-
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 -
British Journal of Pharmacology Mar 2022Corpus cavernosum smooth muscle (CCSM) exhibits phasic contractions that are coordinated by ion channels. Mouse models are commonly used to study erectile dysfunction,...
BACKGROUND AND PURPOSE
Corpus cavernosum smooth muscle (CCSM) exhibits phasic contractions that are coordinated by ion channels. Mouse models are commonly used to study erectile dysfunction, but there are few published electrophysiological studies of mouse CCSM. We describe the voltage-dependent sodium (Na ) currents in mouse CCSM and investigate their function.
EXPERIMENTAL APPROACH
We used electrophysiological, pharmacological and immunocytochemical methods to study the Na currents in isolated CCSM cells from C57BL/6 mice. Tension measurements were carried out using crural sections of the corpus cavernosum in whole tissue.
KEY RESULTS
Fast, voltage-dependent, sodium currents in mouse CCSM were induced by depolarising steps. Steady-state activation and inactivation curves revealed a window current between -60 and -30 mV. Two populations of Na currents, 'TTX-sensitive' and 'TTX-insensitive', were identified. TTX-sensitive currents showed 48% block with the Na channel subtype-specific blockers ICA-121431 (Na 1.1-1.3), PF-05089771 (Na 1.7) and 4,9-anhydro-TTX (Na 1.6). TTX-insensitive currents were resistant to blockade by A803467, specific for Na 1.8 channels. Immunocytochemistry confirmed expression of Na 1.5 and Na 1.4 in freshly dispersed CCSM cells. Veratridine, a Na channel activator, reduced time-dependent inactivation of Na currents and increased duration of evoked action potentials. Veratridine induced phasic contractions in CCSM strips, reversible with TTX and nifedipine but not KB-R7943.
CONCLUSION AND IMPLICATIONS
There are fast, voltage-dependent, sodium currents in mouse CCSM. Stimulation of these currents increased contractility of CCSM in vitro, suggesting an involvement in detumescence and potentially providing a clinically relevant target in erectile dysfunction. Further work will be necessary to define its role.
Topics: Animals; Erectile Dysfunction; Humans; Male; Mice; Mice, Inbred C57BL; Myocytes, Smooth Muscle; Sodium; Sodium Channel Blockers; Tetrodotoxin; Veratridine
PubMed: 34767251
DOI: 10.1111/bph.15728 -
American Journal of Physiology. Cell... Sep 2022Isolated smooth muscle cells (SMCs) from mouse bronchus were studied using the whole cell patch-clamp technique at ∼21°C. Stepping from -100 mV to -20 mV evoked...
Isolated smooth muscle cells (SMCs) from mouse bronchus were studied using the whole cell patch-clamp technique at ∼21°C. Stepping from -100 mV to -20 mV evoked inward currents of mean amplitude -275 pA. These inactivated (tau = 1.1 ms) and were abolished when external Na was substituted with -Methyl-d-glucamine. In current-voltage protocols, current peaked at -10 mV and reversed between +20 and +30 mV. The Vs of activation and inactivation were -25 and -86 mV, respectively. The current was highly sensitive to tetrodotoxin (IC = 1.5 nM) and the Na1.7 subtype-selective blocker, PF-05089771 (IC = 8.6 nM), consistent with Na1.7 as the underlying pore-forming α subunit. Two Na1.7-selective antibodies caused membrane-delineated staining of isolated SMC, as did a nonselective pan-Na antibody. RT-PCR, performed on groups of ∼15 isolated SMCs, revealed transcripts for Na1.7 in 7/8 samples. Veratridine (30 µM), a nonselective Na channel activator, reduced peak current evoked by depolarization but induced a sustained current of 40 pA. Both effects were reversed by tetrodotoxin (100 nM). In tension experiments, veratridine (10 µM) induced contractions that were entirely blocked by atropine (1 µM). However, in the presence of atropine, veratridine was able to modulate the pattern of activity induced by a combination of U-46619 (a thromboxane A2 mimetic) and PGE (prostaglandin E), by eliminating bursts in favor of sustained phasic contractions. These effects were readily reversed to control-like activity by tetrodotoxin (100 nM). In conclusion, mouse bronchial SMCs functionally express Na1.7 channels that are capable of modulating contractile activity, at least under experimental conditions.
Topics: Animals; Atropine Derivatives; Bronchi; Mice; Myocytes, Smooth Muscle; Sodium; Tetrodotoxin; Veratridine
PubMed: 35876287
DOI: 10.1152/ajpcell.00011.2022 -
Journal of Ethnopharmacology Jan 2020Bulbus Fritillaria cirrhosa D. Don (BFC) is a Chinese traditional herbal medicine that has long been used as an indispensable component in herbal prescriptions for...
ETHNOPHARMACOLOGICAL RELEVANCE
Bulbus Fritillaria cirrhosa D. Don (BFC) is a Chinese traditional herbal medicine that has long been used as an indispensable component in herbal prescriptions for bronchopulmonary diseases due to its well-established strong anti-inflammation and pulmonary harmonizing effects. Interestingly, there are few case reports in traditional Chinese medicine available where they found it to contribute in anti-tumor therapies. Imperialine is one of the most favored active substances extracted from BFC and has been widely recognized as an anti-inflammatory agent.
AIM OF THE STUDY
The aim of the current work is to provide first-hand evidences both in vitro and in vivo showing that imperialine exerts anti-cancer effects against non-small cell lung cancer (NSCLC), and to explore the molecular mechanism of this anti-tumor activity. It is also necessary to examine its systemic toxicity, and to investigate how to develop strategies for feasible clinical translation of imperialine.
MATERIALS AND METHODS
To investigate anti-NSCLC efficacy of imperialine using both in vitro and in vivo methods where A549 cell line were chosen as in vitro model NSCLC cells and A549 tumor-bearing mouse model was constructed for in vivo study. The detailed underlying anti-cancer mechanism has been systematically explored for the first time through a comprehensive set of molecular biology methods mainly including immunohistochemistry, western blot and enzyme-linked immunosorbent assays. The toxicity profile of imperialine treatments were evaluated using healthy nude mice by examining hemogram and histopathology. An imperialine-loaded liposomal drug delivery system was developed using thin film hydration method to evaluate target specific delivery.
RESULTS
The results showed that imperialine could suppress both NSCLC tumor and associated inflammation through an inflammation-cancer feedback loop in which NF-κB activity was dramatically inhibited by imperialine. The NSCLC-targeting liposomal system was successfully developed for targeted drug delivery. The developed platform could favorably enhance imperialine cellular uptake and in vivo accumulation at tumor sites, thus improving overall anti-tumor effect. The toxicity assays revealed imperialine treatments did not significantly disturb blood cell counts in mice or exert any significant damage to the main organs.
CONCLUSIONS
Imperialine exerts anti-cancer effects against NSCLC both in vitro and in vivo, and this previously unknown function is related to NF-κB centered inflammation-cancer feedback loop. Imperialine mediated anti-cancer activity is not through cytotoxicity and exhibit robust systemic safety. Furthermore, the liposome-based system we commenced would dramatically enhance therapeutic effects of imperialine while exhibiting extremely low side effects both on cellular and in NSCLC model. This work has identified imperialine as a promising novel anti-cancer compound and offered an efficient target-delivery solution that greatly facilitate practical use of imperialine.
Topics: A549 Cells; Alkaloids; Animals; Blood Cell Count; Carcinoma, Non-Small-Cell Lung; Cevanes; Drugs, Chinese Herbal; Feedback, Physiological; Fritillaria; Humans; Liposomes; Lung Neoplasms; Male; Mice; NF-kappa B; Toxicity Tests; Xenograft Model Antitumor Assays
PubMed: 31605736
DOI: 10.1016/j.jep.2019.112283 -
International Immunopharmacology Jan 2020Isosteroid alkaloids, natural products from Fritillariae Cirrhosae Bulbus, are well known for its antitussive, expectorant, anti-asthmatic and anti-inflammatory...
Isosteroid alkaloids with different chemical structures from Fritillariae cirrhosae bulbus alleviate LPS-induced inflammatory response in RAW 264.7 cells by MAPK signaling pathway.
Isosteroid alkaloids, natural products from Fritillariae Cirrhosae Bulbus, are well known for its antitussive, expectorant, anti-asthmatic and anti-inflammatory properties. However, the anti-inflammatory effect and its mechanism have not been fully explored. In this study, the anti-inflammatory activitives and the potential mechanisms of five isosteroid alkaloids from F. Cirrhosae Bulbus were investigated in lipopolysaccharide (LPS)-induced RAW264.7 macrophage cells. The pro-inflammatory mediators and cytokines were measured by Griess reagent, ELISA and qRT-PCR. The expression of MAPKs was investigated by western blotting. Treatment with the five isosteroid alkaloids in appropriate concentrations could reduce the production of nitric oxide (NO), tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) in supernatant, and suppressed the mRNA expressions of TNF-α and IL-6. Meanwhile, the five isosteroid alkaloids significantly inhibited the phosphorylated activation of mitogen activated protein kinase (MAPK) signaling pathways, including extracellular signal-regulated kinase (ERK1/2), p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). These results demonstrated that isosteroid alkaloids from F. Cirrhosae Bulbus exert anti-inflammatory effects by down-regulating the level of inflammatory mediators via mediation of MAPK phosphorylation in LPS-induced RAW264.7 macrophages, thus could be candidates for the prevention and treatment of inflammatory diseases.
Topics: Alkaloids; Animals; Anti-Inflammatory Agents; Cevanes; Drugs, Chinese Herbal; Fritillaria; Inflammation; Interleukin-6; Lipopolysaccharides; MAP Kinase Signaling System; Mice; Phosphorylation; Plant Roots; RAW 264.7 Cells; Structure-Activity Relationship; Tumor Necrosis Factor-alpha
PubMed: 31816576
DOI: 10.1016/j.intimp.2019.106047 -
International Journal of Molecular... Aug 2023The apical dendrite of a cortical projection neuron (CPN) is generated from the leading process of the migrating neuron as the neuron completes migration. This...
The apical dendrite of a cortical projection neuron (CPN) is generated from the leading process of the migrating neuron as the neuron completes migration. This transformation occurs in the cortical marginal zone (MZ), a layer that contains the Cajal-Retzius neurons and their axonal projections. Cajal-Retzius neurons (CRNs) are well known for their critical role in secreting Reelin, a glycoprotein that controls dendritogenesis and cell positioning in many regions of the developing brain. In this study, we examine the possibility that CRNs in the MZ may provide additional signals to arriving CPNs, that may promote the maturation of CPNs and thus shape the development of the cortex. We use whole embryonic hemisphere explants and multiphoton microscopy to confirm that CRNs display intracellular calcium transients of <1-min duration and high amplitude during early corticogenesis. In contrast, developing CPNs do not show high-amplitude calcium transients, but instead show a steady increase in intracellular calcium that begins at the time of dendritic initiation, when the leading process of the migrating CPN is encountering the MZ. The possible existence of CRN to CPN communication was revealed by the application of veratridine, a sodium channel activator, which has been shown to preferentially stimulate more mature cells in the MZ at an early developmental time. Surprisingly, veratridine application also triggers large calcium transients in CPNs, which can be partially blocked by a cocktail of antagonists that block glutamate and glycine receptor activation. These findings outline a model in which CRN spontaneous activity triggers the release of glutamate and glycine, neurotransmitters that can trigger intracellular calcium elevations in CPNs. These elevations begin as CPNs initiate dendritogenesis and continue as waves in the post-migratory cells. Moreover, we show that the pharmacological blockade of glutamatergic signaling disrupts migration, while forced expression of a bacterial voltage-gated calcium channel (CavMr) in the migrating neurons promotes dendritic growth and migration arrest. The identification of CRN to CPN signaling during early development provides insight into the observation that many autism-linked genes encode synaptic proteins that, paradoxically, are expressed in the developing cortex well before the appearance of synapses and the establishment of functional circuits.
Topics: Calcium Signaling; Calcium; Veratridine; Neurons; Dendrites; Calcium, Dietary; Glutamic Acid
PubMed: 37629145
DOI: 10.3390/ijms241612965 -
Organic Letters Apr 2020An efficient synthetic strategy for three natural -type cholestane alkaloids isolated from the plants, based on commercially available naturally occurring and abundant...
An efficient synthetic strategy for three natural -type cholestane alkaloids isolated from the plants, based on commercially available naturally occurring and abundant (-)-diosgenin (), as exemplified in the concise asymmetric synthesis of (-)-verazine (), (-)-veramiline () (proposed structure), and its 22-epimer, (-)-oblonginine (), is presented. This work highlights the application of a cascade ring-switching process of (-)-diosgenin to achieve the E-ring opening and construction of chiral six-membered lactone challenges in -type cholestane alkaloid synthesis. This approach enables the synthesis of related natural and nature-like novel cholestane alkaloids, opening up opportunities for more extensive exploration of cholestane alkaloid biology.
Topics: Molecular Conformation; Stereoisomerism; Veratrum; Veratrum Alkaloids
PubMed: 32202118
DOI: 10.1021/acs.orglett.0c00747 -
Frontiers in Endocrinology 2021Osteoclasts (OCs) play an important role in osteoporosis, a disease that is mainly characterized by bone loss. In our research, we aimed to identify novel approach for...
Osteoclasts (OCs) play an important role in osteoporosis, a disease that is mainly characterized by bone loss. In our research, we aimed to identify novel approach for regulating osteoclastogenesis and thereby treating osteoporosis. Previous studies have set a precedent for screening traditional Chinese herbal extracts for effective inhibitors. Peiminine is an alkaloid extracted from the bulb of that reportedly has anticancer and anti-inflammatory effects. Thus, the potential inhibitory effect of peiminine on OC differentiation was investigated a series of experiments. According to the results, peiminine downregulated the levels of specific genes and proteins and consequently suppressed OC differentiation and function. Based on these findings, we further investigated the underlying molecular mechanisms and identified the NF-κB and ERK1/2 signaling pathways as potential targets of peiminine. , peiminine alleviated bone loss in an ovariectomized mouse model.
Topics: Animals; Cevanes; Extracellular Signal-Regulated MAP Kinases; Female; Femur; Mice; NF-kappa B; NFATC Transcription Factors; Osteoclasts; Osteogenesis; Ovariectomy; RANK Ligand; Signal Transduction
PubMed: 34630331
DOI: 10.3389/fendo.2021.736863 -
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 -
International Journal of Molecular... Oct 2021bulbs are used in Traditional Chinese Medicine to treat several illnesses. Peimine (Pm), an anti-inflammatory compound from , is known to inhibit some voltage-dependent...
bulbs are used in Traditional Chinese Medicine to treat several illnesses. Peimine (Pm), an anti-inflammatory compound from , is known to inhibit some voltage-dependent ion channels and muscarinic receptors, but its interaction with ligand-gated ion channels remains unexplored. We have studied if Pm affects nicotinic acetylcholine receptors (nAChRs), since they play broad functional roles, both in the nervous system and non-neuronal tissues. Muscle-type nAChRs were incorporated to oocytes and the action of Pm on the membrane currents elicited by ACh (s) was assessed. Functional studies were combined with virtual docking and molecular dynamics assays. Co-application of ACh and Pm reversibly blocked , with an IC in the low micromolar range. Pm inhibited nAChR by: (i) open-channel blockade, evidenced by the voltage-dependent inhibition of , (ii) enhancement of nAChR desensitization, revealed by both an accelerated decay and a decelerated deactivation, and (iii) resting-nAChR blockade, deduced from the inhibition elicited by Pm when applied before ACh superfusion. In good concordance, virtual docking and molecular dynamics assays demonstrated that Pm binds to different sites at the nAChR, mostly at the transmembrane domain. Thus, Pm from bulbs, considered therapeutic herbs, targets nAChRs with high affinity, which might account for its anti-inflammatory actions.
Topics: Animals; Anti-Inflammatory Agents; Cevanes; Drugs, Chinese Herbal; Gene Expression Regulation; Muscles; Oocytes; Plant Extracts; Receptors, Nicotinic; Xenopus laevis
PubMed: 34681946
DOI: 10.3390/ijms222011287