ABSTRACT
Traditional technology of cell disruption has become one of the bottlenecks restricting the industrialization of genetic engineering products due to its high cost and low efficiency. In this study, a novel bioprocess of phage lysis coupled with salting-out extraction (SOE) was evaluated. The lysis effect of T7 phage on genetically engineered Escherichia coli expressing κ-carrageenase was investigated at different multiplicity of infection (MOI), meanwhile the phage and enzyme released into the lysate were separated by SOE. It was found that T7 phage could lyse 99.9% of host cells at MOI = 1 and release more than 90.0% of enzyme within 90 min. After phage lysis, 87.1% of T7 phage and 71.2% of κ-carrageenase could be distributed at the middle phase and the bottom phase, respectively, in the SOE system composed of 16% ammonium sulfate and 20% ethyl acetate (w/w). Furthermore, κ-carrageenase in the bottom phase could be salted out by ammonium sulfate with a yield of 40.1%. Phage lysis exhibits some advantages, such as mild operation conditions and low cost. While SOE can efficiently separate phage and intracellular products. Therefore, phage lysis coupled with SOE is expected to become a viable alternative to the classical cell disruption and intracellular product recovery.
ABSTRACT
Melanoma differentiation associated gene-7 (MDA-7)/interleukin24 (IL-24) has been considered as a tumor-suppressor gene, which suppresses the growth and induces the apoptosis of cancer cells. In the present study, we investigated the effect and mechanisms of MDA-7/IL-24 regarding the inhibition of metastasis of HepG2 and BEL-7402 human hepatocellular carcinoma (HCC) cells in vitro. We established MDA-7/IL-24-overexpressing HepG2 and BEL-7402 cell lines and found that MDA-7/IL-24 overexpression inhibited tumor cell adhesion and invasion, and induced G2/M arrest in tumor cells. To explore its mechanism of action, western blotting and real-time-PCR assay were used to investigate the expression of E-cadherin, CD44, ICAM-1, matrix metalloproteinase (MMP)-2 and -9, CyclinB, Twist, survivin, p-ERK and p-Akt. ELISA assay was used to measure the secretion of TGF-ß, and a reporter gene assay was used to detected the transcriptional activity of NF-κB and AP-1 in HepG2 and BEL-7402 cells. The results showed that MDA-7/IL-24 overexpression decreased the expression of CD44, ICAM-1, MMP-2/-9, CyclinB, Twist, survivin, TGF-ß and p-Akt, transcriptional activity of NF-κB, and increased the expression of E-cadherin and p-ERK and transcriptional activity of AP-1 in HepG2 and BEL-7402 cells. Our results revealed that MDA-7/IL-24 mediated the inhibition of adhesion and invasion in HepG2 and BEL-7402 cells by suppressing metastasis-related gene expression. Thus, MDA-7/IL-24 may be used as a novel cancer-suppressor gene for the therapy of human HCC.
Subject(s)
Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Cell Adhesion/genetics , Interleukins/genetics , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Cell Cycle Checkpoints/genetics , Cell Division/genetics , Cell Line, Tumor , Down-Regulation , G2 Phase/genetics , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Hep G2 Cells , Humans , Interleukins/metabolism , NF-kappa B/genetics , Neoplasm Invasiveness , Transcription Factor AP-1/genetics , Transcription, Genetic , Transcriptional Activation , Transforming Growth Factor beta/geneticsABSTRACT
Lipase from Burkholderia cepacia was encapsulated inside zirconia particles by biomimetic mineralization of K2ZrF6 induced with protamine, a natural cationic protein. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FT-IR) were employed for the characterization of the novel immobilized lipase. SEM and TEM images showed that both the zirconia particles with and without lipase have good spherical structures with average particle sizes of 150 nm. Fluorescence microscopy demonstrated that the lipase was indeed encapsulated inside the zirconia particles. The maximum immobilization capacity of the zirconia particles was 0.15 units/mg under optimum immobilization conditions. Biochemical characterization showed that the encapsulated lipase could retain most of its initial activity. Compared with free lipase, the encapsulated lipase exhibited improved thermal, pH, and recycling stabilities. After 8 weeks of storage, no substantial loss in catalytic activity was observed for the encapsulated lipase. The conversion of the kinetic resolution of (R,S)-1-phenylethanol with vinyl acetate as acetyl donor catalyzed by zirconia-immobilized lipase reached 49.9% with higher ee(s) of 99.9% under the following optimal conditions: octane as solvent, 0.1M (R,S)-1-phenylethanol, 70 mg immobilized lipase, 180 rpm, 50 °C for 48 h. After 6 cycles (288 h), the conversion and ee(s) were still 43% and 85%, respectively.
Subject(s)
Benzyl Alcohols/isolation & purification , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Lipase/chemistry , Lipase/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Benzyl Alcohols/chemistry , Biomimetic Materials , Burkholderia cepacia/enzymology , Enzyme Stability , Kinetics , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Particle Size , Spectroscopy, Fourier Transform Infrared , Stereoisomerism , Vinyl Compounds , ZirconiumABSTRACT
Outward delayed rectifier potassium channel and outward transient potassium channel have multiple important roles in maintaining the excitability of hippocampal neurons. The present study investigated the effects of two bufadienolides, Resibufogenin (RBG) and Cinobufagin (CBG), on the outward delayed rectifier potassium current (IK) and outward transient potassium current (IA) in rat hippocampal neurons. RBG and CBG have similar structures and both were isolated from the venom gland of toad skin. RBG inhibited both IK and IA, whereas CBG inhibited IK without noticeable effect on IA. Moreover, at 1 µM concentration both RBG and CBG could alter some channel kinetics and gating properties of IK, such as steady-state activation and inactivation curves, open probability and time constants. These findings suggested that IK is probably a target of bufadienolides, which may explain the mechanisms of bufadienolides' pathological effects on central nervous system.
Subject(s)
Amphibian Venoms/pharmacology , Bufanolides/pharmacology , Potassium Channels/drug effects , Pyramidal Cells/drug effects , Animals , CA1 Region, Hippocampal/cytology , Cells, Cultured , Membrane Potentials/drug effects , Patch-Clamp Techniques , Potassium Channels/physiology , Pyramidal Cells/physiology , Rats , Rats, Sprague-DawleyABSTRACT
Voltage-gated sodium channels (VGSCs) play important roles in maintaining the excitability of hippocampal neurons. The present study investigated the effects of resibufogenin (RBG, a main component of bufadienolides) on voltage-gated sodium channel currents (I(Na)) in rat hippocampal neurons using whole-cell patch clamp recording. According to the results, RBG activated I(Na) in a concentration-dependent manner. RBG at 1 µM concentration could alter some channel kinetics of I(Na), such as activation thresholds, steady-state activation and inactivation curves, time constant of recovery, and activity-dependent attenuation of I(Na). RBG influenced peak amplitude, overshoot and half-width of the evoked single action potential, and simultaneously lessened the firing rate of evoked repetitive firing. These findings suggested that I(Na) is probably a target of RBG, which may explain the mechanisms for the pathological effects of RBG on central nervous system.
Subject(s)
Bufanolides/toxicity , Hippocampus/cytology , Neurons/drug effects , Neurons/physiology , Sodium Channel Agonists , Sodium Channels/physiology , Action Potentials/drug effects , Animals , Cells, Cultured , Ion Channel Gating/drug effects , Ion Channel Gating/physiology , Medicine, Chinese Traditional/methods , Neurons/cytology , Patch-Clamp Techniques/methods , Rats , Respiratory Mechanics/drug effectsABSTRACT
OBJECTIVE: To further investigate the neuroprotective effects of five isoflavonoids from Astragalus mongholicus on xanthine (XA)/ xanthine oxidase (XO)-induced injury to PC12 cells. METHODS: PC12 cells were damaged by XA/XO. The activities of antioxidant enzymes, MTT, LDH, and GSH assays were used to evaluate the protection of these five isoflavonoids. Contents of Bcl-2 family proteins were determined with flow cytometry. RESULTS: Among the five isoflavonoids including formononetin, ononin, 9, 10-dimethoxypterocarpan-3-O-beta-D-glucoside, calycosin and calycosin-7-O-glucoside, calycosin and calycosin-7-O-glucoside were found to inhibit XA/ XO-induced injury to PC12 cells. Their EC50 values of formononetin and calycosin were 0.05 microg/mL. Moreover, treatment with these three isoflavonoids prevented a decrease in the activities of antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), while formononetin and calycosin could prevent a significant deletion of GSH. In addition, only calycosin and calycosin-7-O-glucoside were shown to inhibit XO activity in cell-free system, with an approximate IC50 value of 10 microg/mL and 50 microg/mL. Formononetin and calycosin had no significant influence on Bcl-2 or Bax protein contents. CONCLUSION: Neuroprotection of formononetin, calycosin and calycosin-7-O-glucoside may be mediated by increasing endogenous antioxidants, rather by inhibiting XO activities or by scavenging free radicals.
Subject(s)
Astragalus Plant/chemistry , Isoflavones/pharmacology , Protective Agents/pharmacology , Xanthine Oxidase/pharmacology , Animals , Glucosides/chemistry , Glucosides/isolation & purification , Glucosides/pharmacology , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Isoflavones/chemistry , Isoflavones/isolation & purification , PC12 Cells , Protective Agents/isolation & purification , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Superoxide Dismutase/metabolism , Xanthine Oxidase/antagonists & inhibitorsABSTRACT
The neuroprotective effects of catalpol, an iridoid glycoside isolated from the fresh rehmannia roots, on the behavior and brain energy metabolism in senescent mice induced by d-galactose were assessed. Except control group, mice were subcutaneously injected with d-galactose (150 mg/kg body weight) for 6 weeks. From the fifth week, drug group mice were treated with catalpol (2.5, 5, 10 mg/kg body weight) and piracetam (300 mg/kg body weight) for the last 2 weeks. Behavioral changes including open field test and passive avoidance were examined after drug administration. To determine the brain damage, pathological alterations were measured by hematoxylin and eosin (HE) staining. The activities of lactate dehydrogenase (LDH), glutathione S-transferase (GSH-ST), glutamine synthetase (GS), creatine kinase (CK) in brain cortex and hippocampus were determined using different biochemical methods. Consistent with the cognition deficits, the activities of GSH-ST, GS and CK decreased while the activity of LDH increased in aging mice brain. Administration of catalpol for 2-weeks not only ameliorated cognition deficit, but also reversed the biochemical markers mentioned above and reduced the histological lesions in mouse brain. These results suggest that catalpol has protective effects on memory damage and energy metabolism failure in aging model mice and is worth testing for further preclinical study aimed for senescence or neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD).
Subject(s)
Energy Metabolism/drug effects , Galactose/antagonists & inhibitors , Galactose/toxicity , Glucosides/pharmacology , Iridoids/pharmacology , Memory Disorders/chemically induced , Aging/physiology , Aging/psychology , Animals , Avoidance Learning/drug effects , Brain/pathology , Brain Chemistry/drug effects , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Cognition/drug effects , Creatine Kinase/metabolism , Female , Glutathione/metabolism , Glutathione Transferase/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Indicators and Reagents , Iridoid Glucosides , L-Lactate Dehydrogenase/metabolism , Male , Memory Disorders/psychology , Mice , Motor Activity/drug effects , Plant Roots/chemistry , Rehmannia/chemistryABSTRACT
To characterize the roles played by surface-charged residue Asp44 in the structure stability of horse heart myoglobin, the code of Asp44, GAT, in the gene of horse heart myoglobin was changed into AAA for Lys by PCR site-directed mutagenesis. The mutant gene was ligated into PstI/BamHI-cut pGYM and the resulting plasmid was transformed into E. coli BL21. The mutant protein (D44K) was expressed in BL21 successfully. The bacteria containing mutant myoglobin were treated with lysozyme. Then the mutant protein was purified by ammonium sulfate precipitation, ion-exchange chromatography and gel filtration. Circular dichroism spectra were employed to monitor the kinetic behaviors of wild-type and mutant myoglobins' denaturation at different pHs or upon heating, and the "two-state" model was used to simulate the kinetic process of wild-type and mutant myoglobins' denaturation upon heating to determine the unfolding thermodynamic parameters of Mb and its mutant (D44K). The results show that the mutation of the surface-charged residue Asp44 to Lys44 can increase the protein's stability on its resistance to heat, resulting in the increase in the protein's denaturing mid-temperature by about 4 degrees C, from 71.9 to 75.1 degrees C, but shows little effect on its resistance to acid denaturation.
Subject(s)
Circular Dichroism/methods , Mutation , Myoglobin/analysis , Animals , Aspartic Acid/chemistry , Aspartic Acid/genetics , Horses , Hydrogen-Ion Concentration , Myoglobin/chemistry , Myoglobin/genetics , Myoglobin/metabolism , Protein Folding , Protein Stability , ThermodynamicsABSTRACT
A glia-mediated inflammation plays an important role in the pathogenesis of Alzheimer's disease (AD). In vitro, besides a direct neurotoxic effect on neurons, Abeta activates glia to produce an array of inflammatory factors including tumor necrosis factor-alpha (TNF-alpha), reactive oxygen species (ROS), nitric oxide (NO) and inducible nitric oxide synthase (iNOS), which accelerate the progression of AD. Catalpol, an iridoid glycoside, isolated from the root of Rehmannia glutinosa, protects neuronal cells from damage caused by a variety of toxic stimulus. In the present study, the effect of catalpol against Abeta(1-42)-induced neurotoxicity in primary cortical neuron-glia cultures as well as its mechanism acting on cells was further investigated. Pretreatment with catalpol at the dosage of 500 microM for 30 min prior to 5 microM Abeta(1-42) not only attenuated the Abeta(1-42)-triggered neurotoxicity to neurons but also inhibited the glial activation to some extent, which was examined by inspecting the morphological changes and measuring the release of the above mentioned inflammatory factors. Therefore, the results demonstrated that catalpol might be a promising anti-inflammatory agent in the therapy or prevention of neurodegenerative diseases associated with inflammation.
Subject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Anti-Inflammatory Agents/pharmacology , Cerebral Cortex/drug effects , Neurons/drug effects , Neuroprotective Agents/pharmacology , Peptide Fragments/antagonists & inhibitors , Quaternary Ammonium Compounds/pharmacology , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Amyloid beta-Peptides/toxicity , Animals , Animals, Newborn , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/therapeutic use , Cells, Cultured , Cerebral Cortex/metabolism , Cerebral Cortex/physiopathology , Coculture Techniques , Dose-Response Relationship, Drug , Encephalitis/drug therapy , Encephalitis/metabolism , Encephalitis/physiopathology , Gliosis/drug therapy , Gliosis/metabolism , Gliosis/physiopathology , Inflammation Mediators/metabolism , Mice , Molecular Structure , Neuroglia/drug effects , Neuroglia/metabolism , Neurons/metabolism , Neuroprotective Agents/chemistry , Neuroprotective Agents/therapeutic use , Nitric Oxide/metabolism , Peptide Fragments/toxicity , Quaternary Ammonium Compounds/chemistry , Quaternary Ammonium Compounds/therapeutic use , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia (A.) oxyphylla, showed the significant neuroprotective effects on hydrogen peroxide (H2O2) or MPP+-induced apoptosis in cultured PC12 cells. However, the mechanism how PCA suppresses rotenone-induced neurotoxicity in cultured PC12 cells remains unclear. In this study, we investigated the protective effects of PCA in PC12 cells exposed to rotenone as an in vitro model of mitochondrial dysfunction and apoptotic cell death. The apoptosis in rotenone-induced PC12 cells was accompanied by the loss of mitochondrial membrane potential, the formation of reactive oxygen species (ROS), the total glutathione depletion, activation of caspase-3 and down-regulation of Bcl-2. In contrast, PCA markedly attenuated the above-mentioned mitochondrial dysfunction in a dose-dependent manner. Taken together, these results suggest that treatment of PC12 cells with PCA can block rotenone-induced apoptosis via ameliorating the mitochondrial dysfunction.
Subject(s)
Anticarcinogenic Agents/pharmacology , Apoptosis/drug effects , Hydroxybenzoates/pharmacology , Mitochondria/drug effects , Rotenone/toxicity , Uncoupling Agents/toxicity , Animals , Caspase 3/metabolism , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , Cell Survival/drug effects , Dopamine/physiology , Flow Cytometry , Genes, bcl-2/drug effects , Genes, bcl-2/genetics , Glutathione/metabolism , Membrane Potentials/drug effects , Necrosis , Neurotoxicity Syndromes/pathology , PC12 Cells , Rats , Reactive Oxygen Species/metabolismABSTRACT
A new quinone compound, p-hydroxymethoxybenzobijuglone (HMBBJ), isolated from Juglans mandshurica by bioassay-guided fractionation, showed cytotoxic activity against HeLa cell line. Its chemical structure was determined by NMR and HREIMS spectra. In this paper, its ability to induce apoptosis in HeLa cells was studied for the first time. After treated with HMBBJ, the growth of HeLa cells was inhibited and cells displayed typical morphological apoptotic characteristics. Data from flow cytometry analysis showed that the HeLa cell cycle was arrested in the G2/M phase by HMBBJ, and the apoptotic rate of HeLa cells increased in a dose-dependent manner. Meanwhile, HMBBJ increased the expression of caspase-8, -3 and Bax, decreased the expression of Bcl-2, and lowered the DeltaPsi(m). These findings reveal that HMBBJ could efficiently induce HeLa cells apoptosis through mitochondria dependent pathway and activation of the caspase cascade, and it may be a potential chemotherapeutic candidate for the treatment of cancer.
Subject(s)
Apoptosis/drug effects , Benzoquinones/pharmacology , Caspase 3/metabolism , Caspase 8/metabolism , Female , Flow Cytometry , HeLa Cells , Humans , Membrane Potentials/drug effects , Mitochondria/drug effects , Mitochondria/physiology , Proto-Oncogene Proteins c-bcl-2/metabolism , Uterine Cervical Neoplasms/pathology , bcl-2-Associated X Protein/metabolismABSTRACT
The neuroprotective effects of catalpol, an iridoid glycoside present in the roots of Rehmannia glutinosa, on 1-methyl-4-phenylpyridinium (MPP(+))-induced oxidative stress in cultured mesencephalic neurons, especially dopaminergic neurons, were investigated. Exposure of mesencephalic neurons to 10microM MPP(+) induced a leakage of lactate dehydrogenase (LDH) and decreased cell viability, measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Catalpol increased neuron viability and markedly attenuated MPP(+)-induced dopaminergic neuron death in a dose-dependent manner. In order to clarify the neuroprotective mechanism of catalpol, mitochondrial function, the activities of endogenous antioxidants and the lipid peroxide content were measured. The results indicated that catalpol prevented the MPP(+)-induced inhibition of complex I activity and the loss of mitochondrial membrane potential. In addition, catalpol reduced the content of lipid peroxide and increased the activity of glutathione peroxidase and superoxide dismutase. Taken together, the above results suggest that catalpol may be a candidate drug for the treatment of oxidative stress-induced neurodegenerative disease.
Subject(s)
1-Methyl-4-phenylpyridinium , Glucosides/pharmacology , Iridoids/pharmacology , Neurons/drug effects , Neuroprotective Agents/pharmacology , Animals , Glutathione Peroxidase/metabolism , Iridoid Glucosides , Membrane Potential, Mitochondrial/drug effects , Mesencephalon/embryology , Mesencephalon/pathology , Mesencephalon/physiology , Mice , Neurons/pathology , Neurons/physiology , Oxidative Stress/drug effects , Superoxide Dismutase/metabolismABSTRACT
Rehmannia, a traditional Chinese medical herb, has a long history in age-related disease therapy. Previous work has indicated that catalpol is a main active ingredient performing neuroprotective effect in rehmannia, while the mechanism underlying the effect remains poorly understood. In this study, we attempt to investigate the effect of catalpol on presynaptic proteins and explore a potential mechanism. The hippocampal levels of GAP-43 and synaptophysin in 3 groups of 4 months (young group), 22-24 months (aged group) and catalpol-treated 22-24 months (catalpol-treated group) rats were evaluated by western blotting. Results clearly showed a significant decrease in synaptophysin (46.6%) and GAP-43 (61.4%) levels in the aged group against the young animals and an increase (45.0% and 31.8% respectively) in the catalpol-treated aged rats in comparison with the untreated aged group. In particular, synaptophysin immunoreactivity (OD) in the dentate granule layer of the hippocampus was increased 0.0251 in the catalpol-treated group as compared with the aged group. The study also revealed a catalpol-associated increase of PKC and BDNF in the hippocampus of the catalpol-treated group in comparison with the aged rats and highly correlated with synaptophysin and GAP-43. Such positive correlations between presynaptic proteins and signaling molecules also existed in the young group. These results suggested that catalpol could increase presynaptic proteins and up-regulate relative signaling molecules in the hippocampus of the aged rats. Consequently, it seemed to indicate that catalpol might ameliorate age-related neuroplasticity loss by "normalizing" presynaptic proteins and their relative signaling pathways in the aged rats.
Subject(s)
Aging/metabolism , Brain-Derived Neurotrophic Factor/drug effects , Glucosides/pharmacology , Hippocampus/drug effects , Iridoids/pharmacology , Neuronal Plasticity/drug effects , Protein Kinase C/drug effects , Aging/drug effects , Analysis of Variance , Animals , Brain-Derived Neurotrophic Factor/metabolism , Drugs, Chinese Herbal/pharmacology , GAP-43 Protein/drug effects , GAP-43 Protein/metabolism , Hippocampus/cytology , Hippocampus/metabolism , Iridoid Glucosides , Male , Neuroprotective Agents/pharmacology , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley , Statistics, Nonparametric , Synaptophysin/drug effects , Synaptophysin/metabolism , Up-RegulationABSTRACT
Our previous study described the neuroprotective effects of catalpol in gerbil ischemic model, in which catalpol was shown to prevent hippocampal neurons from death and ameliorate the cognitive ability of the animals. In the study, we focused on investigating the neuroprotective mechanism of catalpol. Animals were randomly assigned three groups as sham-operated, ischemia-treated with saline and ischemia-treated with catalpol. Transient global ischemia was produced by a 5 min occlusion of the bilateral common carotid arteries. Catalpol was intraperitoneally injected at the dose of 5 mg/kg immediately after reperfusion and repeatedly at 12, 24, 48 and 72 h. Histology as well as immunohistochemistry and TUNEL (the terminal deoxynucleotidyl transferase-mediated UTP nick end label) analysis were performed on serial slices through the dorsal hippocampus after gerbils were sacrificed. The results showed that 5 min transient global ischemia followed by 4 days reperfusion caused significant increases in TUNEL-positive and Bax-positive cells in hippocampal CA1 subfield. Catalpol not only significantly reduced TUNEL-positive and Bax-positive cells but also significantly increased Bcl-2-positive cells. All these suggested that catalpol could effectively inhibit apoptosis by modulating the expressions of Bcl-2 and Bax genes.
Subject(s)
Apoptosis/drug effects , Brain Ischemia/drug therapy , Glucosides/pharmacology , Iridoids/pharmacology , Neuroprotective Agents/pharmacology , Proto-Oncogene Proteins c-bcl-2/drug effects , bcl-2-Associated X Protein/drug effects , Animals , Apoptosis/physiology , Brain Infarction/drug therapy , Brain Infarction/enzymology , Brain Infarction/physiopathology , Brain Ischemia/enzymology , Brain Ischemia/physiopathology , Disease Models, Animal , Drug Administration Schedule , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/therapeutic use , Female , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/physiology , Gerbillinae , Glucosides/therapeutic use , Hippocampus/drug effects , Hippocampus/enzymology , Hippocampus/physiopathology , Immunohistochemistry , In Situ Nick-End Labeling , Iridoid Glucosides , Iridoids/therapeutic use , Male , Nerve Degeneration/drug therapy , Nerve Degeneration/enzymology , Nerve Degeneration/physiopathology , Neuroprotective Agents/therapeutic use , Proto-Oncogene Proteins c-bcl-2/metabolism , Reperfusion Injury/drug therapy , Reperfusion Injury/enzymology , Reperfusion Injury/physiopathology , Treatment Outcome , bcl-2-Associated X Protein/metabolismABSTRACT
In this study, the neuroprotective effects of Alpinia protocatechuic acid (PCA), a phenolic compound isolated from the dried fruits of Alpinia Oxyphylla Miq. was found. The protective effect of Alpinia PCA against H2O2-induced oxidative damage on PC12 cells was investigated by measuring cell viability via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Rats were injected intraperitoneally with Alpinia PCA at a dose of 5mg/kg per day for 7 days, behavioral testing was performed in Y-maze. In order to make clear the neuroprotective mechanism of Alpinia PCA, the activities of endogenous antioxidants and the content of lipid peroxide in brain were assayed. The results proved that Alpinia PCA significantly prevented the H2O2-induced reduction in cell survival, improved the cognition of aged rats, reduced the content of lipid peroxide, increased the activity of glutathione peroxidase and superoxide dismutase. All these suggested that Alpinia PCA was a potential neuroprotective agent and its neuroprotective effects were achieved at least partly by promoting endogenous antioxidant enzymatic activities and inhibiting free radical generation.
Subject(s)
Alpinia/chemistry , Antioxidants/pharmacology , Hydroxybenzoates/pharmacology , Neuroprotective Agents/pharmacology , Oxidative Stress/drug effects , Animals , Antioxidants/metabolism , Avoidance Learning/drug effects , Brain/metabolism , Cell Survival/drug effects , Drugs, Chinese Herbal/pharmacology , Glutathione Peroxidase/metabolism , Hydrogen Peroxide/toxicity , Lipid Peroxides/metabolism , Male , Maze Learning/drug effects , Oxidation-Reduction , PC12 Cells , Rats , Rats, Sprague-Dawley , Superoxide Dismutase/metabolismABSTRACT
The neuroprotective effects of protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia oxyphylla, on hydrogen peroxide (H(2)O(2))-induced apoptosis and oxidative stress in cultured PC12 cells were investigated. Exposure of PC12 cells to 0.4 mM H(2)O(2) induced a leakage of lactate dehydrogenase (LDH) and decreased cell viability denoted by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PCA increased PC12 cellular viability and markedly attenuated H(2)O(2)-induced apoptotic cell death in a dose-dependent manner. By flow cytometric analysis, PCA showed its significant effect on protecting PC12 cells against H(2)O(2)-induced apoptosis. In these cells, the levels of glutathione (GSH) and activity of catalase were augmented, while glutathione peroxidase activity remained unchanged. In addition, PCA also protected against cell damage induced by H(2)O(2) and Fe(2+), which generated hydroxyl radicals (OH) by the Fenton reaction. These results suggest that PCA may be a candidate chemical for the treatment of oxidative stress-induced neurodegenerative disease.
Subject(s)
Alpinia/chemistry , Apoptosis/drug effects , Hydrogen Peroxide/toxicity , Hydroxybenzoates/pharmacology , Analysis of Variance , Animals , Catalase/metabolism , Cell Survival/drug effects , DNA/metabolism , Dose-Response Relationship, Drug , Ferrous Compounds/toxicity , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Hydroxybenzoates/chemistry , Molecular Structure , PC12 Cells , Plant Extracts/chemistry , Plant Extracts/pharmacology , RatsABSTRACT
OBJECTIVE: To study the protective effects of flavonoids from Cuscuta chinensis (CF) on oxidative stress in cultured PC12 cells and investigate the mechanism of the effects. METHODS: The cell viability was analyzed by MTT method and the radical scavenging activity of CF was examined by DPPH (1, 1-diphenyl-2-picrylhydrazyl). The morphological changes were observed by Hoechst 33258 staining assay, and the apoptosis rate of PC12 cells was detected by propidium iodide stain flow cytometry (FCM). RESULTS: Application with 0.3-0.5 mM H2O2 induced a dose and time dependent viability loss in PC12 cells; Treatment with 0.5 mM H2O2 for 24 h was shown to cause nearly 50% viabliity loss and apoptosis in PC12 cells; Pretreatment with different concentrations of CF for 0.5 h increased the survival rate of PC12 cells, inhibited apoptosis induced by H2O2; CF had the activity of scavenging free radicals generated by DPPH in a dose-dependent manner. CONCLUSION: CF can protect PC12 cells against oxidative stress. The mechanism of it may be the ability of scavenging ROS and increasing the activity of antioxidant enzyme.
Subject(s)
Apoptosis/drug effects , Cuscuta/chemistry , Flavonoids/pharmacology , Free Radical Scavengers/pharmacology , PC12 Cells/drug effects , Animals , Cell Survival/drug effects , Dose-Response Relationship, Drug , Flavonoids/administration & dosage , Flow Cytometry , Free Radicals/metabolism , Hydrogen Peroxide , Oxidative Stress/drug effects , PC12 Cells/pathology , Plants, Medicinal/chemistry , RatsABSTRACT
OBJECTIVE: To evaluate the antioxidant activities of different chemical constituents from Astragalus mongholicus Bunge and their protection against xanthine (XA)/xanthine oxidase (XO)-induced toxicity in PC12 cells. METHODS: The compounds of Astragalus mongholicus Bunge were isolated by chromatography and the structures were elucidated on the basis of spectral data interpretation. Their antioxidant activities were detected by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities in a cell-free system. Meanwhile, the effects against XA/XO-induced toxicity were assessed using MTT assay in PC12 cells. RESULTS: Ten principal constituents were isolated and identified as formononetin (I), ononin (II), calycosin (III), calycosin-7-O-beta-D-glucoside (IV), 9,10-dimethoxypterocarpan-3-O-beta-D-glucoside (V), adenosine (VI), pinitol (VII), daucosterol (VIII), beta-sitoster (IX) and saccharose (X) from Astragalus mongholicus Bunge. The compounds I, III, and IV scavenged DPPH free radicals in vitro. Formononetin and calycosin were found to inhibit XA/XO-induced cell injury significantly, with an estimated EC50 of 50 ng/mL. CONCLUSION: Compound II, VI, and VII are first reported in this plant. Calycosin exhibits the most potent antioxidant activity both in the cell-free system and in the cell system.
Subject(s)
Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Free Radical Scavengers/chemistry , Free Radical Scavengers/pharmacology , Animals , Astragalus Plant/chemistry , Astragalus propinquus , Free Radicals/metabolism , Isoflavones/chemistry , Isoflavones/pharmacology , PC12 Cells , Rats , Xanthine/toxicity , Xanthine Oxidase/toxicityABSTRACT
Catalpol, an iridoid glycoside, contained richly in the roots of Rehmannia glutinosa, was found for the first time to be of neuroprotection in gerbils subjected to transient global cerebral ischemia. Catalpol (1 mg/kg ip) used immediately after reperfusion and repeatedly at 12, 24, 48 and 72 h significantly rescued neurons in hippocampal CA1 subfield and reduced working errors during behavioral testing. The neuroprotective efficacy of catalpol became more evident when the doses of catalpol were increased to 5 and 10mg/kg. In addition, it was exciting that the significant neuroprotection by catalpol was also evident when catalpol was applied up to 3 h after ischemia. But the neuroprotective efficacy of catalpol became weak when catalpol was given at 6h after ischemia. Of great encouragement was the finding that the neuroprotection of catalpol could be seen not only in a short post-ischemic period (12 days) but also in a long period (35 days). All these indicated that catalpol was truly neuroprotective rather than simply delayed the onset of neuronal damage and might be of therapeutic value for the treatment of global cerebral ischemia.
Subject(s)
Behavioral Symptoms/drug therapy , Glucosides/therapeutic use , Hippocampus/cytology , Iridoids/therapeutic use , Neurons/drug effects , Neuroprotective Agents/therapeutic use , Reperfusion Injury/drug therapy , Analysis of Variance , Animals , Behavioral Symptoms/physiopathology , Dose-Response Relationship, Drug , Gerbillinae , Glucosides/chemistry , Glucosides/pharmacology , Hippocampus/drug effects , Iridoid Glucosides , Iridoids/chemistry , Iridoids/pharmacology , Neuroprotective Agents/chemistry , Neuroprotective Agents/pharmacology , Reperfusion Injury/physiopathology , Time FactorsABSTRACT
A azoreductase gene with 537 bp was obtained by PCR amplification from Rhodobacter sphaeroides AS1.1737. The enzyme, with a molecular weight of 18.7 kD, was efficiently expressed in Escherichia coli and its biodegradation characteristics for azo dyes were investigated. Furthermore, the reaction kinetics and mechanism of azo dyes catalyzed by the genetically engineered azoreductase were studied in detail. The presence of a hydrazo-intermediate was identified, which provided a convincing evidence for the assumption that azo dyes were degraded via an incomplete reduction stage.