ABSTRACT
Febrile seizures (FS) are the most common seizure disorders in children aged 6 months to 5 years. Children suffering from complex FS have a high risk of developing subsequent temporal lobe epilepsy (TLE). Neuroinflammation is involved in the pathogenesis of FS although the mechanism remains unknown. Our previous study using the Whole Rat Genome Oligo Microarray determined that Dipeptidyl peptidase IV (DPP4) is potentially a related gene in FS rats. In this study, we demonstrated that DPP4 expression was significantly increased at both the protein and mRNA levels after hyperthermia induction. Sitagliptin, a specific enzyme inhibitor of DPP4, remarkably attenuated the severity of seizures in FS rats, and hyperthermia-induced astrocytosis was suppressed after DPP4 inhibition. Furthermore, sitagliptin significantly decreased the levels of the inflammatory cytokines IL-1ß, TNF-α, and IL-6 but not IL-10. In addition, sitagliptin prevented NF-κB activation by decreasing phosphorylation of the p65 subunit. Taken together, our findings demonstrate that DPP4 functions as a critical regulator of neuroinflammation in hyperthermia-induced seizures and the DPP4 inhibitor may be a viable option for FS therapeutics.
Subject(s)
Dipeptidyl Peptidase 4/immunology , Dipeptidyl-Peptidase IV Inhibitors/therapeutic use , Inflammation/drug therapy , Inflammation/immunology , Seizures, Febrile/drug therapy , Seizures, Febrile/immunology , Sitagliptin Phosphate/therapeutic use , Animals , Dipeptidyl Peptidase 4/genetics , Disease Models, Animal , Hyperthermia, Induced/adverse effects , Inflammation/etiology , Inflammation/genetics , NF-kappa B/immunology , Rats , Rats, Sprague-Dawley , Seizures, Febrile/etiology , Seizures, Febrile/genetics , Up-Regulation/drug effectsABSTRACT
Epigenetic modifications including histone modifications are associated with seizure development and epileptogenesis; however, its underlying mechanism remains to be elucidated. Dipeptidyl peptidase 4 (DPP4) and IL6 are identified as febrile seizure (FS)-related genes using gene microarray analysis in hyperthermia prone (HP) rats. This purpose of the study focused on exploring whether epigenetic modifications marker histone H3 lysine 27 trimethylation (H3K27me3)-regulated DPP4 and IL6 expression further affected seizures development. Herein, we reported broad between-group differences in the global levels of H3K27me3 with increased seizure severity in vivo. Using chromatin immunoprecipitation (ChIP), we identified markedly decreased H3K27me3 enrichment at their promoters of DPP4 and IL6 in vivo. We further showed that hyperthermia significantly decreased protein levels of H3K27me3, increased mRNA levels of DPP4 and IL6 by decreasing H3K27me3 enrichment at their promoters of DPP4 and IL6 in vitro. Importantly, H3K27me3 loss via enhancer of zeste homolog 2 (EZH2) knockdown promoted expression of DPP4 and IL6 via the same mechanism in vitro. EZH2 knockdown also increased neuronal firing frequency in vitro and FS susceptibility in vivo companied with upregulation expression of DPP4 and IL6. Taken together, our study provided the first evidence that hyperthermia-induced decreased of H3K27me3 promoted seizure susceptibility via regulating the expression pattern of DPP4 and IL6. These findings suggested that the methylation level of H3K27me3 might be a key regulator of seizure susceptibility.
Subject(s)
Genetic Predisposition to Disease/genetics , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Seizures/genetics , Seizures/metabolism , Animals , Cell Line , Cells, Cultured , Female , Hyperthermia, Induced/adverse effects , Hyperthermia, Induced/trends , Male , Methylation , Rats , Rats, Sprague-Dawley , Seizures/etiologyABSTRACT
Previous studies have demonstrated that the early suppression of HIF-1α after hypoxia-ischemia (HI) injury provides neuroprotection. Vitexin (5, 7, 4-trihydroxyflavone-8-glucoside), an HIF-1α inhibitor, is a c-glycosylated flavone that has been identified in medicinal plants. Therefore, we hypothesized that treatment with vitexin would protect against HI brain injury. Newborn rat pups were subjected to unilateral carotid artery ligation followed by 2.5 h of hypoxia (8% O2 at 37 °C). Vitexin (30, 45 or 60 mg/kg) was administered intraperitoneally at 5 min or 3 h after HI. Vitexin, administered 5 min after HI, was neuroprotective as seen by decreased infarct volume evaluated at 48 h post-HI. This neuroprotection was removed when vitexin was administered 3 h after HI. Neuronal cell death, blood-brain barrier (BBB) integrity, brain edema, HIF-1α and VEGF protein levels were evaluated using a combination of Nissl staining, IgG staining, brain water content, immunohistochemistry and Western blot at 24 and 48 h after HI. The long-term effects of vitexin were evaluated by brain atrophy measurement, Nissl staining and neurobehavioral tests. Vitexin (45 mg/kg) ameliorated brain edema, BBB disruption and neuronal cell death; Upregulation of HIF-1α by dimethyloxalylglycine (DMOG) increased the BBB permeability and brain edema compared to HI alone. Vitexin attenuated the increase in HIF-1α and VEGF. Vitexin also had long-term effects of protecting against the loss of ipsilateral brain and improveing neurobehavioral outcomes. In conclusion, our data indicate early HIF-1α inhibition with vitexin provides both acute and long-term neuroprotection in the developing brain after neonatal HI injury.
Subject(s)
Apigenin/pharmacology , Brain/drug effects , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Ischemia, Brain/drug therapy , Neuroprotective Agents/pharmacology , Animals , Animals, Newborn , Apigenin/chemistry , Atrophy/drug therapy , Atrophy/physiopathology , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/pathology , Blood-Brain Barrier/physiopathology , Brain/pathology , Brain/physiopathology , Brain Edema/drug therapy , Brain Edema/pathology , Brain Edema/physiopathology , Capillary Permeability/drug effects , Capillary Permeability/physiology , Cell Death/drug effects , Cell Death/physiology , Disease Models, Animal , Drug Evaluation, Preclinical , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Ischemia, Brain/pathology , Hypoxia-Ischemia, Brain/physiopathology , Maze Learning/drug effects , Maze Learning/physiology , Neurons/drug effects , Neurons/pathology , Neurons/physiology , Neuroprotective Agents/chemistry , Random Allocation , Rats, Sprague-Dawley , Treatment Outcome , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Febrile seizure (FS) is the most common seizure disorder in children, and children with FS are regarded as a high risk for the eventual development of epilepsy. Brain inflammation may be implicated in the mechanism of FS. Transient receptor potential vanilloid 1 (TRPV1) is believed to act as a monitor and regulator of body temperature. The role of inflammation in synaptic plasticity mediation indicates that TRPV1 is relevant to several nervous system diseases, such as epilepsy. Here, we report a critical role for TRPV1 in a febrile seizure mouse model and reveal increased levels of pro-inflammatory factors in the immature brain. Animals were subjected to hyperthermia for 30 min, which generates seizures lasting approximately 20 min, and then were used for experiments. To invoke frequently repetitive febrile seizures, mice are exposed to hyperthermia for three times daily at an interval of 4h between every time induced seizure, and a total of 4 days to induce. Behavioral testing for febrile seizures revealed that a TRPV1 knock-out mouse model demonstrated a prolonged onset latency and a shortened duration and seizure grade of febrile seizure when compared with wild type (WT) mice. The expression levels of both TRPV1 mRNA and protein increased after a hyperthermia-induced febrile seizure in WT mice. Notably, TRPV1 activation resulted in a significant elevation in the expression of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and HMGB1) in the hippocampus and cortex. These data indicate that the reduction of TRPV1 expression parallels a decreased susceptibility to febrile seizures. Thus, preventative strategies might be developed for use during febrile seizures.
Subject(s)
Brain/metabolism , Cytokines/metabolism , Hyperthermia, Induced , Seizures, Febrile/metabolism , TRPV Cation Channels/metabolism , Animals , Brain/immunology , Cell Line , Disease Models, Animal , Hippocampus/immunology , Hippocampus/metabolism , Mice , Mice, Knockout , Seizures, Febrile/immunology , TRPV Cation Channels/geneticsABSTRACT
Artemisinin, a natural product from the Chinese medicinal plant, Artemisia annua L., is commonly used in the treatment of malaria, and has recently been reported to have potent anticancer activity in various types of human tumors. Yet, the effect of artemisinin on neuroblastoma is still unclear. In the present study, we aimed to investigate the effects of artemisinin on neuroblastoma cells. We observed that artemisinin significantly inhibited cell growth and proliferation, and caused cell cycle arrest in the G1 phase in neuroblastoma cell lines. Annexin V-FITC/PI staining assay revealed that artemisinin markedly induced apoptosis. Soft agar assays revealed that artemisinin suppressed the ability of clonogenic formation of neuroblastoma cells and a xenograft study in NOD/SCID mice showed that artemisinin inhibited tumor growth and development in vivo. Therefore, our results suggest that the Chinese medicine artemisinin could serve as a novel potential therapeutic agent in the treatment of neuroblastoma.
Subject(s)
Antineoplastic Agents/administration & dosage , Artemisinins/administration & dosage , Neuroblastoma/drug therapy , Neuroblastoma/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Artemisinins/pharmacology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Mice , Mice, SCID , Neuroblastoma/pathology , Xenograft Model Antitumor AssaysABSTRACT
Astragalus polysaccharide (APS) has been reported to increase insulin sensitization and to ameliorate diabetes in animal models, and studies have demonstrated that this effect may be correlated with its anti-inflammatory roles in vivo and in vitro. However, the potential pharmacological mechanisms of APS in anti-inflammatory regulation are still poorly understood. Herein, RAW264.7 cells treated with APS showed anti-inflammatory effects. Interleukin (IL)-10 protein levels and expression of most of the anti-inflammatory genes, including IL-10, macrophage mannose receptor (MMR), arginase, Dectin-1, YM-1 and YM-2, were significantly increased after treatment with APS for 24 h. Furthermore, to determine whether APS plays a potential role in RAW264.7 cell inflammation, we pretreated RAW264.7 cells with APS in the presence of palmitate. The results showed that APS markedly recovered the impairment of AMPK activity induced by palmitate. Furthermore, APS induced IL-10 protein production and anti-inflammatory gene expression of IL-10, MMR, Dectin-1, arginase, YM-1 and YM-2. Additionally, APS inhibited IL-1ß protein production and expression of most of the pro-inflammatory genes, such as IL-1ß, iNOS, MCP-1, IL-6 and CD11c but not tumor necrosis factor (TNF)-α. Notably, the effect of APS on inflammatory genes, except for TNF-α, was abrogated when AMPK activity was inhibited using a DN-AMPK plasmid. These results suggest that APS effectively ameliorates palmitate-induced pro-inflammatory responses through AMPK activity.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Anti-Inflammatory Agents/pharmacology , Astragalus Plant/chemistry , Polysaccharides/pharmacology , Animals , Cell Line , Cytokines/genetics , Cytokines/metabolism , Enzyme Activation/drug effects , Gene Expression Regulation/drug effects , Inflammation/genetics , Inflammation/metabolism , Macrophages/drug effects , Macrophages/metabolism , Mice , Palmitates/pharmacology , Plant Extracts/chemistry , Plant Extracts/pharmacologyABSTRACT
To design a releasable PEGylated TNF-α (rPEG-TNF-α), a cathepsin B-sensitive dipeptide (Val-Cit moiety) was inserted into conventional PEG-modified TNF-α (PEG-TNF-α), facilitating its clinical use for anti-tumor therapy. Comparative pharmacokinetic and pharmacodynamic studies showed that the half-lives of both PEGylated forms of TNF-α were â¼60-fold greater than that of unmodified TNF-α. In addition, the in vitro bioactivity of rPEG-TNF-α was greater than that of PEG-TNF-α with the same degree of PEG modification. Release of TNF-α from rPEG-TNF-α in vitro was dependent on the presence of cathepsin B and was inhibited by a cathepsin B inhibitor. Despite the potent cytotoxicity of unmodified TNF-α against normal cells, its PEGylated forms at higher TNF-α concentrations showed low cytotoxic activity against these cells. In contrast, both forms of PEGylated TNF-α showed potent cytotoxic activity against the B16 and L929 cell lines, with rPEG-TNF-α being 5- and 9-fold more potent, respectively, than PEG-TNF-α. Moreover, rPEG-TNF-α was a more potent in vivo antitumor agent than PEG-TNF-α.
Subject(s)
Dipeptides/chemistry , Neoplasms, Experimental/drug therapy , Polyethylene Glycols/chemistry , Tumor Necrosis Factor-alpha/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Cathepsin B/antagonists & inhibitors , Cathepsin B/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Compounding , Drug Evaluation, Preclinical , Glycopeptides/pharmacology , Humans , Hydrogen-Ion Concentration , Leucine/analogs & derivatives , Leucine/pharmacology , Lysosomes/enzymology , Male , Mice , Mice, Inbred C57BL , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Pepstatins/pharmacology , Protease Inhibitors/pharmacology , Rats , Time Factors , Tumor Burden/drug effects , Tumor Necrosis Factor-alpha/chemistry , Tumor Necrosis Factor-alpha/pharmacokineticsABSTRACT
OBJECTIVE: To study the relative expression of the genes involved in artemisinin biosynthesis in different tissues including roots, stems, leaves and flowers of Artemisia annua, and establish the relationship between gene expression and artemisinin accumulation, eventually leading to discover the mainly effective genes involved in artemisinin biosynthesis. METHOD: The 7 functional genes involved in artemisinin biosynthesis were detected at the level of expression by using qRT-PCR, and simultaneously the content of artemisinin in the 4 investigated tissues was detected in parallel. RESULT: The 3 genes including HMGR, DXR and FPS which were involved in the upstream pathway of artemisinin biosynthesis showed the highest expression levels in flowers, and the 4 functional genes including ADS, CYP71AV1, CPR and AAR which were involved in the artemisinin-specific biosynthetic pathway were found to be expressed in all the 4 detected tissues. The highest expression level of ADS was found in leaves, then followed by flowers, and the lowest expression level of ADS was found in roots and stems. CYP71AV1 had highest expression level in flowers and lowest in leaves. CPR showed highest expression level in flowers, and AAR had lower expression levels in the other 3 artemisinin-specific pathway genes in all the tissues. The highest content of artemisinin was found in leaves (0.343 mg x g(-1)), then followed by flowers (0.152 mg x g(-1)), roots (0.062 mg x g(-1)) and stems (0.060 mg x g(-1)). CONCLUSION: In the biosynthesis of artemisinin, the upstream genes including HMGR from the MVA pathway, DXR from the MEP pathway and the checkpoint gene FPS were much more active in flowers, and this suggested that flowers might be the tissues of artemisinin precursor biosynthesis, and further DXR contributed more to artemisinin biosynthesis. The positive correlation of ADS expression and artemisinin content in tissues demonstrated that ADS played a very important role in artemisinin biosynthesis, which was the ideal target for engineering the artemisinin biosynthetic pathway. In summary, the functional genes involved in artemisinin biosynthesis do not express at the same level but synergistically.
Subject(s)
Artemisia annua/chemistry , Artemisia annua/metabolism , Artemisinins/metabolism , Artemisia annua/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Polymerase Chain ReactionABSTRACT
Human foamy virus (HFV), with its nonpathogenic nature and several unique features for gene transfer, is a promising vector system for neurological disorders gene therapy. The question of whether HFV vectors can be developed for the expression of therapeutic genes in primary astrocytes of the brain may be of interest. First, efficient expression for foreign genes, which is critical for the potentials of HFV-derived vector in gene therapy, was successfully demonstrated in rat-cultured astrocytes by the enhanced green fluorescent protein (EGFP) transduction through an HFV vector bearing an EGFP expression cassette. Second, HFV vectors containing human glutamic acid decarboxylase (GAD) complementary DNA, which encodes an inhibitory neurotransmitter gamma-aminobutyric acid (GABA)-producing enzyme, were used to examine the function of GAD on GABA synthesis in cultured astrocytes. We found that the transduction of GAD vector resulted in isoform-specific expression of GAD, synthesis of a significant amount of GABA and tonical GABA release, and behavioral recovery in rat Parkinson's disease (PD) models. These results suggested that HFV vector had the ability to transduce astrocytes and HFV vector-derived GAD expression in astrocytes provided a potential strategy for the treatment of neurological disorders associated with hyperexcitable or diminished inhibitory activity.