OBJECTIVES: The neurotoxicity of carbon monoxide (CO) to the central nervous system is a key pathogenesis of delayed encephalopathy after acute carbon monoxide poisoning (DEACMP). Our previous study found that retinoic acid (RA) can suppress the neurotoxic effects of CO. This study further explores, in vivo and in vitro, the molecular mechanisms by which RA alleviates CO-induced central nervous system damage. METHODS: A cytotoxic model was established using the mouse hippocampal neuronal cell line HT22 and primary oligodendrocytes exposed to CO, and a DEACMP animal model was established in adult Kunming mice. Cell viability and apoptosis of hippocampal neurons and oligodendrocytes were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Annexin V/propidium iodide (PI) double staining. The transcriptional and protein expression of each gene was detected using real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting. Long noncoding RNA (lncRNA) SNHG15 and LINGO-1 were knocked down or overexpressed to observe changes in neurons and oligodendrocytes. In DEACMP mice, SNHG15 or LINGO-1 were knocked down to assess changes in central nervous tissue and downstream protein expression. RESULTS: RA at 10 and 20 μmol/L significantly reversed CO-induced apoptosis of hippocampal neurons and oligodendrocytes, downregulation of SNHG15 and LINGO-1, and upregulation of brain-derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (TrkB) (all P<0.05). Overexpression of SNHG15 or LINGO-1 weakened the protective effect of RA against CO-induced cytotoxicity (all P<0.05). Knockdown of SNHG15 or LINGO-1 alleviated CO-induced apoptosis of hippocampal neurons and oligodendrocytes and upregulated BDNF and TrkB expression levels (all P<0.05). Experiments in DEACMP model mice showed that knockdown of SNHG15 or LINGO-1 mitigated central nervous system injury in DEACMP (all P<0.05). CONCLUSIONS RA alleviates CO-induced apoptosis of hippocampal neurons and oligodendrocytes, thereby reducing central nervous system injury and exerting neuroprotective effects. LncRNA SNHG15 and LINGO-1 are key molecules mediating RA-induced inhibition of neuronal apoptosis and are associated with the BDNF/TrkB pathway. These findings provide a theoretical framework for optimizing the clinical treatment of DEACMP and lay an experimental foundation for elucidating its molecular mechanisms.
Animals ; RNA, Long Noncoding/physiology* ; Brain-Derived Neurotrophic Factor/genetics* ; Carbon Monoxide Poisoning/complications* ; Mice ; Tretinoin/pharmacology* ; Nerve Tissue Proteins/metabolism* ; Membrane Proteins/metabolism* ; Apoptosis/drug effects* ; Hippocampus/cytology* ; Receptor, trkB/metabolism* ; Neurons/drug effects* ; Male ; Brain Diseases/etiology* ; Oligodendroglia/drug effects* ; Signal Transduction ; Cell Line

The present study was designed to analyze the metabolites of all-trans-retinal (atRal) and compare the cytotoxicity of atRal versus its derivative all-trans-retinoic acid (atRA) in human retinal pigment epithelial (RPE) cells. We confirmed that atRA was produced in normal pig neural retina and RPE. The amount of all-trans-retinol (atROL) converted from atRal was about 2.7 times that of atRal-derived atRA after incubating RPE cells with 10 μmol/L atRal for 24 h, whereas atRA in medium supernatant is more plentiful (91 vs. 29 pmol/mL), suggesting that atRA conversion facilitates elimination of excess atRal in the retina. Moreover, we found that mRNA expression of retinoic acid-specific hydroxylase CYP26b1 was dose-dependently up-regulated by atRal exposure in RPE cells, indicating that atRA inactivation may be also initiated in atRal-accumulated RPE cells. Our data show that atRA-caused viability inhibition was evidently reduced compared with the equal concentration of its precursor atRal. Excess accumulation of atRal provoked intracellular reactive oxygen species (ROS) overproduction, heme oxygenase-1 (HO-1) expression, and increased cleaved poly(ADP-ribose) polymerase 1 (PARP1) expression in RPE cells. In contrast, comparable dosage of atRA-induced oxidative stress was much weaker, and it could not activate apoptosis in RPE cells. These results suggest that atRA generation is an antidotal metabolism pathway for atRal in the retina. Moreover, we found that in the eyes of ABCA4^-/-RDH8^-/- mice, a mouse model with atRal accumulation in the retina, the atRA content was almost the same as that in the wild type. It is possible that atRal accumulation simultaneously and equally promotes atRA synthesis and clearance in eyes of ABCA4^-/-RDH8^-/- mice, thus inhibiting the further increase of atRA in the retina. Our present study provides further insights into atRal clearance in the retina.
ATP-Binding Cassette Transporters/physiology* ; Alcohol Oxidoreductases/physiology* ; Animals ; Cell Survival/drug effects* ; Cells, Cultured ; Humans ; Inactivation, Metabolic ; Mice ; Retina/metabolism* ; Retinal Pigment Epithelium/metabolism* ; Swine ; Tretinoin/pharmacology*

OBJECTIVEThis study aimed to investigate the expression pattern and function of Nuclear receptor subfamily 2 group E member 1 (Nr2e1) in retinoic acid (RA)-induced brain abnormality.

METHODSThe mouse model of brain abnormality was established by administering 28 mg/kg RA, and neural stem cells (NSCs) were isolated from the mouse embryo and cultured in vitro. Nr2e1 expression was detected by whole mount in situ hybridization, RT-PCR, and Western blotting. Nr2e1 function was determined by transducing Nr2e1 shRNA into NSCs, and the effect on the sonic hedgehog (Shh) signaling pathway was assessed in the cells. In addition, the regulation of Nr2e1 expression by RA was also determined in vitro.

RESULTSNr2e1 expression was significantly downregulated in the brain and NSCs of RA-treated mouse embryos, and knockdown of Nr2e1 affected the proliferation of NSCs in vitro. In addition, a similar expression pattern of Nr2e1 and RA receptor (RAR) α was observed after treatment of NSCs with different concentrations of RA.

CONCLUSIONOur study demonstrated that Nr2e1 could be regulated by RA, which would aid a better understanding of the mechanism underlying RA-induced brain abnormality.

Animals ; Brain ; cytology ; embryology ; Cell Proliferation ; Down-Regulation ; Gene Expression Regulation ; Gene Expression Regulation, Developmental ; drug effects ; Mice ; Mice, Inbred C57BL ; Neural Stem Cells ; drug effects ; physiology ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Tretinoin ; pharmacology

All-trans retinoic acid (ATRA) is a vitamin A derivative and plays an important role in the regulation of cell aggregation, differentiation, apoptosis, proliferation, and inflammatory response. In recent years, some progress has been made in the role of ATRA in renal diseases, especially its protective effect on podocytes. This article reviews the research advances in podocyte injury, characteristics of ATRA, podocyte differentiation and regeneration induced by ATRA, and the protective effect of ATRA against proliferation, deposition of fibers, and apoptosis.
Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cytoprotection ; Humans ; Podocytes ; drug effects ; physiology ; Tretinoin ; pharmacology

The purpose of this study was to establish a drug screening method for small molecules extracted from traditional Chinese medicines (TCM) that have neuronal differentiation promoting effects, using P19 embryonic carcinoma cell as a cell-based model. First, the constructed plasmid (pTα1-Luc) was transfected into P19 cells to establish a screening model. Second, several TCMs were screened using the established model and all-trans-retinoic acid as a positive control. Finally, the underlying molecular mechanism was explored using immunofluorescence staining, qT-PCR, and Western blot analysis. Our results indicated that the drug screen model was established successfully and that both honokiol and hyperoside induced P19 differentiation into neurons, with the possible molecular mechanism being modulating the Wnt signaling pathway. In conclusion, the drug screening model developed in the present study provides a rapid, cell-based screening platform for identifying natural compounds with neuronal differentiation effects.
Animals ; Biphenyl Compounds ; pharmacology ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Evaluation, Preclinical ; methods ; Drugs, Chinese Herbal ; pharmacology ; Embryonal Carcinoma Stem Cells ; drug effects ; Lignans ; pharmacology ; Mice ; Neurons ; drug effects ; Quercetin ; analogs & derivatives ; pharmacology ; Tretinoin ; physiology ; Wnt Signaling Pathway

This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.
Ameloblasts ; physiology ; Amelogenesis ; genetics ; Amelogenin ; analysis ; Bone Morphogenetic Protein 4 ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Line ; Cell Lineage ; Embryonic Stem Cells ; drug effects ; physiology ; Epithelial Cells ; drug effects ; physiology ; Fibroblast Growth Factor 8 ; analysis ; Hedgehog Proteins ; analysis ; Homeodomain Proteins ; analysis ; Humans ; Keratins ; analysis ; classification ; Lithium Chloride ; pharmacology ; MSX1 Transcription Factor ; analysis ; Mouth Mucosa ; cytology ; Phenotype ; Regeneration ; physiology ; Skin ; cytology ; Transcription Factors ; analysis ; Tretinoin ; pharmacology

OBJECTIVETo establish an optimized primary drug screen model of neuronal differentiation using P19 embryonal carcinoma cells.

METHODSThe final concentration of retinoid acid (RA), days of suspension culture, manner of adherent culture, suitable cell density and adherent culture medium were tested, respectively. Two stages of neuronal differentiation were examined based on morphological changes and immunocytochemistry analysis of neuronal specific protein β-tubulin III.

RESULTSOn d 8 of differentiation culture, neuron-like cells were observed with final concentration of 1 μmol/L RA. Neuron-like network was formed on d 16 of neuronal differentiation. β-tubulin III was positively stained on both stages, indicating P19 cells were differentiated into neurons.

CONCLUSIONThe model using RA to induce P19 embryonic carcinoma cells to differentiate into neuron-like cells has been successfully established, which may provide a rapid, phenotypic cell-based platform for primary screening of neurogenesis-promoting drugs.

Animals ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; physiology ; Cell Line ; Embryonal Carcinoma Stem Cells ; cytology ; drug effects ; Mice ; Neurogenesis ; drug effects ; Neurons ; cytology ; metabolism ; Phenotype ; Tretinoin ; pharmacology ; Tubulin ; metabolism

OBJECTIVETo study the effects of TrkB-BDNF signal pathway on the synthesis and secretion of vascular endothelial growth factor (VEGF) in human neuroblastoma cells (NB).

METHODSTrkB protein expression in SY5Y cells before and after all-trans-retinoicacid (ATRA) treatment was detected by Western blot. P-TrkB protein expression in SY5Y cells before and after the treatment of ATRA along with BDNF was also detected by Western blot. VEGF concentrations in the SY5Y cell culture supernatants were measured using ELISA after the treatment with ATRA, BDNF, tyrosine kinase inhibitor K252a and PI3k inhibitor LY294002.

RESULTSTrkB protein was undetectable in SY5Y cells before ATRA treatment. After the treatment of 1, 10 and 100 nM/L ATRA for five days, TrkB protein was expressed in SY5Y cells and the TrkB protein level increased with the increasing ATRA concentration. P-TrkB protein was not expressed in SY5Y cells treated only with 10 nM/L ATRA, but it was detectable after the treatment of ATRA along with BDNF. VEGF concentrations in the group treated with ATRA+BDNF were significantly higher than those in the untreated control and the ATRA alone treatment groups (P<0.01). VEGF concentrations in the K252a pretreated ATRA+BDNF group were significantly lower than those in the group treated with ATRA+BDNF (P<0.05). VEGF concentrations in the LY294002 treatment group (ATRA+LY294002+BDNF group) were also significantly lower than those in the group treated with ATRA+BDNF (P<0.01).

CONCLUSIONSActivation of TrkB-BDNF signal pathway may increase the synthesis and secretion of VEGF in human NB cells. The synthesis and secretion of VEGF can be inhibited by blocking TrkB-BDNF signal pathway with K252a or blocking the TrkB-BDNF downstream signal pathway PI3K/Akt with LY294002.

Brain-Derived Neurotrophic Factor ; physiology ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; Humans ; Neuroblastoma ; metabolism ; pathology ; Phosphatidylinositol 3-Kinases ; physiology ; Proto-Oncogene Proteins c-akt ; physiology ; Receptor, trkB ; physiology ; Signal Transduction ; physiology ; Tretinoin ; pharmacology ; Vascular Endothelial Growth Factor A ; analysis ; biosynthesis

OBJECTIVETo study the role of Ezh2 in the all-trans retinoic acid RA induced P19 neural differentiation.

METHODSThe expression of Ngn1 in the RA induced P19 cells was detected at the mRNA and protein levels using real time RT-PCR and Western blot assays. The binding of Ezh2 and H3K27me3 on the Ngn 1 promoter was analyzed using chromatin immunoprecipitation assay.

RESULTIn the RA induced P19 cells, the recruitment of Ezh2 and its methylated substrate H3K27me3 on the promoter of Ngn 1 gene elevated in the first 2 days, and then declined rapidly, followed by the initiation of neuronal differentiation.

CONCLUSIONSEzh2 produces a repressive histone mark H3K27me3 in the early stage of RA induced P12 cells. By avoiding the premature expression of Ngn1 gene, Ezh2 can ensure the normal differentiation of P19 cells.

Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Cell Differentiation ; drug effects ; physiology ; Enhancer of Zeste Homolog 2 Protein ; Histone-Lysine N-Methyltransferase ; genetics ; metabolism ; Histones ; metabolism ; Mice ; Nerve Tissue Proteins ; genetics ; metabolism ; Neurons ; cytology ; drug effects ; metabolism ; Polycomb Repressive Complex 2 ; Tretinoin ; pharmacology ; Tumor Cells, Cultured

OBJECTIVETo study the role of extracellular matrix (ECM) in neural differentiation of mouse embryonic stem cells (ESCs).

METHODSMouse ESCs were incubated in the ESC conditioned medium, and the formation of embryonic bodies (EBs) were induced in bacteriological dishes using high-concentration all-trans retinoic acid (RA). The EBs were seeded on different matrixes (gelatin, fibronectin, and laminin/poly-L-ornithine) to test their impact on neural differentiation of the ESCs using immunofluorescence assay. The effect of laminin/poly-L-ornithine on the growth of neurites was evaluated with fluorescence microscopy.

RESULTSHigh-concentration RA activated and accelerated the differentiation of ESCs toward nestin-positive neural progenitor cells. Fibronectin supplement in the matrix dose-dependently promoted ESC differentiation into neural progenitor cells, while laminin/poly-L-ornithine increased the growth of the neurites and induced the maturation of the differentiated neural cells.

CONCLUSIONECM plays an important role in neural differentiation of mouse ESCs, and application of FN produces the most conspicuous effect during the differentiation of the ESCs into the neural progenitor cells;laminin/poly-L-ornithine is the most effective during their differentiation into neural cells.

Animals ; Cell Differentiation ; drug effects ; Cells, Cultured ; Culture Media ; Embryonic Stem Cells ; cytology ; drug effects ; Extracellular Matrix ; physiology ; Fibronectins ; pharmacology ; Laminin ; pharmacology ; Mice ; Neurons ; cytology ; drug effects ; Peptides ; pharmacology ; Tretinoin ; pharmacology