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Organogenesis 2018To investigate the molecular mechanism underlying taurine-stimulated proliferation and differentiation of cochlear neural stem cells (NSCs) and potential involvement of...
To investigate the molecular mechanism underlying taurine-stimulated proliferation and differentiation of cochlear neural stem cells (NSCs) and potential involvement of Sonic Hedgehog (Shh) pathway. The NSCs were characterized with immunofluorescence stained with nestin antibody. Cell viability was determined by MTT assay. The relative proliferation was measured by BrdU incorporation assay. The morphologic index was measured under light microscope. The relative protein level was determined by immunoblotting. Here we presented our findings that taurine stimulated proliferation and neurite outgrowth of NSCs, which was completely abolished by Shh inhibitor cyclopamine. In addition, cyclopamine antagonized taurine's effect on glutamatergic and GABAergic neuron population via suppressing expressions of Ptc-1, Smo and Gli-1. Our data supported the critical role of Shh pathway underlying the protective effect of taurine on auditory neural system.
Topics: Animals; Cell Differentiation; Cell Proliferation; Cell Survival; Cochlea; GABAergic Neurons; Hedgehog Proteins; Mice, Inbred BALB C; Nerve Tissue Proteins; Neural Stem Cells; Neuronal Outgrowth; Signal Transduction; Spiral Ganglion; Taurine; Veratrum Alkaloids
PubMed: 30102120
DOI: 10.1080/15476278.2018.1477462 -
The American Journal of Pathology Oct 2018Caroli disease represents a hepatic manifestation of autosomal recessive polycystic kidney disease, and belongs to a class of cholangiociliopathies. The role of Hedgehog...
Caroli disease represents a hepatic manifestation of autosomal recessive polycystic kidney disease, and belongs to a class of cholangiociliopathies. The role of Hedgehog signaling, a major pathway regulated by primary cilia, in biliary cystogenesis in Caroli disease remains unknown. Using the polycystic kidney (PCK) rat as an animal model of Caroli disease, this study investigated the involvement of Hedgehog signaling in its pathogenesis. In vitro experiments revealed that PCK cholangiocytes overexpressed Smoothened, Gli1, and Gli1's target molecule cyclin D1. The nuclear expression of Gli1, Gli2, and Gli3 was observed in PCK cholangiocytes by immunocytochemistry. An immunohistochemical analysis using liver sections confirmed the overexpression of Smoothened and cyclin D1, and the nuclear expression of the Gli proteins in the biliary epithelium of PCK rats as well as human Caroli disease. The treatment of PCK cholangiocytes with cyclopamine inhibited cell proliferative activity that was associated with the inhibition of nuclear translocation of Gli1 and Gli2, and reduced cyclin D1 expression. The in vivo administration of cyclopamine to PCK rats decreased abnormally elevated serum liver enzymes, and significantly attenuated bile duct dilation as well as kidney cyst formation. These results suggest that cholangiocyte hyperproliferation is causally associated with the aberrant activation of Hedgehog signaling, and the inhibition of the signaling has potential as a therapeutic strategy for biliary cystogenesis in Caroli disease.
Topics: Animals; Bile Ducts; Caroli Disease; Cell Proliferation; Cells, Cultured; Cyclin D1; Enzyme Inhibitors; Hedgehog Proteins; Male; Polycystic Kidney, Autosomal Recessive; Rats; Signal Transduction; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2; Zinc Finger Protein Gli3
PubMed: 30036521
DOI: 10.1016/j.ajpath.2018.06.014 -
Scientific Reports Jul 2018The objective of this study was to determine if Ihh is required for fracture healing. Fibular fracture was created in adult Col2a1-CreER; Ihh mice. Ihh mice received...
The objective of this study was to determine if Ihh is required for fracture healing. Fibular fracture was created in adult Col2a1-CreER; Ihh mice. Ihh mice received Tamoxifen (TM) to delete Ihh. WT mice received Cyclopamine to inhibit Hh pathway. Callus tissue properties and Ihh pathway were analyzed at 1, 2, and 3 weeks post-fracture by X-ray, micro-CT, mechanical test, RT-PCR and immunohistochemistry. Deleted Ihh was evidenced by the occurrence of growth plate closure in the Ihh mice by X-ray 3 weeks after TM treatment. All mice showed fracture healing at 3 weeks post-operation. Histology analysis indicated that, compared to the control, cartilage area was less in fracture sites from Ihh deficient animals by either genetic deletion or drug inhibition at 1 and 2 weeks post-fracture. Ihh immunostaining and its mRNA level were diminished in the fracture callus in Ihh reduced mice. There was no significant difference in BV/TV, BMD and mechanical test. Interruption to Ihh pathway by either genetic or pharmaceutical approach didn't affect fibular fracture healing in these mice. This surprised finding implicates that the deleted Ihh does not affect fracture healing in this model.
Topics: Animals; Chondrocytes; Collagen Type II; Disease Models, Animal; Down-Regulation; Fibula; Fracture Healing; Fractures, Bone; Hedgehog Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Signal Transduction; Veratrum Alkaloids; X-Ray Microtomography; Zinc Finger Protein GLI1
PubMed: 29985470
DOI: 10.1038/s41598-018-28657-7 -
Molecules (Basel, Switzerland) Jun 2018A simple and high sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous...
A simple and high sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of peimine and peiminine in beagle dog plasma after the oral administration of Maxim and Miq powder. Chromatographic separation was achieved on an ACQUIT UPLC BEH C column (1.7 μm, 2.1 × 100 mm) in a gradient elution way with a mobile phase consisting of acetonitrile and water containing 0.1% formic acid at a flow rate of 0.4 mL/min. The plasma samples were prepared by a liquid⁻liquid extraction (LLE) method with ethyl acetate. The analytes were detected with a triple quadrupole tandem mass spectrometry (MS) in multiple reaction monitoring (MRM) mode and a positive ion electrospray ionization (ESI) of the transitions at / 432.4→414.4 for peimine and / 430.3→412.3 for peiminine. The method was linear for two analytes over the investigated range with all determined correlation coefficients exceeding 0.9900. The lower limit of quantification (LLOQ) was 0.988 ng/mL for peimine and 0.980 ng/mL for peiminine. The mean extraction recoveries of peimine and peiminine at three quality control samples (QC) levels were ranged from 82.56 to 88.71%, and matrix effects ranged from 92.06 to 101.2%. The intra-day and inter-day precision and accuracy were within the acceptable limits at LLOQ and QC levels. The method was effectively and successfully applied to the pharmacokinetics of peimine and peiminine after oral administration of powder to beagle dogs. The obtained results may be help to guide the clinical application of Maxim and Miq.
Topics: Administration, Oral; Animals; Cevanes; Chromatography, Liquid; Dogs; Drugs, Chinese Herbal; Fritillaria; Male; Tandem Mass Spectrometry
PubMed: 29958456
DOI: 10.3390/molecules23071573 -
Acta Pharmacologica Sinica Nov 2018Veratridine is a lipid-soluble neurotoxin derived from plants in the family Liliaceae. It has been broadly investigated for its action as a sodium channel agonist....
Veratridine is a lipid-soluble neurotoxin derived from plants in the family Liliaceae. It has been broadly investigated for its action as a sodium channel agonist. However, the effects of veratridine on subtypes of sodium channels, especially Nav1.7, remain to be studied. Here, we investigated the effects of veratridine on human Nav1.7 ectopically expressed in HEK293A cells and recorded Nav1.7 currents from the cells using whole-cell patch clamp technique. We found that veratridine exerted a dose-dependent inhibitory effect on the peak current of Nav1.7, with the half-maximal inhibition concentration (IC) of 18.39 µM. Meanwhile, veratridine also elicited tail current (linearly) and sustained current [half-maximal concentration (EC): 9.53 µM], also in a dose-dependent manner. Veratridine (75 µM) shifted the half-maximal activation voltage of the Nav1.7 activation curve in the hyperpolarized direction, from -21.64 ± 0.75 mV to -28.14 ± 0.66 mV, and shifted the half-inactivation voltage of the steady-state inactivation curve from -59.39 ± 0.39 mV to -73.78 ± 0.5 mV. An increased frequency of stimulation decreased the peak and tail currents of Nav1.7 for each pulse along with pulse number, and increased the accumulated tail current at the end of train stimulation. These findings reveal the different modulatory effects of veratridine on the Nav1.7 peak current and tail current.
Topics: Dose-Response Relationship, Drug; HEK293 Cells; Humans; Ion Channel Gating; NAV1.7 Voltage-Gated Sodium Channel; Veratridine; Voltage-Gated Sodium Channel Blockers
PubMed: 29950616
DOI: 10.1038/s41401-018-0065-z -
Scientific Reports May 2018Adult stem cells are considered promising candidates for cellular therapies due to their capacity to differentiate and self-renew. Differentiation leads to changes in...
Adult stem cells are considered promising candidates for cellular therapies due to their capacity to differentiate and self-renew. Differentiation leads to changes in the metabolism, structure, and gene expression patterns of cells. Hedgehog is one of the pathways that is involved in the enhancement of osteogenesis and chondrogenesis in adult stem cells, but its mechanisms are poorly understood. In this study, we treated adipose tissue-derived stem cells (ADSC) with two well-characterized drugs, purmorphamine (Hedgehog pathway activator) and cyclopamine (Hedgehog pathway inhibitor), and identified mRNAs associated with polysomes in each treatment group to determine the post transcriptional genetic networks governed by the Hedgehog pathway. Activation of the Hedgehog pathway by purmorphamine results in significant upregulation of mRNAs associated with cellular communication and signal transduction. Furthermore, our experiments show that cyclopamine acts late downregulating GLI1 expression in ADSCs but promotes the upregulation of mRNAs associated with energy pathways and metabolism at early times. Through in silico analysis, we identified some miRNAs, such as miR-355, that could regulate these mRNAs association with polysomes and thereby modulate the Hedgehog pathway. Our results suggest that activation of the Hedgehog pathway by purmorphamine also results in a negative regulation of mRNAs in the protein translation machinery.
Topics: Adipose Tissue; Cell Nucleus; Energy Metabolism; Gene Regulatory Networks; Hedgehog Proteins; High-Throughput Nucleotide Sequencing; Humans; Morpholines; Polyribosomes; Purines; Sequence Analysis, RNA; Stem Cells; Veratrum Alkaloids
PubMed: 29849100
DOI: 10.1038/s41598-018-26533-y -
Cell Jul 2018The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is...
The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition.
Topics: Allosteric Regulation; Animals; Binding Sites; Cell Line; Cholesterol; Crystallography, X-Ray; Flow Cytometry; Hedgehog Proteins; Humans; Mice; Molecular Dynamics Simulation; Protein Binding; Protein Domains; Protein Structure, Tertiary; Signal Transduction; Small Molecule Libraries; Smoothened Receptor; Veratrum Alkaloids; Xenopus Proteins; Xenopus laevis
PubMed: 29804838
DOI: 10.1016/j.cell.2018.04.029 -
International Journal of Molecular... Mar 2018Neuroinflammation, characterized marked by microglial activation, plays a very important role in the pathogenesis of Parkinson's disease (PD). Upon activation,...
Neuroinflammation, characterized marked by microglial activation, plays a very important role in the pathogenesis of Parkinson's disease (PD). Upon activation, pro-inflammatory mediators are produced by microglia, triggering excessive inflammatory responses and ultimately damaging dopaminergic neurons. Therefore, the identification of agents that inhibit neuroinflammation may be an effective approach for developing novel treatments for PD. In this study, we sought to investigate whether peiminine protects dopaminergic neurons by inhibiting neuroinflammation. We evaluated the effects of peiminine on behavioural dysfunction, microglial activation and the loss of dopaminergic neurons in a rat model of lipopolysaccharide (LPS)-induced PD. BV-2 cells were pretreated with peiminine for 1 h and then stimulated with LPS for different times. Then, inflammatory responses and the related signalling pathways were analysed. Peiminine markedly attenuated behavioural dysfunction and inhibited the loss of dopaminergic neurons and microglial activation in the LPS-induced PD rat model. In BV-2 cells, peiminine significantly decreased LPS-induced expression of the pro-inflammatory mediators TNF-α, IL-6 and IL-1β, COX-2 and iNOS by inhibiting the phosphorylation of ERK1/2, AKT and NF-κB p65. Based on these results demonstrated that peiminine has a role in protecting dopaminergic neurons in the LPS-induced PD rat model by inhibiting neuroinflammation.
Topics: Animals; Anti-Inflammatory Agents; Cell Death; Cell Line; Cevanes; Cyclooxygenase 2; Dopaminergic Neurons; Female; Interleukins; Lipopolysaccharides; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Neuroprotective Agents; Nitric Oxide Synthase Type II; Proto-Oncogene Proteins c-akt; Rats, Wistar; Signal Transduction; Tumor Necrosis Factor-alpha
PubMed: 29534526
DOI: 10.3390/ijms19030821 -
Clinical Toxicology (Philadelphia, Pa.) Sep 2018Steroidal alkaloids are found in plants of the genus Veratrum. Their toxicity manifests as gastrointestinal symptoms followed by a Bezold-Jarisch reflex: hypopnea,...
INTRODUCTION
Steroidal alkaloids are found in plants of the genus Veratrum. Their toxicity manifests as gastrointestinal symptoms followed by a Bezold-Jarisch reflex: hypopnea, hypotension, and bradycardia. Some Veratrum steroidal alkaloids are also teratogens interfering with the hedgehog-2 signaling pathway, which causes cyclopsia and holoprosencephaly. We present a case of accidental poisoning from Veratrum parviflorum mistaken for the edible Allium tricoccum (ramps, wild leek).
CASE HISTORY
A 27-year-old man and his 25-year-old wife presented to the emergency department with nausea, vomiting, hypotension, and bradycardia after foraging and ingesting plants that they believed to be a local native species of wild leek.
METHODS
We collected and analyzed the implicated fresh plant material and both patients' serum/plasma. We used liquid chromatography-mass spectroscopy and high-resolution electrospray ionization time of flight tandem mass spectrometry to extract and characterize steroidal alkaloids from the foraged plant and patients' serum.
RESULTS
Our V. parviflorum samples contained verazine, veratramine, veratridine, and cyclopamine.
DISCUSSION
Steroidal alkaloids have been previously isolated from Veratrum viride and Veratrum album and toxicity has been reported mainly from V. album species.
CONCLUSION
V. parviflorum toxicity manifests with gastrointestinal and cardiac symptoms. Treatment is symptomatic and supportive as with previous case reports of toxicity with other Veratrum species.
Topics: Adult; Antiemetics; Female; Gastrointestinal Diseases; Georgia; Humans; Male; Plant Poisoning; Treatment Outcome; Veratrum; Veratrum Alkaloids; Vomiting
PubMed: 29490507
DOI: 10.1080/15563650.2018.1442007 -
ENeuro 2018Axonal spike is an important upstream process of transmitter release, which directly impacts on release probability from the presynaptic terminals. Despite the...
Axonal spike is an important upstream process of transmitter release, which directly impacts on release probability from the presynaptic terminals. Despite the functional significance, possible activity-dependent modulation of axonal spikes has not been studied extensively, partly due to inaccessibility of the small structures of axons for electrophysiological recordings. In this study, we tested the possibility of use-dependent changes in axonal spikes at the hippocampal mossy fibers, where direct recordings from the axon terminals are readily feasible. Hippocampal slices were made from mice of either sex, and loose-patch clamp recordings were obtained from the visually identified giant mossy fiber boutons located in the stratum lucidum of the CA3 region. Stimulation of the granule cell layer of the dentate gyrus elicited axonal spikes at the single bouton which occurred in all or none fashion. Unexpected from the digital nature of spike signaling, the peak amplitude of the second spikes in response to paired stimuli at a 50-ms interval was slightly but reproducibly smaller than the first spikes. Repetitive stimuli at 20 or 100 Hz also caused progressive use-dependent depression during the train. Notably, veratridine, an inhibitor of inactivation of sodium channels, significantly accelerated the depression with minimal effect on the initial spikes. These results suggest that sodium channels contribute to use-dependent depression of axonal spikes at the hippocampal mossy fibers, possibly by shaping the afterdepolarization (ADP) following axonal spikes. Prolonged depolarization during ADP may inactivate a fraction of sodium channels and thereby suppresses the subsequent spikes at the hippocampal mossy fibers.
Topics: 4-Aminopyridine; Action Potentials; Animals; Axons; Mice, Inbred C57BL; Mossy Fibers, Hippocampal; Neuronal Plasticity; Neurotransmitter Agents; Patch-Clamp Techniques; Presynaptic Terminals; Sodium Channels; Tetrodotoxin; Time Factors; Tissue Culture Techniques; Veratridine
PubMed: 29468192
DOI: 10.1523/ENEURO.0415-17.2018