ABSTRACT
Study Design: Pre-clinical animal experiment. Objective: In this study, we investigated therapeutic effects of silibinin in a spinal cord injury (SCI) model. In SCI, loss of cells due to secondary damage mechanisms exceeds that caused by primary damage. Ferroptosis, which is iron-dependent non-apoptotic cell death, is shown to be influential in the pathogenesis of SCI. Methods: The study was conducted as an in vivo experiment using a total of 78 adult male/female Sprague Dawley rats. Groups were as follows: Sham, SCI, deferoxamine (DFO) treatment, and silibinin treatment. There were subgroups with follow-up periods of 24 h, 72 h, and 6 weeks in all groups. Malondialdehyde (MDA), glutathione (GSH), and Fe2+ levels were measured by spectrophotometry. Glutathione peroxidase-4 (GPX4), ferroportin (FPN), transferrin receptor (TfR1), and 4-hydroxynonenal (4-HNE)-modified protein levels were assessed by Western blotting. Functional recovery was assessed using Basso-Beattie-Bresnahan test. Results: Silibinin achieved significant suppression in MDA and 4-HNE levels compared to the SCI both in 72-h and 6 weeks group (p < 0.05). GSH, GPX4, and FNP levels were found to be significantly higher in the silibinin 24 h, 72 h, and 6 weeks group compared to corresponding SCI groups (p < 0.05). Significant reduction in iron levels was observed in silibinin treated rats in 72 h and 6 weeks group (p < 0.05). Silibinin substantially suppressed TfR1 levels in 24 h and 72 h groups (p < 0.05). Significant difference among recovery capacities was observed as follows: Silibinin > DFO > SCI (p < 0.05). Conclusion: Impact of silibinin on iron metabolism and lipid peroxidation, both of which are features of ferroptosis, may contribute to therapeutic activity. Within this context, our findings posit silibinin as a potential therapeutic candidate possessing antiferroptotic properties in SCI model. Therapeutic agents capable of effectively and safely mitigating ferroptotic cell death hold the potential to be critical points of future clinical investigations.
ABSTRACT
BACKGROUND: HCC is among the most common cancer. Ganoderma lucidum (G.lucidum) has been essential in preventing and treating cancer. The Nrf2 signaling cascade is a cell protective mechanism against further damage, such as cancer development. This signaling pathway upregulates the cytoprotective genes and is vital in eliminating xenobiotics and reactive oxygen. This study aimed to show the potential cytotoxic activity of G. lucidum aqueous extract in HCC. METHODS AND RESULTS: MTT assay was used to detect cell viability. Nrf2-related proteins were measured by western blotting, and the flow cytometry method assayed cell population in different cycle phases. Cell viability was 49% and 47% following G. lucidum extract at 100 µg/ml at 24 and 48 h treatments, respectively. G. lucidum extract (aqueous, 100 or 50 µg/ml) treatments for 24, 48, or 72 h were able to significantly change the cytoplasmic/nuclear amount of Nrf2 and HO-1, NQO1 protein levels. Moreover, at both concentrations, arrest of the G0/G1 cell cycle was stimulated in HCC. CONCLUSIONS: The activation of the Nrf2 signaling pathways seems to be among the mechanisms underlining the protective and therapeutic action of G. lucidum against HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Reishi , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Humans , Liver Neoplasms/drug therapy , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Oxygen , Reishi/metabolism , XenobioticsABSTRACT
N-methyl-D-aspartate (NMDA) receptor stimulation may lead to excitotoxicity, which triggers neuronal death in brain disorders. In addition to current clinical therapeutic approaches, treatment strategies by phytochemicals or their derivatives are under investigation for neurodegenerative diseases. In the present study, novel amino and 1,2,3-triazole derivatives of tomentosin were prepared and tested for their protective and anti-apoptotic effects in NMDA-induced excitotoxicity. Amino-tomentosin derivatives were generated through a diastereoselective conjugate addition of several secondary amines to the α-methylene-γ-butyrolactone function, while the 1,2,3-triazolo-tomentosin was prepared by a regioselective Michael-type addition carried out in the presence of trimethylsilyl azide (TMSN3) and the α-methylene-γ-lactone function. The intermediate key thus obtained underwent 1,3-dipolar Huisgen cycloaddition using a wide range of terminal alkynes. The possible effects of the derivatives on cell viability and free-radical production following NMDA treatment were measured by Water-Soluble Tetrazolium Salts (WST-1) and Dichlorofluorescein Diacetate (DCF-DA) assays, respectively. The alterations in apoptosis-related proteins were examined by Western blot technique. Our study provides evidence that synthesized triazolo- and amino-tomentosin derivatives show neuroprotective effects by increasing cellular viability, decreasing ROS production, and increasing the Bcl-2/Bax ratio in NMDA-induced excitotoxicity. The findings highlight particularly 2e, 2g, and 6d as potential regulators and neuroprotective agents in NMDA overactivation.
ABSTRACT
Neurodegenerative diseases affect millions of people. Major reasons behind the onset and progression of these diseases are still under investigation. Therefore, any approach that would treat/prevent progression is important. In this study, we aimed to investigate the potential protective effects of Psephellus pyrrhoblepharus (Boiss.) Wagenitz extracts in MPP+-induced dopaminergic cell damage and compare the effectiveness of different extracts (methanol:water (1:1), chloroform and n-hexane). The cells were pretreated with four different concentrations (10, 50, 100, and 200 µg/ml) of methanol:water (1:1), chloroform and n-hexane extracts of P. pyrrhoblepharus following MPP+ treatment for 12 or 24 h. The changes in cell viability were determined using the MTT assay. Additionally, antioxidant activities and total phenolic/flavonoid contents of the extracts were determined with radical scavenging capacity, Folin-Ciocalteu and aluminum chloride assays, respectively. The extracts at selected concentrations were found to be protective in a dose-dependent manner at 12 and 24 h. Nevertheless, the methanol extract of the plant showed the highest protection both at 100 and 200 µg/ml (115.13%±3.98, 121.87%±1.66; p < 0.05) against dopaminergic damage at 24 h. The results showed that selected concentrations were not toxic and did not affect cell proliferation rate. Besides, the chloroform extract was found to have higher antioxidant activity than the other extracts (p < 0.05). The total phenolic and total flavonoid contents were found consistent with antioxidant activities. Our findings support the neuroprotective and antioxidant potential of P. pyrrhoblepharus. However, further studies on identifying the presence of chemicals in P. pyrrhoblepharus extracts which are responsible for protection should be carried out to confirm their therapeutic potential.
Subject(s)
Cytoprotection , Plant Extracts , Antioxidants/pharmacology , Flavonoids/pharmacology , Humans , Phenols/toxicity , Plant Extracts/pharmacologyABSTRACT
The loss of neurons is strongly correlated with aging and aging-associated disorders. In this study, cell viability assays and mitochondrial function were performed to evaluate the effect of new spiro-pyrazole derivatives, prepared from aldehydes and 3-amino-1-phenyl-2-pyrazolin-5-one, on neuroprotection in an in vitro model of dopaminergic cell death induced by 1-methyl-4-phenylpyridinium (MPP+). The percentages of neuroprotection by derivatives were found between 21.26% and 52.67% at selected concentrations (10-50 µM) with compound 4d exerting the best neuroprotective effect. The results show that the studied spiropyrazolones perform important roles in dopaminergic neuroprotection and can be used for potential new therapies in the treatment of neurodegenerative disorders including Parkinson's disease.
Subject(s)
Neuroprotective Agents/pharmacology , Pyrazoles/pharmacology , Spiro Compounds/pharmacology , Cell Death/drug effects , Cell Survival/drug effects , Cytoprotection/drug effects , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Dose-Response Relationship, Drug , Humans , Molecular Structure , Neuroprotective Agents/chemical synthesis , Neuroprotective Agents/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Spiro Compounds/chemical synthesis , Spiro Compounds/chemistry , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
PURPOSE: The present study was conducted to determine oxidative stress and cell viability after contact with resin composites polymerized for different times. METHODS: Disk-shaped specimens of Admira Fusion, Ceram X One Universal, Solare x and Filtek Z550 (n = 12) were prepared, and two subgroups with polymerization times of 20 and 40 s were employed. The specimens were incubated with mouse fibroblast cells for 48 and 72 h, and changes in reactive oxygen species (ROS) production and cellular viability were determined by an assay with a cell-permeable fluorescent dye, 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA), and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, respectively. RESULTS: At 72 h, ROS production in the presence of Admira Fusion polymerized for 40 s was reduced relative to that in the presence of Admira Fusion polymerized for 20 s (P < 0.05). Cell viability was maximal in the Admira Fusion and Solare x groups and there was no difference relative to the control group at 48 h. Cell viability was higher in the Admira Fusion and Solare x groups polymerized for 40 s than for the same materials polymerized for 20 s at 72 h (P < 0.05). CONCLUSION: Extension of the polymerizaton time has a material-specific effect and may be used as a strategy to increase the biocompability of resin composites.
Subject(s)
Composite Resins , Animals , Cell Survival , Materials Testing , Mice , Polymerization , Reactive Oxygen SpeciesABSTRACT
Recently, nuclear translocation and stability of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) have gained increasing attention in the prevention of oxidative stress. The present study was aimed to evaluate the regulatory role of glycogen synthase kinase-3ß (GSK-3ß) inhibition by tideglusib through the Nrf2 pathway in a cellular damage model. Gene silencing (siRNA-mediated) was performed to examine the responses of Nrf2-target genes (i.e., heme oxygenase-1, NAD(P)H:quinone oxidoreductase1) to siRNA depletion of Nrf2 in MPPâº-induced dopaminergic cell death. Nrf2 and its downstream regulated genes/proteins were analyzed using Real-time PCR and Western Blotting techniques, respectively. Moreover, free radical production, the changes in mitochondrial membrane potential, total glutathione, and glutathione-S-transferase were examined. The possible contribution of peroxisome proliferator-activated receptor gamma (PPARγ) to tideglusib-mediated neuroprotection was evaluated. The number of viable cells and mitochondrial membrane potential were increased following GSK-3ß enzyme inhibition against MPPâº. HO-1, NQO1 mRNA/protein expressions and Nrf2 nuclear translocation significantly triggered by tideglusib. Moreover, the neuroprotection by tideglusib was not observed in the presence of siRNA Nrf2. Our study supports the idea that GSK-3ß enzyme inhibition may modulate the Nrf2/ARE pathway in cellular damage and the inhibitory role of tideglusib on GSK-3ß along with PPARγ activation may be responsible for neuroprotection.
Subject(s)
1-Methyl-4-phenylpyridinium/adverse effects , Neurons/cytology , Signal Transduction/drug effects , Thiadiazoles/pharmacology , Cell Death/drug effects , Cell Line , Glycogen Synthase Kinase 3 beta/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta/metabolism , Heme Oxygenase-1/genetics , Heme Oxygenase-1/metabolism , Humans , Membrane Potential, Mitochondrial/drug effects , NAD(P)H Dehydrogenase (Quinone)/genetics , NAD(P)H Dehydrogenase (Quinone)/metabolism , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Neurons/drug effects , Neurons/metabolism , Pioglitazone/pharmacologyABSTRACT
OBJECTIVE: The aim of this in vitro study is to evaluate the effects of resveratrol (RES) addition on the cytotoxicity and microtensile bond strength (µTBS) of different adhesives. MATERIALS AND METHODS: Five self-etching adhesives (G-aenial Bond-GC, Optibond All in One-Kerr, Gluma Self Etch-Kulzer, Clearfil S3 Bond-Kuraray, and Nova Compo-B Plus-Imicryl) were tested. They were applied to L-929 cell culture by the extract method. In the test groups, 0.5 µM RES (Sigma-Aldrich) was added into the medium. Cell viability was assessed by MTT assay after 24 h. Human extracted third molars were used for µTBS test (n = 7). The adhesives with or without 0.5 µM RES addition were applied on dentin surfaces. A composite build-up was constructed. Then, the specimens were sectioned into multiple beams with the non-trimming version of the microtensile test and subjected to microtensile forces. Statistical analysis was performed using ANOVA and post hoc Tukey test (p Ë 0.05). RESULTS: The extracts of all adhesives decreased the cell viability. However, RES addition increased the cell viability in all groups (p Ë 0.05). RES addition did not cause any decrease in µTBS values of the adhesives compared to baseline. Optibond All in One showed the highest µTBS after RES addition. It was followed by Clerafil S3 Bond and Nova Compo-B Plus. No difference was determined between the Optibond All in One and Clearfil S3 Bond. There was difference between Optibond All in One and Nova Compo-B Plus (p Ë 0.05). CONCLUSION: RES addition may improve the biocompatibility without causing negative influence on µTBS of the adhesives. CLINICAL RELEVANCE: RES addition has clinical applicable potential to overcome the adverse biocompatibility of adhesives.
Subject(s)
Dental Bonding , Dental Cements , Resveratrol , Adhesives , Composite Resins , Dental Cements/pharmacology , Dentin , Dentin-Bonding Agents , Humans , Materials Testing , Resin Cements , Resveratrol/pharmacology , Tensile StrengthABSTRACT
Spinal cord injury (SCI) leads to vascular damage and disruption of blood-spinal cord barrier which participates in secondary nerve injury. Epidermal growth factor (EGF) is an endogenous protein which regulates cell proliferation, growth and differention. Previous studies reported that EGF exerts neuroprotective effect in spinal cord after SCI. However, the molecular mechanisms underlying EGF-mediated protection in different regions of nervous system have not shown yet. In this study, we aimed to examine possible anti-apoptotic and protective roles of EGF not only in spinal cord but also in brain following SCI. Twenty-eight adult rats were divided into four groups of seven animals each as follows: sham, trauma (SCI), SCIâ¯+â¯EGF and SCIâ¯+â¯methylprednisolone (MP) groups. The functional neurological deficits due to the SCI were assessed by behavioral analysis using the Basso, Beattie and Bresnahan (BBB) open-field locomotor test. The alterations in pro-/anti-apoptotic protein levels and antioxidant enzyme activities were measured in spinal cord and frontal cortex. In our study, EGF promoted locomotor recovery and motor neuron survival of SCI rats. EGF treatment significantly decreased Bax and increased Bcl-2 protein expressions both in spinal cord and brain when compared to SCI group. Moreover, antioxidant enzyme activities including catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were increased following EGF treatment similar to MP treatment. Our experiment also suggests that alteration of the ratio of Bcl-2 to Bax may result from decreased apoptosis following EGF treatment. As a conclusion, these results show, for the first time, that administration of EGF exerts its protection via regulating apoptotic and oxidative pathways in response to spinal cord injury in different regions of central nervous system.
Subject(s)
Apoptosis/physiology , Epidermal Growth Factor/pharmacology , Frontal Lobe/physiopathology , Neuroprotective Agents/pharmacology , Oxidative Stress/physiology , Spinal Cord Injuries/physiopathology , Spinal Cord/physiopathology , Animals , Blotting, Western , Catalase/metabolism , Disease Models, Animal , Epidermal Growth Factor/metabolism , Frontal Lobe/metabolism , Male , Neuroprotective Agents/metabolism , Rats , Rats, Sprague-Dawley , Recovery of Function , Spinal Cord/metabolism , Spinal Cord Injuries/immunology , Superoxide Dismutase/metabolismABSTRACT
Oxidative stress and apoptosis are both associated with various acute and chronic disorders. Thus, the aim of the present study is to synthesize imidazo[2,1-c][1,2,4]triazines derivatives and to evaluate their effects in H2O2-induced oxidative stress in human neuroblastoma cell line (SH-SY5Y cells). The effects of the compounds on cell viability were measured by MTT assay and the changes in stress and apoptosis-related proteins were investigated by PathScan® Stress and Apoptosis Signaling Antibody Array kit and Western Blot technique. In particular, four compounds were found to protect SH-SY5Y cells from H2O2-induced toxicity by increasing Bcl-2/Bax ratio, regulating PI3-K/Akt cascade and inhibiting the ERK pathway.
Subject(s)
Hydrogen Peroxide/antagonists & inhibitors , Imidazoles/pharmacology , Triazines/pharmacology , Apoptosis/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Hydrogen Peroxide/pharmacology , Imidazoles/chemical synthesis , Imidazoles/chemistry , Molecular Structure , Oxidative Stress/drug effects , Stereoisomerism , Structure-Activity Relationship , Triazines/chemical synthesis , Triazines/chemistry , Tumor Cells, CulturedABSTRACT
STUDY DESIGN: Experimental animal study investigating the efficacy of C-terminal domain of tetanus toxin application as neuroprotective effects on rat brain in a model of spinal cord injury (SCI). OBJECTIVE: The aim of the present study was to investigate the possible role of C-terminal domain of tetanus toxin (Hc-TeTx) on cell death mechanisms including apoptosis and autophagy following SCI. SUMMARY OF BACKGROUND DATA: Traumatic SCI can lead to posttraumatic inflammation, oxidative stress, motor neuron apoptosis, necrosis, and autophagy of tissue. To promote and enhance recovery after SCI, recent development of devices and therapeutic interventions are needed. METHODS: Twenty-eight adult rats were divided into four groups (nâ=â7 each) as follows: sham, trauma (SCI), SCI + Hc-TeTx, and SCI + methylprednisolone groups. The functional neurological deficits due to the SCI were assessed by behavioral analysis using the Basso, Beattie and Bresnahan (BBB) open-field locomotor test. The alterations in pro-/anti-apoptotic and autophagy related-protein levels were measured by Western blotting technique. RESULTS: In this study, Hc-TeTx promotes locomotor recovery and motor neuron survival of SCI rats. Hc-TeTx also decreased expression of bax, bad, bak, cleaved caspase-3, Ask1, and autophagy-related proteins including Atg5 and LC3II in brain. Our study provides an evidence that cell death mechanisms play critical roles in SCI and that the nontoxic peptides including Hc-TeTx may exert protective effect and decrease cell death following SCI. CONCLUSION: Our preliminary findings suggest a possible therapeutic agent to improve survival after spinal cord trauma, but further analysis are still needed to evaluate the difference between acute and chronic injuries. LEVEL OF EVIDENCE: N/A.
Subject(s)
Motor Neurons/drug effects , Neuroprotective Agents/pharmacology , Spinal Cord Injuries/drug therapy , Tetanus Toxin/pharmacology , Animals , Apoptosis/drug effects , Autophagy/drug effects , Brain/drug effects , Brain/metabolism , Cell Survival/drug effects , Disease Models, Animal , Methylprednisolone/pharmacology , Rats , Spinal Cord/drug effects , Spinal Cord/physiopathology , Spinal Cord Injuries/physiopathologyABSTRACT
Neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and Huntington's disease affect millions of people in the world. Thus several new approaches to treat brain disorders are under development. The aim of the present study is to synthesize potential neuroprotective heterocyclic compounds based on pyrazolopyridine derivatives and then to evaluate their effects in MPP+-induced neurodegeneration in human neuroblastoma cell line (SH-SY5Y cells). The effects of the compounds on cell viability were measured by MTT assay and the changes in apoptosis-related proteins including bax, Bcl-2, Bcl-xl and caspase-3 were investigated by western blot technique. Based on the cell viability results obtained by MTT assay, the percentage of neuroprotection-induced by compounds against MPP+-induced neurotoxicity in SH-SY5Y cells was between 20% and 30% at 5 µM concentrations of all synthesized compounds. Moreover, the downregulation in pro-apoptotic proteins including bax and caspase-3 were found following the novel synthesized compounds treatments and these effects were observed in a dose-dependent manner. Our results provide an evidence that these heterocyclic compounds based on pyrazolopyridine derivatives may have a role on dopaminergic neuroprotection via antiapoptotic pathways.
Subject(s)
Heterocyclic Compounds/pharmacology , Neurodegenerative Diseases/drug therapy , Neuroprotective Agents/chemical synthesis , Pyrazoles/pharmacology , Pyridines/pharmacology , Apoptosis Regulatory Proteins/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Heterocyclic Compounds/chemistry , Humans , Neuroblastoma/pathology , Neurodegenerative Diseases/chemically induced , Neuroprotective Agents/pharmacology , Pyrazoles/chemistry , Pyridines/chemistryABSTRACT
This study was designed to determine the in vivo performance of three different materials as scaffolds for dental pulp stem cells (DPSC) undergoing induced odontogenic differentiation. An odontogenic medium modified by the addition of recombinant human bone morphogenetic protein 2 was used in the experimental groups to induce differentiation. Mesenchymal stem cell medium was used in the control groups. DPSC were transplanted onto the backs of mice via three scaffolds: copolymer of L-lactide and DL-lactide (PLDL), copolymer of DL-lactide (PDL) and hydroxyapatite tricalcium phosphate (HA/TCP). The expression levels of dentin sialo-phosphoprotein (DSPP), dentin matrix protein-1 (DMP1), enamelysin/matrix metalloproteinase 20 (MMP20) and phosphate-regulating gene with homologies to endopeptidases on X chromosome (PHEX) were analysed using RT-PCR. The expressions in the experimental groups were compared to those in the control groups. The transcript expressions at 6 and 12 weeks were significantly different for all scaffolds (p < 0.05), except for the expression of DSPP in the PLDL group with regard to the time variable. Although there was a decrease in the expression of enamelysin/MMP20 in PLDL and HA/TCP at 12 weeks, all other expressions increased and reached their highest level at 12 weeks. The highest DSPP expression was in the PDL group (p < 0.05). The highest expression of DMP1 was detected in the HA/TCP group (p < 0.05). The highest expression of PHEX was in the PLDL group (p < 0.05). Consequently, PLDL and PDL seemed to be promising scaffold candidates for odontogenic regeneration at least as HA-TCP, when they were applied with the DPSC induced for odontogenic differentiation.
Subject(s)
Cell Differentiation/physiology , Dental Pulp/cytology , Polymers/chemistry , Stem Cells/physiology , Tissue Scaffolds/chemistry , Animals , Biocompatible Materials/chemistry , Calcium Phosphates/chemistry , Cell Culture Techniques , Dental Enamel/chemistry , Dentin/chemistry , Dioxanes/chemistry , Durapatite/chemistry , Extracellular Matrix Proteins/analysis , Gene Expression , Humans , Matrix Metalloproteinase 20/analysis , Mice , PHEX Phosphate Regulating Neutral Endopeptidase/analysis , Phosphoproteins/analysis , Reproducibility of Results , Sialoglycoproteins/analysis , Time FactorsABSTRACT
Increased concentration of manganese (Mn) in the brain is known to be associated with excitotoxicity and neuroinflammation. Vinpocetine, an alkaloid derived from the plant Vinca minor L., basically shows its effect via phosphodiesterase inhibition and voltage-dependent Na+ channels. Vasoactive intestinal peptide (VIP) has gastrointestinal, vasomotor, muscular, and neuroprotective effects. The aim of this study was to examine the potential protective effects of vinpocetine and VIP against Mn toxicity in NE-4C neural stem cells (NSCs). VIP treatment at 1 µM and vinpocetine treatment at 2 µM concentrations were sufficient to yield maximum protection, and these concentrations were adopted in the following experiments. In this study, Mn treatment significantly increased lactate dehydrogenase (LDH) leakage, reactive oxygen species (ROS) production, and triggered cell death in NE-4C cultures. However, significant reduction in LDH release was observed following vinpocetine or VIP treatments when compared with control. Similar to these findings, vinpocetine or VIP treatments significantly reduced membrane degradation induced by Mn (p < 0.001). Moreover, vinpocetine attenuated Mn-induced decrease of mitochondrial membrane potential. Similarly, proapoptotic protein bax and ROS production significantly decreased in cells after incubation with vinpocetine (p = 0.01) or VIP in the presence of Mn (p < 0.001). Our study provides the evidence that both vinpocetine and VIP may exert protective effects via modulating oxidative stress and apoptosis in Mn-induced neurodegeneration in NE-4C cells.
Subject(s)
Manganese/toxicity , Membrane Potential, Mitochondrial/drug effects , Reactive Oxygen Species/metabolism , Vasoactive Intestinal Peptide/pharmacology , Vinca Alkaloids/pharmacology , Cell Death/drug effects , Cell Line , Dose-Response Relationship, Drug , Humans , Neural Stem CellsABSTRACT
The aim of this study was to evaluate whether medium modification improves the odontogenic differentiation of human dental pulp stem cells (DPSC) in vitro and in vivo. DPSC isolated from human impacted third molar teeth were analysed for clusters of differentiation with flow cytometry. Odontogenic differentiation was stimulated by medium modification with the addition of bone morphogenetic protein 2 (BMP2). The expression of dentin sialophosphoprotein, dentin matrix protein 1, enamelysin/matrix metalloproteinase 20 and the phosphate-regulating gene with homologies to endopeptidases on the X chromosome of the cells were analysed with RT-PCR at 7, 14 and 21 days. Then, DPSC were transplanted on the back of immunocompromised mice via a hydroxyapatite tricalcium phosphate scaffold, and the structure of the formed tissue was investigated. The cells were identified as mesenchymal stem cells with a 98.3% CD73 and CD90 double-positive cell rate. The increase in mineralization capacity and expression of human enamel-dentin specific transcripts proportional to the culture period were determined after differentiation. Six weeks after transplantation, an osteo-dentin matrix was formed in the group in which odontogenic differentiation was stimulated, and the odontogenic characteristics of the matrix were confirmed by histological examination and RT-PCR analysis. Odontogenic differentiation of the isolated and characterized human DPSC was improved with medium modification by the addition of BMP2 in vitro and in vivo. The defined medium and applied technique have a potential use for forming reparative dentin in the future, but the effects of the method should be investigated in long-term studies.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Cell Differentiation/drug effects , Culture Media/chemistry , Dental Pulp/cytology , Stem Cells/cytology , Actins/analysis , Adult , Animals , Bone Morphogenetic Protein 2/chemistry , Cell Differentiation/physiology , Cell Proliferation/drug effects , Cell Proliferation/physiology , Cells, Cultured , Extracellular Matrix Proteins/analysis , Flow Cytometry , Humans , Matrix Metalloproteinase 20/analysis , Mice , Odontogenesis/drug effects , Odontogenesis/physiology , PHEX Phosphate Regulating Neutral Endopeptidase/analysis , Phosphoproteins/analysis , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Sialoglycoproteins/analysis , Stem Cell Transplantation/methods , Time Factors , Young AdultABSTRACT
Abstract The aim of this study was to evaluate whether medium modification improves the odontogenic differentiation of human dental pulp stem cells (DPSC) in vitro and in vivo. DPSC isolated from human impacted third molar teeth were analysed for clusters of differentiation with flow cytometry. Odontogenic differentiation was stimulated by medium modification with the addition of bone morphogenetic protein 2 (BMP2). The expression of dentin sialophosphoprotein, dentin matrix protein 1, enamelysin/matrix metalloproteinase 20 and the phosphate-regulating gene with homologies to endopeptidases on the X chromosome of the cells were analysed with RT-PCR at 7, 14 and 21 days. Then, DPSC were transplanted on the back of immunocompromised mice via a hydroxyapatite tricalcium phosphate scaffold, and the structure of the formed tissue was investigated. The cells were identified as mesenchymal stem cells with a 98.3% CD73 and CD90 double-positive cell rate. The increase in mineralization capacity and expression of human enamel-dentin specific transcripts proportional to the culture period were determined after differentiation. Six weeks after transplantation, an osteo-dentin matrix was formed in the group in which odontogenic differentiation was stimulated, and the odontogenic characteristics of the matrix were confirmed by histological examination and RT-PCR analysis. Odontogenic differentiation of the isolated and characterized human DPSC was improved with medium modification by the addition of BMP2 in vitro and in vivo. The defined medium and applied technique have a potential use for forming reparative dentin in the future, but the effects of the method should be investigated in long-term studies.
Subject(s)
Humans , Animals , Adult , Mice , Young Adult , Stem Cells/cytology , Cell Differentiation/drug effects , Culture Media/chemistry , Dental Pulp/cytology , Bone Morphogenetic Protein 2/pharmacology , Phosphoproteins/analysis , Sialoglycoproteins/analysis , Time Factors , Cell Differentiation/physiology , Cells, Cultured , Reproducibility of Results , Extracellular Matrix Proteins/analysis , Actins/analysis , Reverse Transcriptase Polymerase Chain Reaction , Stem Cell Transplantation/methods , Cell Proliferation/drug effects , Cell Proliferation/physiology , Matrix Metalloproteinase 20/analysis , PHEX Phosphate Regulating Neutral Endopeptidase/analysis , Bone Morphogenetic Protein 2/chemistry , Flow Cytometry , Odontogenesis/drug effects , Odontogenesis/physiologyABSTRACT
Abstract This study was designed to determine the in vivo performance of three different materials as scaffolds for dental pulp stem cells (DPSC) undergoing induced odontogenic differentiation. An odontogenic medium modified by the addition of recombinant human bone morphogenetic protein 2 was used in the experimental groups to induce differentiation. Mesenchymal stem cell medium was used in the control groups. DPSC were transplanted onto the backs of mice via three scaffolds: copolymer of L-lactide and DL-lactide (PLDL), copolymer of DL-lactide (PDL) and hydroxyapatite tricalcium phosphate (HA/TCP). The expression levels of dentin sialo-phosphoprotein (DSPP), dentin matrix protein-1 (DMP1), enamelysin/matrix metalloproteinase 20 (MMP20) and phosphate-regulating gene with homologies to endopeptidases on X chromosome (PHEX) were analysed using RT-PCR. The expressions in the experimental groups were compared to those in the control groups. The transcript expressions at 6 and 12 weeks were significantly different for all scaffolds (p < 0.05), except for the expression of DSPP in the PLDL group with regard to the time variable. Although there was a decrease in the expression of enamelysin/MMP20 in PLDL and HA/TCP at 12 weeks, all other expressions increased and reached their highest level at 12 weeks. The highest DSPP expression was in the PDL group (p < 0.05). The highest expression of DMP1 was detected in the HA/TCP group (p < 0.05). The highest expression of PHEX was in the PLDL group (p < 0.05). Consequently, PLDL and PDL seemed to be promising scaffold candidates for odontogenic regeneration at least as HA-TCP, when they were applied with the DPSC induced for odontogenic differentiation.
Subject(s)
Humans , Animals , Polymers/chemistry , Stem Cells/physiology , Cell Differentiation/physiology , Dental Pulp/cytology , Tissue Scaffolds/chemistry , Phosphoproteins/analysis , Sialoglycoproteins/analysis , Time Factors , Biocompatible Materials/chemistry , Calcium Phosphates/chemistry , Gene Expression , Reproducibility of Results , Extracellular Matrix Proteins/analysis , Durapatite/chemistry , Cell Culture Techniques , Dental Enamel/chemistry , Dentin/chemistry , Dioxanes/chemistry , Matrix Metalloproteinase 20/analysis , PHEX Phosphate Regulating Neutral Endopeptidase/analysisABSTRACT
This study was designed to evaluate the cytotoxicity of four dentin bonding agents and the effects of an antioxidant addition. Group A: G-aenial Bond, Group B: Optibond All in One, Group C: Gluma Self Etch and Group D: Clearfil S(3) Bond were added to the medium using extract method. The cells were cultured with or without resveratrol (RES) addition. MTT, reactive oxygen species (ROS), DCF, Comet and 8-OHdG measurements were performed. The agents had a dose-dependent (1:1>1:10>1:20) cytotoxic effect. Considering 1:10 concentration; Group D at 1 h (p<0.01) and Group B and D at 24 h had the weakest cytotoxic effect (p<0.05). After RES addition, the highest cell viability was determined in Groups B+RES and D+RES at 1 h and in Groups A+RES and B+RES at 24 h (p<0.01). The dentin bonding agents induced ROS production and DNA damage regarding to their composition. However, RES addition decreased the indicated parameters.
Subject(s)
Cytotoxins/toxicity , Dentin-Bonding Agents/toxicity , Stilbenes/pharmacology , Animals , Cell Culture Techniques , Cell Survival/drug effects , DNA Damage , Dental Bonding , Dose-Response Relationship, Drug , Fibroblasts , Glutaral , Methacrylates , Mice , Oxidative Stress , Polymethacrylic Acids , Reactive Oxygen Species , Resin Cements , ResveratrolABSTRACT
BACKGROUND: N-methyl-d-aspartate (NMDA) receptors are major pharmacological targets to prevent or reduce the progression of neurodegenerative diseases. Successful therapy with NMDA receptor antagonists in humans has been limited by the severe side effects of complete receptor blockade. The aim of the present study was to investigate the possible protective effects of tideglusib against NMDA receptor overactivation in neural stem cells. METHODS: We measured the alteration in membrane integrity, free radical generation, intracellular Ca(2+) accumulation, mitochondrial membrane potential (MMP)/mitochondrial morphology and glycogen synthase kinase-3 (α/ß isoforms and phospho-GSK-3α/ß) protein expression levels following treatments. RESULTS: NMDA treatment, with or without d-serine, significantly increased LDH leakage and triggered cell death in neural stem cells. Reactive oxygen species (ROS) formation and intracellular Ca(2+) levels were increased following NMDA receptor overactivation. The significant reduction in MMP was found in NMDA/d-serine-treated cells. Tideglusib significantly decreased ROS production and membrane degradation, but did not change intracellular Ca(2+) levels following NMDA receptor activation. Both in the presence or in the absence of NMDA/d-serine, tideglusib increased MMP and the levels of phospho-GSK-3ß in NSCs. Moreover, GW9662 (a peroxisome-proliferator-activated receptor gamma (PPARγ) antagonist) treatment significantly inhibited the protective effect of tideglusib in NMDA/d-serine-treated cells. CONCLUSION: Our study provides the evidence that GSK-3ß and PPARγ may be directly involved in pathways leading to NMDA receptor-induced cell death and that the inhibitors including tideglusib may exert neuroprotective effect against these receptor overactivation.