaic acid (OA), a protein phosphatase inhibitor, 20 ng in a 0.5 microl volume, was injected into the frontal cortex of rat brain and immunostaining was used to observe the phosphorylation of tau protein and the expression of excitatory amino acid transporter 1 (EAAT1) in the brain following the injection. The results showed that (1) the neurons in the center of the injection region displayed cytoplasmic shrinkage, swelling, nuclear pyknosis, and dislocation at the early stage, and necrosis appeared 3 d after the injection. However, most neurons in the peri-injected areas showed normal morphological characters with immuno positive reaction for AT8, a tau phosphorylated marker; (2) morphological analysis showed that tau hyperphosphorylation caused by OA treatment was mainly observed in the axons and dendrites of neuronal cells at 6 h in the cell body at 1 d, which brought about dystrophic neurites and neurofibrillary tangle (NFT)-like pathological changes; (3) the induction of glutamate transporter EAAT1 was observed in the involved areas corresponding to that with AT8 immunopositive staining, and the number of EAAT1-positive staining cells markedly increased at 12 h (P<0.01), peaked at 1 d (P<0.001), then decreased at 3 d following the injection. Combined with a confocal laser scanning microscopic analysis, double fluorescent immunostaining showed that EAAT1 positive staining appeared in neurons as well as astrocytes in the peri-injected areas of the frontal cortex. These results demonstrate that OA increases glutamate transporter EAAT1 expression in neurons while it induces tau hyperphosphorylation. However, the mechanism and significance of the induction of glutamate transporter EAAT1 expression remain to be further elucidated.
Animals
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Astrocytes
;
drug effects
;
metabolism
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Axons
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drug effects
;
metabolism
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Brain
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cytology
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Dendrites
;
drug effects
;
metabolism
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Excitatory Amino Acid Transporter 1
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metabolism
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Neurofibrillary Tangles
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pathology
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Neurons
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drug effects
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metabolism
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Okadaic Acid
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pharmacology
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Phosphorylation
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Rats
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tau Proteins
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metabolism
4.Effect of thrombin on cultured rat cerebral astrocyte injured by hypoxia/reoxygenation and its relationship with iNOS.
Li-juan YANG ; Li-xiang WU ; Fa-yi LIU ; Xuan ZHOU ; Fang HE ; Yan ZHANG ; Bo-sheng HUANG
Journal of Central South University(Medical Sciences) 2007;32(5):831-835
OBJECTIVE:
To observe the effect of thrombin on the cytotoxicity of astrocytes injured by hypoxia/reoxygenation(H/R) and to explore its relationship with inducible nitric oxide synthase (iNOS).
METHODS:
Primary astrocytes were cultured in DMEM with 10% approximately 15% calf serum and divided into 6 groups: a control group, a Tm control group, an H/R group, a Tm+H/R group, a hirudin (HR) control group, and a Tm+HR+ H/R group. The cell damage and viability were detected by the 3-(4, 5-di-methyl-thazol-2-yl)-2, 5 diphenyl-tetrazol-iumbromide (MTT) conversion method. The NO level in the cultured cell supernatant was assayed by Griess reagent. The flow cytometry was performed to evaluate the apoptosis rate of astrocytes. The iNOS mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Immunocytochemistry was used to observe the expression of iNOS protein.
RESULTS:
The cell viability injured by H/R was lower than that of the control group, the NO production and apoptosis rate in the cell of H/R group were higher than those of the control group. Incubation of H/R cell with 10kU/L Tm enhanced the cytotoxicity of H/R stimulation compared with the cells injured by H/R. Hirudin can reverse the effect of thrombin. RT-PCR and immunocytochemistry analysis demonstrated that the levels of iNOS mRNA and iNOS protein increased in the cells treated by H/R. Tm enhanced the expression of iNOS mRNA and iNOS protein in the cells treated by H/R. Hirudin blocked the effect of Tm.
CONCLUSION
Increasing the level of iNOS and enhancing the production of NO may be the mechanism of thrombin cytotoxicity in astrocytes injured by H/R.
Animals
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Apoptosis
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drug effects
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Astrocytes
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cytology
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drug effects
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Cell Hypoxia
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Cells, Cultured
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Nitric Oxide
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metabolism
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Nitric Oxide Synthase Type II
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metabolism
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Rats
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Rats, Sprague-Dawley
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Thrombin
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pharmacology
5.Synergistic effect of schwann cells and retinoic acid on differentiation and synaptogenesis of hippocampal neural stem cells in vitro.
Xue-Bao ZHANG ; Yuan-Shan ZENG ; Wei ZHANG ; Ya-Yun CHEN ; Wei ZHANG ; Yi XIONG ; Sui-Jun CHEN
Biomedical and Environmental Sciences 2006;19(3):219-224
OBJECTIVETo investigate the synergistic effect of Schwann cells (SCs) and retinoic acid (RA) on differentiation and synaptogenesis of neural stem cells (NSCs) derived from hippocampus of neonatal rats.
METHODSThe classical method for 2x2 factorial analysis experiment was used to assess synergistic action of SCs and RA. NSCs were treated with RA, SCs, and SCs + RA in DMEM/F12 with 0.5% fetal bovine serum for six days, respectively. Double immunofluorescent staining was used to detect the differentiation of NSCs including nestin, glial fibrillary acidic protein (GFAP) and Map2. The expression of PSD95 was used to demonstrate synaptogenesis.
RESULTSAfter NSCs were treated with RA or SCs, the expression of nestin and GFAP was significantly decreased while the expression of Map2 and PSD95 was significantly increased in comparison with the control. Factorial ANOVA showed that interactions between SCs and RA could induce the expression of Map2 and PSD95.
CONCLUSIONSCs and RA could promote synergistically the neuronal differentiation and synaptogenesis of hippocampal neural stem cells in vitro while they decreased the astrocytes and nestin positive NSCs.
Animals ; Animals, Newborn ; Astrocytes ; cytology ; metabolism ; Cell Differentiation ; drug effects ; physiology ; Cells, Cultured ; Drug Synergism ; Fluorescent Antibody Technique ; Glial Fibrillary Acidic Protein ; metabolism ; Hippocampus ; cytology ; drug effects ; ultrastructure ; Intermediate Filament Proteins ; metabolism ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neurons ; cytology ; drug effects ; ultrastructure ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; metabolism ; Stem Cells ; cytology ; drug effects ; ultrastructure ; Synapses ; drug effects ; physiology ; Tretinoin ; pharmacology
6.Human Brain Astrocytes Mediate TRAIL-mediated Apoptosis after Treatment with IFN-gamma.
Jeonggi LEE ; Jeon Soo SHIN ; In Hong CHOI
Yonsei Medical Journal 2006;47(3):354-358
TNF-related apoptosis inducing ligand (TRAIL) expressions were studied in primary human brain astrocytes in response to pro-inflammatory cytokines. When astrocytes were treated with IL-1beta TNF-alphaor IFN-gamma TRAIL was induced in cultured fetal astrocytes. In particular, IFN-gammainduced the highest levels of TRAIL in cultured astrocytes. When astrocytes were pre-reated with IFN-gamma they induced apoptosis in TRAIL-sensitive Peer cells. Our results suggest that IFN-gamma modulates the expression of TRAIL in astrocytes, which may enhance cytotoxic sensitivity of infiltrating immune cells or brain cells other than astrocytes during inflammation of brain.
Tumor Necrosis Factor-alpha/genetics/*metabolism
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TNF-Related Apoptosis-Inducing Ligand
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Membrane Glycoproteins/genetics/*metabolism
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Interferon Type II/*pharmacology
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Humans
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Cells, Cultured
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Astrocytes/*cytology/drug effects/metabolism
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Apoptosis Regulatory Proteins/genetics/*metabolism
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Apoptosis/*drug effects/physiology
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Antineoplastic Agents/*pharmacology
7.Human Brain Astrocytes Mediate TRAIL-mediated Apoptosis after Treatment with IFN-gamma.
Jeonggi LEE ; Jeon Soo SHIN ; In Hong CHOI
Yonsei Medical Journal 2006;47(3):354-358
TNF-related apoptosis inducing ligand (TRAIL) expressions were studied in primary human brain astrocytes in response to pro-inflammatory cytokines. When astrocytes were treated with IL-1beta TNF-alphaor IFN-gamma TRAIL was induced in cultured fetal astrocytes. In particular, IFN-gammainduced the highest levels of TRAIL in cultured astrocytes. When astrocytes were pre-reated with IFN-gamma they induced apoptosis in TRAIL-sensitive Peer cells. Our results suggest that IFN-gamma modulates the expression of TRAIL in astrocytes, which may enhance cytotoxic sensitivity of infiltrating immune cells or brain cells other than astrocytes during inflammation of brain.
Tumor Necrosis Factor-alpha/genetics/*metabolism
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TNF-Related Apoptosis-Inducing Ligand
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Membrane Glycoproteins/genetics/*metabolism
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Interferon Type II/*pharmacology
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Humans
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Cells, Cultured
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Astrocytes/*cytology/drug effects/metabolism
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Apoptosis Regulatory Proteins/genetics/*metabolism
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Apoptosis/*drug effects/physiology
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Antineoplastic Agents/*pharmacology
8.Effect of shentong zhuyu decoction on pain behavior and spinal cord astrocytes model of osteocarcinoma pain.
Bing-xu REN ; Zheng-liang MA ; Yan-qing JIN
Chinese Journal of Integrated Traditional and Western Medicine 2011;31(3):381-385
OBJECTIVETo study the analgesic effect of shentong zhuyu decoction (SZD) and its effect on the expression of the spinal cord glial fibrillary acidic protein (GFAP).
METHODSOne hundred C3H/HeNCrlVr male mice were randomly divided into the normal group (n=8), the sham operation group (n=30), the model group (n=30), the Chinese medicine (CM) group 1 (n=8), the CM group 2 (n=8), the CM group 3 (n=8), and the vehicle group (n=8). 0.1 g crude drug of SZD/0.4 mL, 0.3 g crude drug of SZD/0.4 mL, 0.9 g crude drug of SZD/0.4 mL, and 0.4 mL normal saline were respectively given by gastrogavage to mice in CM 1, 2, 3 groups and the vehicle group, once daily for seven days starting from Day 14. The paw withdrawal thermal latency (PWTL), as the behavior indicator, was assessed in mice using radiant thermal stimulator. The lumbar enlargement of the spinal cord was taken after the behavioral test on Day 21. GFAP mRNA and protein expressions were detected using real-time quantitative RT-PCR and Western blot.
RESULTSCompared with the normal group (Day 0) (PWTL: 15.91 +/- 1.65 s) and the sham operation group (PWTL: Day 4: 13.33 +/- 1.44 s; Day 7: 11.28 +/- 0.61 s; Day 10: 15.47 +/- 2.46 s; Day 14: 15.69 +/- 1.98 s; Day 21: 15.69 +/- 1.68 s), the PWTL value in the model group (Day 4: 13.24 +/- 1.02 s; Day 7: 11.30 +/- 1.09 s; Day 10: 9.12 +/- 0.54 s; Day 14: 7.79 +/- 0.77 s; Day 21: 6.36 +/- 0.59 s) progressively decreased (P < 0.05) as time went by, while the spinal cord GFAP mRNA and protein expressions gradually increased. Compared with the normal group (Day 0) and the sham operation group (Day 14), the PWTL value in the CM groups and the vehicle group obviously decreased on Day 14 (P < 0.05). The PWTL value was not significantly different among the model group, CM groups, and the vehicle group on Day 14 (P > 0.05). On Day 21 the PWTL value of CM group 2 and 3 increased and the spinal cord GFAP mRNA and protein expression levels decreased when compared with the model group and the vehicle group (P < 0.05). But no significant difference in the PWTL value or GFAP expression levels was shown among the CM 1 group, the vehicle group, and the model group (P > 0.05).
CONCLUSIONSZD had analgesic effect. Inhibition of the proliferation and activation of the spinal cord astrocytes might be one of its mechanisms.
Animals ; Astrocytes ; cytology ; drug effects ; metabolism ; Bone Neoplasms ; complications ; psychology ; Drugs, Chinese Herbal ; pharmacology ; Glial Fibrillary Acidic Protein ; metabolism ; Male ; Mice ; Mice, Inbred C3H ; Osteosarcoma ; complications ; psychology ; Pain ; etiology ; metabolism ; Spinal Cord ; cytology ; metabolism
9.Morphine treatment enhances extracellular ATP enzymolysis and adenosine generation in rat astrocytes.
Wei LIU ; Zhan-li YANG ; Le-quan ZHOU ; Xiao-ying LI ; Fu-man YAN ; Li GUAN ; Hai-mei LIU ; Jian-qiang FENG
Acta Physiologica Sinica 2011;63(1):20-24
Recent studies have shown that astrocytes play important roles in ATP degradation and adenosine (a well known analgesic molecule) generation, which are closely related to pain signaling pathway. The aim of this study was to investigate whether morphine, a well known analgesic drug, could affect the speeds of ATP enzymolysis and adenosine generation in rat astrocytes. Intracellular calcium concentration ([Ca(2+)](i)) of astrocyte was measured by flow cytometry, and the time points that morphine exerted notable effects were determined for subsequent experiments. Cultured astrocytes were pre-incubated with morphine (1 μmol/L) and then were incubated with substrates, ATP and AMP, for 30 min. The speeds of ATP enzymolysis and adenosine generation were measured by high performance liquid chromatography (HPLC). The results showed that both 1.5 and 48 h of morphine pre-incubation induced maximal ATP enzymolysis speed in astrocytes among all the time points, and there was no statistical difference of ATP enzymolysis speed between morphine treatments for 1.5 and 48 h. As to adenosine, morphine pre-incubation for 1.5 h statistically increased adenosine generation, which was degraded from AMP, in cultured astrocytes compared with control group. However, no difference of adenosine generation was observed after 48 h of morphine pre-incubation. These results indicate that treatment of morphine in vitro dynamically changes the concentrations of ATP and adenosine in extracellular milieu of astrocytic cells. In addition, astrocyte can be regarded as at least one of the target cells of morphine to induce changes of ATP and adenosine levels in central nervous system.
Adenosine
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biosynthesis
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Adenosine Triphosphate
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metabolism
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Analgesics, Opioid
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pharmacology
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Animals
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Animals, Newborn
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Astrocytes
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cytology
;
drug effects
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metabolism
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Calcium
;
analysis
;
metabolism
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Cells, Cultured
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Cerebral Cortex
;
cytology
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Morphine
;
pharmacology
;
Rats
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Rats, Sprague-Dawley
10.Protective effect of musk extract on rat's cerebral cortical neurons with inflammatory injury.
Jin-Feng SHI ; Bo-Ai ZHANG ; Yan-Jie JIA
Chinese Journal of Integrated Traditional and Western Medicine 2010;30(6):625-629
OBJECTIVETo investigate the protective effects of musk extract (ME) and its possible mechanism on rat's cerebral cortical neurons with inflammatory injury induced by lipopolysaccharide (LPS).
METHODSNeurons and astrocytes from newborn rat cerebral cortex were cultured in vitro respectively, and the astrocyte conditioned medium (ACM), obtained by treating astrocytes with 10 mg/L LPS and different concentrations of ME for 24 h, was added in the culture fluid of neurons. The survival rate and apoptotic rate of neurons were measured by MTT method and AO/EB stain; and the changes of inflammatory factors in the ACM were determined by ELISA.
RESULTSThe survival rate (%) of neurons treated by ACM with ME in concentrations of 18 mg/L, 36 mg/L, 72 mg/L and 144 mg/L was 52.55 +/- 3.52, 55.77 +/- 2.36, 64.89 +/- 3.45 and 73.67 +/- 1.80, respectively, significantly higher than that in the model neurons (43.62 +/- 4. 51, P < 0.05), while the apoptotic rate (%) in them, 68.11 +/- 2.16, 44.27 +/- 3.68, 32.56 +/- 2.14 and 21.89 +/- 2.46, respectively, was significantly lower than that in model neurons (71.33 +/- 3.25, P < 0.05 or P < 0.01). Level of IL-6 was decreasing along with the raising of ME concentration in the ACM, showing a concentration-dependent state.
CONCLUSIONME shows apparent protective effect on neurons against inflammatory injury, especially in a high concentration (144 mg/L), which may be associated with the reduction of IL-6 secreted by astrocytes.
Animals ; Animals, Newborn ; Astrocytes ; cytology ; metabolism ; Cell Survival ; drug effects ; Cells, Cultured ; Cerebral Cortex ; cytology ; Fatty Acids, Monounsaturated ; chemistry ; Inflammation ; chemically induced ; prevention & control ; Interleukin-6 ; secretion ; Lipopolysaccharides ; Male ; Materia Medica ; pharmacology ; Neurons ; cytology ; Protective Agents ; pharmacology ; Rats ; Rats, Wistar