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Frontiers in Immunology 2020Activated fibroblast-like synoviocytes (FLSs) play a central role in the formation of synovial pannus and joint destruction in rheumatoid arthritis (RA). Targeting FLSs...
Activated fibroblast-like synoviocytes (FLSs) play a central role in the formation of synovial pannus and joint destruction in rheumatoid arthritis (RA). Targeting FLSs could be a potential therapeutic strategy. The objective of this study is to explore the role of c-Jun N-terminal kinase (JNK) in proliferation, migration and invasion of FLSs promoted by the sonic hedeghog (SHH) signaling pathway in patients with RA. Activation of SHH signaling was evaluated by real-time PCR and Western Blot. Levels of phosphorylation of JNK and c-Jun were detected by Western Blot. FLSs proliferation was quantified by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Cell migration and invasion were assessed by wound healing assay and Transwell chamber assay. Invasiveness of FLSs was evaluated using a humanized synovitis animal model. We observed that treatment of SHH agonist (SAG) significantly increased the levels of phosphorylation of JNK and c-Jun, while SHH antagonist (cyclopamine) significantly decreased the expression of phospho-JNK and phospho-c-Jun in FLSs. The elevated level of phospho-c-Jun stimulated by SAG was decreased in the presence of JNK inhibitor (SP600125) ( < 0.001). FLSs proliferation, migration and invasion were promoted by SHH agonist ( < 0.05). However, the enhanced aggressiveness of FLSs was abolished in the presence of JNK inhibitor ( < 0.05). study showed that the invasion of FLSs into cartilage was increased by SHH overexpression and the excessive invasiveness was inhibited by blockade of JNK signaling ( < 0.01). These results suggest that JNK is one of the downstream molecules mediating the effect of SHH signaling in FLSs. These findings indicate that SHH-JNK signaling could be a potential therapeutic target to suppress the aggressiveness of FLSs and prevent articular damage of RA.
Topics: Arthritis, Rheumatoid; Biomarkers; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Cytokines; Female; Flow Cytometry; Hedgehog Proteins; Humans; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Male; Matrix Metalloproteinase 1; Middle Aged; Synoviocytes; Veratrum Alkaloids
PubMed: 32670287
DOI: 10.3389/fimmu.2020.01300 -
Molecular Medicine Reports Aug 2020The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH...
The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH signaling pathway in the development of the ossification of ligamentum flavum (OLF) at the cellular and tissue levels. The expression levels and localization of the osteogenic genes Runt-related transcription factor 2 (RUNX2), Osterix, alkaline phosphatase (ALP), osteocalcin (OCN) and IHH were evaluated in OLF tissues by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. Non-ossified ligamentum flavum (LF) sections were used as control samples. The tissue explant method was used to obtain cultured LF cells. In addition, OLF cells were subjected to cyclic stretch application for 0, 6, 12 or 24 h. The expression levels of osteogenic genes, and the IHH signaling pathway genes IHH, Smoothened (SMO), GLI family zinc finger 1 (GLI1), GLI2 and GLI3 were evaluated with RT-qPCR and western blotting. Osteogenic differentiation was further evaluated by assessing ALP activity and staining. Moreover, the effect of cyclopamine (Cpn), an IHH signaling inhibitor, on osteogenic differentiation was examined. The RT-qPCR and immunohistochemical results indicated that the mRNA and protein expression levels of RUNX2, Osterix, ALP, OCN and IHH were significantly higher in the OLF group compared with the LF group. Furthermore, application of cyclic stretch to OLF cells resulted in greater ALP activity, and significant increases in mRNA and protein expression levels of RUNX2, Osterix, ALP and OCN in a time-d00ependent manner. Cyclic stretch application also led to significant increases in IHH signaling pathway genes, including IHH, SMO, GLI1 and GLI2, while no significant effect was found on GLI3 expression level. In addition, it was found that Cpn significantly reversed the effect of cyclic stretch on the ALP activity, and the expression levels of RUNX2, Osterix, ALP, OCN, GLI1 and GLI2. Collectively, the present results suggested that the IHH signaling pathway may mediate the effect of cyclic stretch on the OLF cells.
Topics: Adult; Aged; Alkaline Phosphatase; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Female; Hedgehog Proteins; Humans; Ligamentum Flavum; Male; Middle Aged; Nerve Tissue Proteins; Nuclear Proteins; Ossification, Heterotopic; Osteocalcin; Signal Transduction; Smoothened Receptor; Sp7 Transcription Factor; Stress, Mechanical; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2; Zinc Finger Protein Gli3
PubMed: 32626952
DOI: 10.3892/mmr.2020.11200 -
Scientific Reports Jul 2020Voltage-gated Na (Na) channels regulate homeostasis in bacteria and control membrane electrical excitability in mammals. Compared to their mammalian counterparts,...
Voltage-gated Na (Na) channels regulate homeostasis in bacteria and control membrane electrical excitability in mammals. Compared to their mammalian counterparts, bacterial Na channels possess a simpler, fourfold symmetric structure and have facilitated studies of the structural basis of channel gating. However, the pharmacology of bacterial Na remains largely unexplored. Here we systematically screened 39 Na modulators on a bacterial channel (NaChBac) and characterized a selection of compounds on NaChBac and a mammalian channel (human Na1.7). We found that while many compounds interact with both channels, they exhibit distinct functional effects. For example, the local anesthetics ambroxol and lidocaine block both Na1.7 and NaChBac but affect activation and inactivation of the two channels to different extents. The voltage-sensing domain targeting toxin BDS-I increases Na1.7 but decreases NaChBac peak currents. The pore binding toxins aconitine and veratridine block peak currents of Na1.7 and shift activation (aconitine) and inactivation (veratridine) respectively. In NaChBac, they block the peak current by binding to the pore residue F224. Nonetheless, aconitine has no effect on activation or inactivation, while veratridine only modulates activation of NaChBac. The conservation and divergence in the pharmacology of bacterial and mammalian Na channels provide insights into the molecular basis of channel gating and will facilitate organism-specific drug discovery.
Topics: Aconitine; Anesthetics, Local; Bacterial Proteins; Drug Interactions; Electrophysiological Phenomena; HEK293 Cells; Humans; Ion Channel Gating; NAV1.7 Voltage-Gated Sodium Channel; Small Molecule Libraries; Sodium Channels; Toxins, Biological; Veratridine; Voltage-Gated Sodium Channel Agonists
PubMed: 32612253
DOI: 10.1038/s41598-020-67761-5 -
Life (Basel, Switzerland) Jun 2020Veratrum-type steroidal alkaloids (VSA) are the major bioactive ingredients that strongly determine the pharmacological activities of . Biosynthesis of VSA at the...
Veratrum-type steroidal alkaloids (VSA) are the major bioactive ingredients that strongly determine the pharmacological activities of . Biosynthesis of VSA at the molecular and genetic levels is not well understood. Next-generation sequencing of representational difference analysis (RDA) products after elicitation and precursor feeding was applied to identify candidate genes involved in VSA biosynthesis. A total of 12,048 contigs with a median length of 280 bases were received in three RDA libraries obtained after application of methyl jasmonate, squalene and cholesterol. The comparative analysis of annotated sequences was effective in identifying candidate genes. GABAT2 transaminase and hydroxylases active at C-22, C-26, C-11, and C-16 positions in late stages of jervine biosynthesis were selected. Moreover, genes coding pyrroline-5-carboxylate reductase and enzymes from the short-chain dehydrogenases/reductases family (SDR) associated with the reduction reactions of the VSA biosynthesis process were proposed. The data collected contribute to better understanding of jervine biosynthesis and may accelerate implementation of biotechnological methods of VSA biosynthesis.
PubMed: 32575579
DOI: 10.3390/life10060088 -
Molecular Autism Jun 2020Fragile X syndrome (FXS), a neurodevelopmental disorder, is a leading monogenetic cause of intellectual disability and autism spectrum disorder. Notwithstanding the...
BACKGROUND
Fragile X syndrome (FXS), a neurodevelopmental disorder, is a leading monogenetic cause of intellectual disability and autism spectrum disorder. Notwithstanding the extensive studies using rodent and other pre-clinical models of FXS, which have provided detailed mechanistic insights into the pathophysiology of this disorder, it is only relatively recently that human stem cell-derived neurons have been employed as a model system to further our understanding of the pathophysiological events that may underlie FXS. Our study assesses the physiological properties of human pluripotent stem cell-derived cortical neurons lacking fragile X mental retardation protein (FMRP).
METHODS
Electrophysiological whole-cell voltage- and current-clamp recordings were performed on two control and three FXS patient lines of human cortical neurons derived from induced pluripotent stem cells. In addition, we also describe the properties of an isogenic pair of lines in one of which FMR1 gene expression has been silenced.
RESULTS
Neurons lacking FMRP displayed bursts of spontaneous action potential firing that were more frequent but shorter in duration compared to those recorded from neurons expressing FMRP. Inhibition of large conductance Ca-activated K currents and the persistent Na current in control neurons phenocopies action potential bursting observed in neurons lacking FMRP, while in neurons lacking FMRP pharmacological potentiation of voltage-dependent Na channels phenocopies action potential bursting observed in control neurons. Notwithstanding the changes in spontaneous action potential firing, we did not observe any differences in the intrinsic properties of neurons in any of the lines examined. Moreover, we did not detect any differences in the properties of miniature excitatory postsynaptic currents in any of the lines.
CONCLUSIONS
Pharmacological manipulations can alter the action potential burst profiles in both control and FMRP-null human cortical neurons, making them appear like their genetic counterpart. Our studies indicate that FMRP targets that have been found in rodent models of FXS are also potential targets in a human-based model system, and we suggest potential mechanisms by which activity is altered.
Topics: Action Potentials; Adolescent; Animals; Cell Differentiation; Cerebral Cortex; Child, Preschool; Fragile X Mental Retardation Protein; Humans; Indoles; Induced Pluripotent Stem Cells; Male; Mice; Neurons; Riluzole; Sodium Channels; Veratridine; Young Adult
PubMed: 32560741
DOI: 10.1186/s13229-020-00351-4 -
Oncology Reports Aug 2020Liver cancer is the second leading cause of cancer‑related deaths. Traditional therapeutic strategies, such as chemotherapy, targeted therapy and interventional...
Liver cancer is the second leading cause of cancer‑related deaths. Traditional therapeutic strategies, such as chemotherapy, targeted therapy and interventional therapy, are inefficient and are accompanied by severe side effects for patients with advanced liver cancer. Therefore, it is crucial to develop a safer more effective drug to treat liver cancer. Veratramine, a known natural steroidal alkaloid derived from plants of the lily family, exerts anticancer activity in vitro. However, the underlying mechanism and whether it has an antitumor effect in vivo remain unknown. In the present study, the data revealed that veratramine significantly inhibited HepG2 cell proliferation, migration and invasion in vitro. Moreover, it was revealed that veratramine induced autophagy‑mediated apoptosis by inhibiting the PI3K/Akt/mTOR signaling pathway, which partly explained the underlying mechanism behind its antitumor activity. Notably, the results of in vivo experiments also revealed that veratramine treatment (2 mg/kg, 3 times a week for 4 weeks) significantly inhibited subcutaneous tumor growth of liver cancer cells, with a low systemic toxicity. Collectively, the results of the present study indicated that veratramine efficiently suppressed liver cancer HepG2 cell growth in vitro and in vivo by blocking the PI3K/Akt/mTOR signaling pathway to induce autophagic cell death. Veratramine could be a potential therapeutic agent for the treatment of liver cancer.
Topics: Animals; Autophagic Cell Death; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Veratrum Alkaloids; Xenograft Model Antitumor Assays
PubMed: 32468056
DOI: 10.3892/or.2020.7622 -
Toxins May 2020Ciguatoxins (CTXs) are a group of neurotoxins responsible for the syndrome ciguatera fish poisoning (CFP) as a result of the consumption of contaminated fish. The...
Ciguatoxins (CTXs) are a group of neurotoxins responsible for the syndrome ciguatera fish poisoning (CFP) as a result of the consumption of contaminated fish. The presence of these toxins has been detected around the Pacific, Caribbean and Indian coasts. Recent reports indicate the emergence of CFP in other geographic areas, in particular in European coasts, of the Canary Islands (Spain) and Madeira (Portugal). A neuroblastoma cell line of murine origin (N2a) has been applied to assay different groups of neurotoxins, acting on voltage-gated sodium channel (VGSC) of excitable cells, N2a-MTT. The great potential of N2a-MTT as a sensitive tool for the CTXs screening is clearly recognized, notably because it allows the detection of these toxins at levels below recommended as security levels. However, the complexity of the matrix is a critical point on the application of N2a-MTT, which needs to be evaluated. The aim of this work is to provide recommendations for an implemented N2a-MTT method for CTXs determination in fish that avoids matrix effects, particularly those related to high lipid content.
Topics: Animals; Biological Assay; Cell Line, Tumor; Cell Survival; Ciguatoxins; Fishes; Membrane Potentials; Mice; Neurons; Ouabain; Veratridine; Voltage-Gated Sodium Channel Agonists; Voltage-Gated Sodium Channels
PubMed: 32397386
DOI: 10.3390/toxins12050308 -
The Journal of Organic Chemistry May 2020The synthesis of the stereotriad core in the eastern portion of the alkaloids jervine (), cyclopamine (), and veratramine () is reported. Starting from a known...
The synthesis of the stereotriad core in the eastern portion of the alkaloids jervine (), cyclopamine (), and veratramine () is reported. Starting from a known β-methyltyrosine derivative (), the route utilizes a diastereoselective substrate-controlled 1,2-reduction to establish the stereochemistry of the vicinal amino alcohol motif embedded within the targets. Oxidative dearomatization is demonstrated to be a viable approach for the synthesis of the spirocyclic DE ring junction found in jervine and cyclopamine.
Topics: Veratrum; Veratrum Alkaloids
PubMed: 32352768
DOI: 10.1021/acs.joc.0c00685 -
Toxins Apr 2020The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet a consensus protocol is still lacking. In this...
The neuroblastoma cell-based assay (CBA-N2a) is widely used for the detection of marine biotoxins in seafood products, yet a consensus protocol is still lacking. In this study, six key parameters of CBA-N2a were revisited: cell seeding densities, cell layer viability after 26 h growth, MTT incubation time, Ouabain and Veratridine treatment and solvent and matrix effects. A step-by-step protocol was defined identifying five viability controls for the validation of CBA-N2a results. Specific detection of two voltage gated sodium channel activators, pacific ciguatoxin (P-CTX3C) and brevetoxin (PbTx3) and two inhibitors, saxitoxin (STX) and decarbamoylsaxitoxin (dc-STX) was achieved, with EC values of 1.7 ± 0.35 pg/mL, 5.8 ± 0.9 ng/mL, 3 ± 0.5 ng/mL and 15.8 ± 3 ng/mL, respectively. When applied to the detection of ciguatoxin (CTX)-like toxicity in fish samples, limit of detection (LOD) and limit of quantification (LOQ) values were 0.031 ± 0.008 and 0.064 ± 0.016 ng P-CTX3C eq/g of flesh, respectively. Intra and inter-assays comparisons of viability controls, LOD, LOQ and toxicity in fish samples gave coefficients of variation (CVs) ranging from 3% to 29%. This improved test adaptable to either high throughput screening or composite toxicity estimation is a useful starting point for a standardization of the CBA-N2a in the field of marine toxin detection.
Topics: Animals; Biological Assay; Cell Line, Tumor; Cell Proliferation; Cell Survival; Ciguatoxins; Dose-Response Relationship, Drug; Fishes; Limit of Detection; Marine Toxins; Mice; Neuroblastoma; Neurons; Ouabain; Oxocins; Reproducibility of Results; Saxitoxin; Time Factors; Veratridine; Voltage-Gated Sodium Channel Agonists; Voltage-Gated Sodium Channels
PubMed: 32349302
DOI: 10.3390/toxins12050281 -
The Turkish Journal of Gastroenterology... Mar 2020To investigate the effect and the possible mechanism of lanthanum citrate on the proliferation and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721...
BACKGROUND/AIMS
To investigate the effect and the possible mechanism of lanthanum citrate on the proliferation and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 through the Hedgehog (Hh) signaling pathway.
MATERIALS AND METHODS
Different concentrations of lanthanum citrate and KAAD-cyclopamine (the Hh signaling pathway representative inhibitor) were used to treat SMMC-7721 cells. Cell proliferation was detected using Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected using flow cytometry analysis of Annexin V-FITC/ propidium iodide (PI). The protein expressions of regulatory genes, such as cell cycle protein D1 (CyclinD1), cyclin-dependent kinase inhibitor 1 (p21), cysteinyl aspartate specific proteinase 3 (Caspase-3), B-cell lymphoma-2 (Bcl-2), glioma-associated oncogene homolog 1 (Gli1), and sonic hedgehog (Shh) were quantified using Western blot assays. The mRNA expressions of Gli1 and Shh were tested using quantitative real-time polymerase chain reaction (qRT-PCR) assays and the protein expressions of Gli1 and Shh were determined using immunofluorescence assays.
RESULTS
The Annexin V-FITC and PI double staining results revealed that the 0.1 mM lanthanum citrate group and the 15 µM KAAD-cyclopamine group had both increased the apoptosis rate of SMMC-7721 cells. Both lanthanum citrate and KAAD-cyclopamine downregulated the protein expressions of CyclinD1, Bcl-2, Gli1, and Shh and upregulated the protein expressions of p21 and Caspase-3. Additionally, the immunofluorescence results revealed that the protein expressions of Gli1 and Shh were significantly decreased in both the lanthanum citrate group and the KAAD-cyclopamine group compared to the control group.
CONCLUSION
Lanthanum citrate inhibits proliferation and promotes apoptosis in HCC SMMC-7721 cells by suppressing the Hh signaling pathway.
Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cinnamates; Citrates; Hedgehog Proteins; Humans; Liver Neoplasms; Signal Transduction; Veratrum Alkaloids
PubMed: 32343239
DOI: 10.5152/tjg.2020.18800