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Proceedings of the National Academy of... Jan 2018Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs,...
Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the Nav1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non-pore-blocking mechanisms and discovered Nav1.7-selective chemical scaffolds. Hence, we address a major hurdle in Nav1.7 drug discovery, and this mechanistic approach to assay design is applicable to Cav3.1, Kv1.3, and many other ion channels to facilitate drug discovery.
Topics: Animals; Drug Discovery; High-Throughput Screening Assays; Humans; Insect Proteins; Membrane Potentials; Molecular Targeted Therapy; NAV1.7 Voltage-Gated Sodium Channel; Rats; Veratridine; Voltage-Gated Sodium Channel Blockers; Wasp Venoms
PubMed: 29311306
DOI: 10.1073/pnas.1713701115 -
Life Sciences Mar 2018The sperm plasma membrane contains specific ion channels and transporters that initiate changes in Ca, Na, K and H ions in the sperm cytoplasm. Ion channels are key...
AIMS
The sperm plasma membrane contains specific ion channels and transporters that initiate changes in Ca, Na, K and H ions in the sperm cytoplasm. Ion channels are key regulators of the sperm membrane potential, cytoplasmic Ca and intracellular pH (pH), which leads to regulate motility, capacitation, acrosome reaction and other physiological processes crucial for successful fertilization. Expression of epithelial sodium channels (ENaC) and voltage-gated sodium channels (Na) in human spermatozoa has been reported, but the role of Na fluxes sodium channels in the regulation of sperm cell function remains poorly understood. In this context, we aimed to analyze the physiological role of Na channels in human sperm.
MAIN METHODS
Motility and hyperactivation analysis was conducted by CASA analysis. Flow cytometry and spectrophotometry approaches were carried out to measure Capacitation, Acrosome reaction, immunohistochemistry for Tyr-residues phosporylation, [Ca] levels and membrane potential.
KEY FINDINGS
Functional studies showed that veratridine, a voltage-gated sodium channel activator, increased sperm progressive motility without producing hyperactivation while the Na antagonist lidocaine did induce hyperactivated motility. Veratridine increased protein tyrosine phosphorylation, an event occurring during capacitation, and its effects were inhibited in the presence of lidocaine and tetrodotoxin. Veratridine had no effect on the acrosome reaction by itself, but was able to block the progesterone-induced acrosome reaction. Moreover, veratridine caused a membrane depolarization and modified the effect of progesterone on [Ca] and sperm membrane potential.
SIGNIFICANCE
Our results suggest that veratridine-sensitive Na channels are involved on human sperm fertility acquisition regulating motility, capacitation and the progesterone-induced acrosome reaction in human sperm.
Topics: Acrosome Reaction; Adolescent; Adult; Female; Fertilization; Humans; Immunohistochemistry; In Vitro Techniques; Lidocaine; Male; Membrane Potentials; Progesterone; Receptors, Androgen; Semen; Sodium; Sodium Channel Agonists; Sodium Channel Blockers; Sodium Channels; Sperm Capacitation; Sperm Motility; Spermatozoa; Veratridine; Young Adult
PubMed: 29307525
DOI: 10.1016/j.lfs.2018.01.004 -
International Journal of Molecular... Feb 2019The present study aimed to investigate the effects of astragaloside IV on osteoblast‑like cell proliferation and migration, in addition to the underlying signaling...
The present study aimed to investigate the effects of astragaloside IV on osteoblast‑like cell proliferation and migration, in addition to the underlying signaling pathway. In order to observe the effect on proliferation, a Cell Counting Kit‑8 assay and flow cytometry were used. To detect cell migration ability, cell scratch and Transwell cell migration assays were performed. The RNA and protein expression levels of hedgehog signaling molecules, including Sonic hedgehog (SHH) and GLI family zinc finger 1 (GLI1), were examined by reverse transcription‑quantitative polymerase chain reaction and western blot analyses. To inhibit the hedgehog signaling pathway, cyclopamine was used. Astragaloside IV, at a dosage of 1x10‑2 µg/ml in MG‑63 cells and 1x10‑3 µg/ml in U‑2OS cells, resulted in the enhanced proliferation and migration of cells, and the gene expression levels of the SHH and GLI1 were significantly increased. The combination of astragaloside IV and cyclopamine reduced MG‑63 and U‑2OS cell proliferation and migration, and inhibited the gene expression of SHH and GLI1. Astragaloside IV enhanced the proliferation and migration of human osteoblast‑like cells through activating the hedgehog signaling pathway. The results of the present study provide a rational for the mechanistic link in astragaloside IV promoting the proliferation and migration of osteoblasts via the hedgehog signaling pathway.
Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Antagonism; Drugs, Chinese Herbal; Hedgehog Proteins; Humans; Molecular Targeted Therapy; Osseointegration; Osteoblasts; Osteogenesis; Saponins; Signal Transduction; Triterpenes; Veratrum Alkaloids; Zinc Finger Protein GLI1
PubMed: 30535481
DOI: 10.3892/ijmm.2018.4013 -
International Immunopharmacology Oct 2022Peimine (PM), a main isosterol alkaloid component isolated from the bulbs of traditional Chinese herb Fritillaria cirrhosa D. Don, has been demonstrated to exhibit...
BACKGROUND AND PURPOSE
Peimine (PM), a main isosterol alkaloid component isolated from the bulbs of traditional Chinese herb Fritillaria cirrhosa D. Don, has been demonstrated to exhibit multiple pharmacological properties, including anti-inflammation, anti-cancer and pain suppression. However, its effect on rheumatoid arthritis (RA) remains unknown. In the present study, we investigated the effect of PM on collagen-induced arthritis (CIA) rats in vivo and its inhibition on destructive behaviors of arthritic fibroblast-like synoviocytes (FLSs) in vitro.
METHODS
Arthritis was induced in rats by chicken type II collagen. Arthritis score, radiological evaluation, and histopathological assessment were used to evaluate the therapeutic effects of PM on CIA rats. EdU assay, wound healing assay and real-time PCR were used to examine the inhibitory effect of PM on proliferation, migration, and over-expression of pro-inflammatory cytokines in TNFα-induced arthritic FLSs. TRAP staining and scanning electron microscopy were used to analyze the effect of PM on osteoclastogensis and bone resorption. Western blot was used to reveal PM's molecular mechanism of action on RA.
RESULTS
PM significantly suppressed synovitis and bone destruction in CIA rats. In vitro experiments showed that PM treatment significantly inhibited TNFα-induced destructive behaviors of arthritic FLSs, including over-proliferation, migration and over-expression of pro-inflammatory cytokines. Additionally, RANKL-induced osteoclast formation and bone-resorpting function were also inhibited by PM. Further molecular mechanism studies revealed that PM treatment significantly suppressed TNFα-induced activations of MAPKs (ERK, JNK and p38) in arthritic FLSs.
CONCLUSION
Our findings provide strong evidence that PM has the potential to be developed as a therapeutic agent for patients with RA.
Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Cell Proliferation; Cells, Cultured; Cevanes; Cytokines; Fibroblasts; Rats; Synoviocytes; Tumor Necrosis Factor-alpha
PubMed: 36027853
DOI: 10.1016/j.intimp.2022.109181 -
Biopharmaceutics & Drug Disposition Jul 2015Veratramine, a steroidal alkaloid originating from Veratrum nigrum L., has demonstrated distinct anti-tumor and anti-hypertension effects, however, its metabolism has...
Veratramine, a steroidal alkaloid originating from Veratrum nigrum L., has demonstrated distinct anti-tumor and anti-hypertension effects, however, its metabolism has rarely been explored. The objective of the current study was to provide a comprehensive investigation of its metabolic pathways. The in vitro metabolic profiles of veratramine were evaluated by incubating it with liver microsomes and cytosols. The in vivo metabolic profiles in plasma, bile, urine and feces were monitored by UPLC-MS/MS after oral (20 mg/kg) and i.v. (50 µg/kg) administration in rats. Meanwhile, related P450s inhibitors and recombinant P450s and SULTs were used to identify the isozymes responsible for its metabolism. Eleven metabolites of veratramine, including seven hydroxylated, two sulfated and two glucuronidated metabolites, were characterized. Unlike most alkaloids, the major reactive sites of veratramine were on ring A and B instead of on the amine moiety. CYP2D6 was the major isozyme mediating hydroxylation, and substrate inhibition was observed with a Vmax , Ki and Clint of 2.05 ± 0.53 nmol/min/mg, 33.08 ± 10.13 µ m and 13.58 ± 1.27 µL/min/mg. SULT2A1, with Km , Vmax and Clint values of 19.37 ± 0.87 µ m, 1.51 ± 0.02 nmol/min/mg and 78.19 ± 8.57 µL/min/mg, was identified as the major isozyme contributing to its sulfation. In conclusion, CYP2D6 and SULT2A1 mediating hydroxylation and sulfation were identified as the major biotransformation for veratramine.
Topics: Animals; Arylsulfotransferase; Bile; Cytochrome P-450 Enzyme System; Cytosol; Feces; Humans; Isoenzymes; Liver; Male; Microsomes, Liver; Rats, Sprague-Dawley; Veratrum Alkaloids
PubMed: 25765359
DOI: 10.1002/bdd.1942 -
Virus Research Jan 2024Porcine epidemic diarrhea (PED) is a contagious intestinal disease caused by α-coronavirus porcine epidemic diarrhea virus (PEDV). At present, no effective vaccine is...
Porcine epidemic diarrhea (PED) is a contagious intestinal disease caused by α-coronavirus porcine epidemic diarrhea virus (PEDV). At present, no effective vaccine is available to prevent the disease. Therefore, research for novel antivirals is important. This study aimed to identify the antiviral mechanism of Veratramine (VAM), which actively inhibits PEDV replication with a 50 % inhibitory concentration (IC) of ∼5 µM. Upon VAM treatment, both PEDV-nucleocapsid (N) protein level and virus titer decreased significantly. The time-of-addition assay results showed that VAM could inhibit PEDV replication by blocking viral entry. Importantly, VAM could inhibit PEDV-induced phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) activity and further suppress micropinocytosis, which is required for PEDV entry. In addition, PI3K inhibitor LY294002 showed anti-PEDV activity by blocking viral entry as well. Taken together, VAM possessed anti-PEDV properties against the entry stage of PEDV by inhibiting the macropinocytosis pathway by suppressing the PI3K/Akt pathway. VAM could be considered as a lead compound for the development of anti-PEDV drugs and may be used during the viral entry stage of PEDV infection.
Topics: Animals; Chlorocebus aethiops; Coronavirus Infections; Phosphatidylinositol 3-Kinases; Porcine epidemic diarrhea virus; Proto-Oncogene Proteins c-akt; Swine; Swine Diseases; Veratrum Alkaloids; Vero Cells; Virus Internalization
PubMed: 37923169
DOI: 10.1016/j.virusres.2023.199260 -
ACS Chemical Neuroscience Feb 2020Voltage-gated sodium ion channels (Nas) are integral to both neuronal and muscular signaling and are a primary target for a number of proteinaceous and small molecule...
Voltage-gated sodium ion channels (Nas) are integral to both neuronal and muscular signaling and are a primary target for a number of proteinaceous and small molecule toxins. Included among these neurotoxins is veratridine (VTD), a C-nor-D homosteroidal alkaloid from the seeds of members of the genus. VTD binds to Na within the pore region, causing a hyperpolarizing shift in the activation threshold in addition to reducing peak current. We have characterized the activity of VTD against heterologously expressed rat Na1.4 and have demonstrated that VTD acts on the channel as either an agonist or antagonist depending on the nature of the electrophysiological stimulation protocol. Structure-activity studies with VTD and VTD derivatives against Na mutants show that the functional duality of VTD can be decoupled. These findings suggest that the dichotomous activity of VTD may derive from two distinct, use-dependent binding orientations of the toxin.
Topics: Animals; Neurons; Neurotoxins; Rats; Sodium; Veratridine; Voltage-Gated Sodium Channels
PubMed: 31951114
DOI: 10.1021/acschemneuro.9b00621 -
Bioorganic & Medicinal Chemistry Letters Oct 2016Three new steroidal alkaloids 1-3, together with four known compounds 4-7, were isolated from the ethanol extract of Veratrum grandiflorum Loes. Their structures were...
Three new steroidal alkaloids 1-3, together with four known compounds 4-7, were isolated from the ethanol extract of Veratrum grandiflorum Loes. Their structures were elucidated by NMR (1D and 2D NMR) and MS spectroscopic data. The inhibition activities on Hedgehog (Hh) pathway were evaluated using a cell-based bioassay system (Shh-LIGHT 2 cells). The results showed that compounds 1-3 and 5 displayed inhibitory activities obviously with the IC50 values of 0.63-3.11μM. Among them, compound 5 showed the most prominent inhibition activity (IC50=0.63±0.02μM). Thus, these active alkaloids may be potent natural compounds as Hh pathway inhibitors for the treatment of various cancers.
Topics: Cell Line; Hedgehog Proteins; Humans; Magnetic Resonance Spectroscopy; Spectrometry, Mass, Electrospray Ionization; Veratrum; Veratrum Alkaloids
PubMed: 27567371
DOI: 10.1016/j.bmcl.2016.08.040 -
Journal of Ethnopharmacology Jan 2022Fritillariae thunbergii Miq (FTM)exhibit versatile biological activities including the significant antitussive and expectorant activities. As a herbal medicine, the...
Chemical profiling of Fritillariae thunbergii Miq prepared by different processing methods reveals two new quality markers: Zhebeininoside and imperialine-3-β-D-glucoside.
ETHNOPHARMACOLOGICAL RELEVANCE
Fritillariae thunbergii Miq (FTM)exhibit versatile biological activities including the significant antitussive and expectorant activities. As a herbal medicine, the therapeutic effects of FTM may be expressed by multi-components which have complicated integration effects on multi-targets. With the time going, the different processing methods of FTM has been changed a lot. Thus,the study described the effect of processing methods to FTM and its quality.
MATERIAL AND METHOD
Studies were undertaken by using UHPLC-LTQ Orbitrap MS and pharmacodynamic models. All reagents were involved of analytical grade. While a HPLC-ELSD's method has been developed and validated, a certified Quality System is conformed to ICH requirements. The experimental animals followed the animal welfare guidelines.
AIM OF THE STUDY
We aimed to found the differences after the different processing methods of FTM, and to demonstrate the changes could be selected as quality control indicators, and established a method for simultaneous determination of these for quality control.
RESULTS
we have previously found two new steroidal alkaloids: zhebeininoside and imperialine-3-β-D-glucoside from the different processing methods of FTM, which is the difference between the different processing methods of FTM, mainly on the steroidal alkaloids. The activity analysis of zhebeininoside, imperialine-3-β-D-glucoside, verticine and verticinone showed that the mouse model of cough expectorant has antitussive effect. The positive drug selected was dextromethorphan syrup. The positive group showed biological activity, but the blank group showed nothing. The model group showed illness which means that the model was effective. There are two ways of the mechanism of action of the expectorant action which can make sputum thin, reduce its viscosity, and be easy to cough up, or can accelerate the movement of mucous cilia in the respiratory tract and promote the discharge of sputum. In our study, the content of phenol red was significantly reduced in the administration group.
CONCLUSIONS
To sum up, our results suggest that zhebeininoside and other three components cloud be selected as quality control indicators, and a method for simultaneous determination of zhebeininoside and other three components was established for quality control.
Topics: Animals; Mice; Ammonia; Antitussive Agents; Cevanes; Cough; Dextromethorphan; Drugs, Chinese Herbal; Fritillaria; Phytotherapy; Plant Stems; Quality Control; Random Allocation
PubMed: 34653522
DOI: 10.1016/j.jep.2021.114670 -
Annals of Clinical and Laboratory... Jan 2020The aim of the present study is to investigate the effect of cyclopamine, a hedgehog signaling pathway inhibitor, on adjuvant arthritis (AA), rat articular chondrocyte...
The aim of the present study is to investigate the effect of cyclopamine, a hedgehog signaling pathway inhibitor, on adjuvant arthritis (AA), rat articular chondrocyte viability, and part mechanisms In this study, an AA rat model was established by Freund's complete adjuvant (FCA). The arthritis index (AI), secondary paw swelling degree, and HE staining were used to evaluate whether the model was successfully established. Chondrocytes of the ankle joint of AA rats were cultured and identified. Cyclopamine (0, 0.03, 0.1, 0.3, 1, 3, 10 and 30 mg/l) was administered to determine chondrocyte viability. Chondrocyte apoptosis was detected by Annexin V-FITC/PI double dye. The expression of hedgehog signaling pathway-related proteins Shh, Ptch1, and Gli1 in chondrocytes was detected by western blotting. The results show that AA was successfully induced by FCA since the AI of AA rats and secondary paw swelling degree increased and the cartilage tissue of the rats' ankle joint was damaged. Thus, the chondrocytes were successfully cultured following the identification of toluidine blue and type II collagen. Cyclopamine (0.03, 0.1, 0.3, 1, 3, 10 and 30 mg/l) could increase the viability of chondrocytes and reduce the apoptotic rate of chondrocytes. As compared with the control group, different doses of cyclopamine (0.3, 3 and 10 mg/l) significantly decreased the expression of Shh, Ptch1 and Gli1 proteins in AA chondrocytes. Therefore, an AA rat model was successfully established in the present study and cyclopamine improved the viability and inhibited the apoptosis of chondrocytes. This is an effect that may be associated with the inhibition of the chondrocyte hedgehog signaling pathway.
Topics: Animals; Arthritis, Experimental; Cartilage, Articular; Cell Survival; Chondrocytes; Hedgehog Proteins; In Vitro Techniques; Male; Rats; Rats, Wistar; Veratrum Alkaloids
PubMed: 32161016
DOI: No ID Found