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Objective To evaluate effects of ascorbic acid on proliferative activity of cultured melanocytes in vitro, as well as on H2O2?induced oxidative injury in melanocytes. Methods The optimal concentration of ascorbic acid solution and median lethal dose of H2O2 solution were determined by CCK?8 assay for the following experiment. Cultured melanocytes were classified into the control group, ascorbic acid group, H2O2 group and combination group. During the first 24 hours, the control group and H2O2 group were treated with M254 medium, while the ascorbic acid group and combination group with ascorbic acid solution. During an additional 24?hour period, the control group and ascorbic acid group were treated with M254 medium, while the H2O2 group and combination group with H2O2 solution at the median lethal dose. After 48?hour treatment, CCK?8 assay and flow cytometry were performed to determine the survival rate and apoptosis rate of melanocytes, respectively, in the 4 groups. Biochemical methods were used to evaluate the superoxide dismutase(SOD)activity and determine the malondialdehyde(MDA)concentration, and fluores?cent staining was conducted to detect the level of reactive oxygen species(ROS)in the control group, H2O2 group and combination group. Results The optimal concentration of ascorbic acid solution was 1 000μmol/L, and the median lethal dose of H2O2 solution was 300 μmol/L. The cell survival rate, apoptosis rate, SOD activity, MDA concentration and ROS fluorescence intensity in the control group were 100% ± 4.99%, 6.90% ± 0.87%, 54.71 ± 4.75 U/mgprot, 263.39 ± 20.17 nmol/mgprot and 342.16 ± 27.36 respectively. Compared with the control group, H2O2 solution could significantly increase the cell apoptosis rate(16.47%± 1.07%), SOD activity(103.62 ± 10.44 U/mgprot), MDA concentration(493.70 ± 31.36 nmol/mgprot)and intracellular ROS fluorescence intensity (782.48 ± 36.25), but decrease the survival rate of melanocytes (39.07% ± 2.94%), while ascorbic acid solution markedly down?regulated the H2O2?induced apoptosis (11.83%± 0.95%), SOD activity(76.73 ± 5.20 U/mgprot), MDA concentration(371.82 ± 23.05 nmol/mgprot) and ROS level (475.64 ± 52.18), but increased the cell survival rate (74.31% ± 5.53%). Conclusion Ascorbic acid solution at the concentration of 1 000 μmol/L can not only promote proliferative activity of melanocytes, but also protect melanocytes from H2O2?induced oxidative injury.
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Neuroinflammation may be one of the causes in the pathogenesis of Alzheimer's disease(AD).Epidemiological studies suggest that long-term use of nonsteroidal anti-inflammatory drugs(NSAIDs)can reduce the risk of AD.Laboratory evidence indicates that the protection of NSAIDs is mediated by inhibition of cyclooxygenase(COX)activity and the non-COX mechanisms.This review summarizes the possible underlying mechanisms in the action.
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Aim To investigate the effect of ultra low molecular weight heparin(ULMWH)to protect primarily cultured rat cortical neurons from the deleterious effects of the neurotoxicant glutamate (Glu)and explore the related mechanism.Methods Cortical neurons of fetal rats were cultured carefully in vitro and treated with 100 ?mol?L-1 Glu so that the protective effects of ULMWH were observed thoroughly. And then the viability of cortical neurons, morphological change together with the number of apoptotic neurons,and the concentration of intracellular free Ca2+([Ca2+]i)were measured by MTT assay, Hoechst33258 staining and Fura-2/AM double wavelength fluoremetry, respectively.Results Brief exposure of cultures to 100 ?mol?L-1Glu led to extensive neuronal death and rapid increase of [Ca2+]i.Pretreatment with ULMWH over the concentration range of 0.01~1 mg?L-1 significantly inhibited the Glu-induced neuronal cell death assessed by a MTT assay and the number of apoptotic nuclei, evidenced by Hoechst 33258 staining.Glu-induced elevation of[Ca2+]i was decreased by pretreatment with 1 mg?L-1 ULMWH, which was indicated by Fura-2/AM not only in assay medium containing Ca2+ but also in Ca2+-free assay medium.Conclusions Those results suggest that ULMWH protects the cultured neurons from Glu-induced neurotoxicity, which may be attributed to its alleviating intracellular free Ca2+ overload via suppressing intracellular Ca2+ release from internal stores induced by Glu.
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Aim To investigate the protective effects of LMWH-SOD(Low molecular weight heparin- Superoxide dismutase Conjugate)on the injuries induced by oxygen and glucose deprivation in cultured neurons. Methods The cortical neurons of fetal rat were cultured in vitro. The antioxidant and protective effects of LMWH-SOD were observed by treating neurons with oxygen and glucose deprivation. Results LMWH-SOD reduced the number of cell death and the efflux of LDH and the content of NO,MDA and increased the membrane fluidity after the injuries of cells. Conclusion LMWH-SOD has protective effects on cerebral cortical neurons through its action of scavenging free radicals.
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This article reviews the proteins and transduction pat hw ay in endotoxic action. Lipopolysaccharide-binding protein(LBP) binds and trans ports the LPS to its receptors, which include CD14 and CD11/CD18. Toll-like rec eptors(TLRs) is closely associated with transducting the signals into cytoplasm. Scavenger receptors are related to hepatic clearance on endotoxin .The intracel lular signal transduction is involved in several paths which finally leads to t he release of cytokines.
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<p><b>OBJECTIVE</b>To investigate the antagonistic effect and mechanism of the effect of cyproheptadine (Cyp) on endotoxic shock in rats.</p><p><b>METHODS</b>Endotoxic shock was produced in rats by i.v. injection of lipopolysaccharides (LPS) (5 mg/kg). Tumor necrosis factor (TNF(alpha)) mRNA expression was assessed by Northern blot. Plasma TNF(alpha) content was measured by radioimmunoassay. Plasma superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured. The intracellular free calcium concentration ([Ca(2+)](i)) in single endothelial cells was determined by laser scanning confocal microscopy (LSCM).</p><p><b>RESULTS</b>Cyp 5 mg/kg injected immediately after i.v. LPS raised the mean arterial blood pressure (MABP) of shocked rats and improved their 24 h survival rate. Meanwhile, Cyp markedly decreased TNF(alpha) mRNA levels in rat liver (18 +/- 10 vs. LPS + saline 38 +/- 10, P < 0.01) as well as plasma TNF(alpha) content [(7.8 +/- 2.4) microg/L vs. LPS + saline (21.5 +/- 3.2) microg/L, P < 0.01)]. It enhanced plasma SOD activity [(1037.2 +/- 112.8) NU/L vs LPS + saline (615.4 +/- 92.6) NU/L, P < 0.01], reduced the MDA content [(5.2 +/- 1.1) micromol/L vs. LPS + saline (9.8 +/- 1.5) micromol/L, P < 0.01], and inhibited TNF(alpha)-induced [Ca(2+)](i) elevation.</p><p><b>CONCLUSION</b>Cyp exerts an anti-endotoxic shock effect by inhibiting TNF(alpha) gene expression, enhancing SOD activity, reducing lipid peroxidation, and preventing [Ca(2+)](i) overload.</p>
Subject(s)
Animals , Rats , Cyproheptadine , Pharmacology , Histamine H1 Antagonists , Pharmacology , Malondialdehyde , Blood , Rats, Wistar , Shock, Septic , Drug Therapy , Metabolism , Superoxide Dismutase , Blood , Tumor Necrosis Factor-alpha , GeneticsABSTRACT
AIM: To study the effects of cyproheptadine (Cyp) and anisodamine (Ani)on the changes of intracellular free Ca 2+ concentration ([Ca 2+ ] i) induced by tumor necrosis factor (TNF ?) in single endothelial cells, and to explore the mechanisms of TNF ?-mediated shock and antishock actions of Cyp and Ani. METHODS: Human umbilical vein endothelial cell strains(ECV304) were seed in 35 mm tissue culture dish with 2 mL DMEM culture medium. The cultured cells were loaded by Fluo-3/AM. The spatial distribution and the dynamic changes of [Ca 2+ ] i in single endothelial cell was determined by laser scanning confocal microscopy(LSCM). RESULTS: [Ca 2+ ] i in single endothelial cell after stimulation of TNF ? rapidly increased in a dose-dependent manner and approached the peak value within 60 seconds, afterwards, decreased and kept above the basal level. The confocal scanning image showed that [Ca 2+ ] i elevation was more obvious in nuclear than in cytoplasma, and decreased slowly. Cyp (3?10 -5 , 6?10 -5 mol/L) and Ani (2?10 -5 , 4?10 -5 mol?L -1 ) markedly inhibited TNF ? (1.2?10 -9 mol?L -1 )-induced [Ca 2+ ] i elevation. CONCLUSIONS: TNF ? markedly induces elevation of [Ca 2+ ] i in single endothelial cell, it may be an important mechanism of TNF ?-induced shock and tissue injury. Cyp and Ani obviously suppress TNF ?-induced [Ca 2+ ] i elevation, which probably is one of the mechanisms of their antishock effects.
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Melatonin (MT) is secreted by the pineal gland and has obvious biological rhythmicity including circadian, season rhythm and life rhythm (aging clock). The reduction of MT secretion is related to body aging, particularly in close relation to brain aging. The hypothesis of aging is involved in pineal calcification, biological clock, neuro-en-docrinoimmunology, and free radical damage. MT is an endogenous free radical scavenger, may anto-gonize the attack of hydroxyl free radical ( ?OH)on organism and glutamate (Glu) excitotoxicity, and has a potent protective effect of central nervous system. In vivo studies showed that the food restriction and exogenous MT could obviously prolong life, postpone aging, and reduce the chances of age-related diseases. Investigating of MT anti-aging effect shows a vast prospect.
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T-type calcium channels present in cardiovascular, neuronal and endocrine systems, and they are now receiving attention as novel therapeutic targets. It plays important roles in multiple cellular functions and genes those encode the T-type calcium channels have been recently reported. Many drugs and compounds non-specifically block T-type calcium channels. We review circumstances of the research of T- type calcium channels in the molecular structure, distribution, function, regulation and the related drugs.
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AIM To observe the effects of melatonin on acute and chronic injuries induced by oxygen and glucose deprivation (OGD) in co-cultured neuron and glia and to explore the probable mechanisms of melatonin in antagonizing the injuries. METHODS The injury model of cultured neuron and co-cultured neuron and glia was made by administration of sodium dithionite and glucose-deprived Earles solution. In neuron and glia co-culture, two different models, acute injury model at the phase of OGD and chronic injury model after 'reperfusion' were established. The levels of nitric oxide (NO) and the activity of lactate dehydrogenase (LDH) were measured by Griess reagent and LDH kits respectively. The content of malondialdehyde (MDA) was determined by TBA method. Cell viability was analyzed using colorimetric MTT assay. RESULTS Melatonin increased the level of NO at the concentration of 10 -6 , 10 -7 mol?L -1 and decreased the level of MDA content elevated by OGD at the concentration of 10 -6 , 10 -7 , 10 -8 mol?L -1 in vitro cultured cortical neurons. In the chronic injury model after 'reperfusion' melatonin (10 -6 , 10 -7 , 10 -8 mol?L -1 ) significantly decreased LDH activity and increased MTT value in neurons and glia co-cultured. But in the acute injury model, melatonin obviously increased LDH activity and decreased MTT value. CONCLUSION Melatonin protection for neuron from injuries induced by oxygen and glucose deprivation may be related to increase in the level of NO and decrease in the content of MDA. Melatonin can antagonize the injury in the chronic injury model after 'reperfusion', but exaggerate the injury in the acute injury model. These may be all related to its antioxidant action. Our results also suggest that melatonin may probably inhibit activation of microglia.
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The mechanism of killer toxin is believed to relate to the inhibition of yeast cell wall ? glucan synthesis. Mannoprotein probably is one of the receptor for HM 1 killer toxin. The resistance is due to the decrease of N glycosylation of the sensitive yeast strain. HM 1 is a hydrophilic protein consisting of 88 amino acids. Two arginine residues located at positions 82 and 86 in the C terminal region of molecule are essential for the action of killer activity of HM 1 toxin. It is very useful to develop a novel anti fungal agent and prevent wild yeast contamination.