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Proceedings of the National Academy of... Aug 2021In this study, we use molecular genetic approaches to clarify the role of the Hedgehog (Hh) pathway in regulating the blood-brain/spinal cord barrier (BBB) in the adult...
In this study, we use molecular genetic approaches to clarify the role of the Hedgehog (Hh) pathway in regulating the blood-brain/spinal cord barrier (BBB) in the adult mouse central nervous system (CNS). Our work confirms and extends prior studies to demonstrate that astrocytes are the predominant cell type in the adult CNS that transduce Hh signaling, revealed by the expression of , a target gene of the canonical pathway that is activated in cells receiving Hh, and other key pathway transduction components. Gli1+ (Hh-responsive) astrocytes are distributed in specific regions of the CNS parenchyma, including layers 4/5/6 of the neocortex, hypothalamus, thalamus, and spinal cord, among others. Notably, although BBB properties in endothelial cells are normally regulated by both paracellular and transcellular mechanisms, conditional inactivation of Hh signaling in astrocytes results in transient, region-specific BBB defects that affect transcytosis but not paracellular diffusion. These findings stand in contrast to prior studies that implicated astrocytes as a source of Sonic hedgehog that limited extravasation via both mechanisms [J. I. Alvarez et al., 334, 1727-1731 (2011)]. Furthermore, using three distinct Cre driver lines as well as pharmacological approaches to inactivate Hh-pathway transduction globally in CNS astrocytes, we find that these specific BBB defects are only detected in the rostral hypothalamus and spinal cord but not the cortex or other regions where Gli1+ astrocytes are found. Together, our data show that Gli1+ Hh-responsive astrocytes have regionally distinct molecular and functional properties and that the pathway is required to maintain BBB properties in specific regions of the adult mammalian CNS.
Topics: Animals; Astrocytes; Blood-Brain Barrier; Brain; Gene Expression Regulation; Gliosis; Hedgehog Proteins; Mice; Mice, Transgenic; Selective Estrogen Receptor Modulators; Smoothened Receptor; Spinal Cord; Tamoxifen; Veratrum Alkaloids
PubMed: 34417306
DOI: 10.1073/pnas.2017779118 -
Pharmaceutical Biology Dec 2021Eriodictyol (EDT) is a flavonoid with strong anti-inflammatory, anti-apoptotic, and antioxidant properties.
CONTEXT
Eriodictyol (EDT) is a flavonoid with strong anti-inflammatory, anti-apoptotic, and antioxidant properties.
OBJECTIVE
To investigate the protective effect and mechanism of EDT in ulcerative colitis (UC).
MATERIALS AND METHODS
UC model was induced by 3% dextran sulphate sodium (DSS) solution for 7 days, meanwhile, EDT and Smoothened (Smo) inhibitor cyclopamine (Cyc) were intraperitoneally injected. In the first experiment, C57BL/6 mice divided into blank control, DSS, DSS + EDT (20 or 40 mg/kg) groups. In second experiment, added Cyc (5 mg/kg) and EDT + Cyc groups. All mice were sacrificed on day 8. Disease activity index (DAI), colon length and colon histology as well as MDA levels, SOD, and GSH-Px activities were measured. The expression of Sonic hedgehog (Shh), Patched, Smo, glioblastoma-1, zonula occludens-1 (ZO-1), occludin, cleaved caspase 3, Bax and Bcl-2 in colon was detected using RT-PCR and Western blotting.
RESULTS
After EDT treatment, compared with the DSS group, DAI (2.33 ± 0.516 vs. 3.67 ± 0.516), colon shortening (5.27 ± 0.476 vs. 4.53 ± 0.528 cm) and histological score (6.67 ± 1.211 vs. 12 ± 1.265) was significantly decreased. EDT also reduced inflammation, oxidative stress and apoptosis in colon. Additionally, EDT increased the expression of the tight junction proteins ZO-1 (35%) and occludin (66.3%). Mechanistically, EDT upregulated the Shh signalling pathway. However, Cyc-mediated inhibition of the Shh pathway partially abolished the effects of EDT.
DISCUSSION AND CONCLUSIONS
These results indicate EDT attenuates DSS-induced colitis by activating the Shh pathway. Further clinical trials are needed to demonstrate its efficacy on UC.
Topics: Animals; Colitis, Ulcerative; Colon; Cytokines; Dextran Sulfate; Flavanones; Hedgehog Proteins; Inflammation; Male; Mice; Mice, Inbred C57BL; Models, Animal; Oxidative Stress; Signal Transduction; Veratrum Alkaloids
PubMed: 34348563
DOI: 10.1080/13880209.2021.1948066 -
Hematology (Amsterdam, Netherlands) Dec 2021Hypomethylating agents (HMAs) have been reported to target the Sonic Hedgehog (Shh) signaling pathway in myelodysplastic syndrome (MDS). However, the synergistic...
Synergistic inhibitory effect of Smo inhibitor jervine and its combination with decitabine can target Hedgehog signaling pathway to inhibit myelodysplastic syndrome cell line.
OBJECTIVE
Hypomethylating agents (HMAs) have been reported to target the Sonic Hedgehog (Shh) signaling pathway in myelodysplastic syndrome (MDS). However, the synergistic inhibitory effect of Smo inhibitor jervine and its combination with decitabine in MUTZ-1 cell lines remains lacking.
METHODS
We used a CCK-8 assay to detect the in-vitro proliferation rate of MUTZ-1 cell lines. Besides, the Annexin V-FITC/PI double staining flow cytometry was utilized to detect the apoptosis rate and cell cycle changes. The expression levels of mRNA were quantified by using qRT-PCR, and the western blot was employed to detect the expression of proteins.
RESULTS
We found that the single-agent jervine or decitabine can significantly inhibit the proliferation rate of MUTZ-1 cell lines, and this inhibitory effect is time-dependent and concentration-dependent. The combined intervention of the jervine and decitabine can more significantly inhibit cell proliferation, induce cell apoptosis, and block the G1 phase of the cell cycle. The combined intervention of the two drugs significantly reduced Smo and G1i-1 mRNA expression in MUTZ-1 cells. Furthermore, after combining both of the drug treatments, the proteins levels of Smo, G1i-1, PI3K, p-AKT, Bcl2, and Cyclin Dl were significantly downregulated, and Caspase-3 is upregulated, indicating that jervine with its combination of decitabine might be effective for controlling the proliferation, apoptosis, and cell cycle.
CONCLUSION
The Smo inhibitor jervine and its combination with decitabine have a synergistic effect on the proliferation, cell cycle, and apoptosis of MUTZ-1 cells, and its mechanism may be achieved by interfering with the Shh signaling pathway.
Topics: Apoptosis; Cell Cycle; Cell Line; Cell Proliferation; Cells, Cultured; Decitabine; Dose-Response Relationship, Drug; Drug Synergism; Gene Expression Regulation; Hedgehog Proteins; Humans; Myelodysplastic Syndromes; Signal Transduction; Smoothened Receptor; Veratrum Alkaloids; Zinc Finger Protein GLI1
PubMed: 34314648
DOI: 10.1080/16078454.2021.1950897 -
Nature Jul 2021Biomolecular condensates have emerged as an important subcellular organizing principle. Replication of many viruses, including human respiratory syncytial virus (RSV),...
Biomolecular condensates have emerged as an important subcellular organizing principle. Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm. IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation. Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers.
Topics: Animals; Antiviral Agents; Biomolecular Condensates; Cell Line; Female; Humans; Inclusion Bodies; Lung; Mice; Mice, Inbred BALB C; Respiratory Syncytial Virus, Human; Transcription Factors; Veratrum Alkaloids; Viral Proteins; Virus Replication
PubMed: 34234347
DOI: 10.1038/s41586-021-03703-z -
Nature Communications Jun 2021The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR...
The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P. Functional comparison of FZD and FZD PF mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO.
Topics: Binding Sites; Bioluminescence Resonance Energy Transfer Techniques; Boron Compounds; Cryoelectron Microscopy; Cyclic AMP-Dependent Protein Kinases; Frizzled Receptors; Humans; Molecular Dynamics Simulation; Mutation; Phosphoproteins; Protein Conformation; Smoothened Receptor; Veratrum Alkaloids
PubMed: 34168128
DOI: 10.1038/s41467-021-24004-z -
Cancer Medicine Jul 2021The Sonic Hedgehog (SHH) signaling pathway plays an important role in various types of human cancers including ovarian cancer; however, its function and underlying...
BACKGROUND
The Sonic Hedgehog (SHH) signaling pathway plays an important role in various types of human cancers including ovarian cancer; however, its function and underlying mechanism in ovarian cancer are still not entirely understood.
METHODS
We detected the expressions of SHH and SQSTM1 in borderline ovarian tumor tissues, epithelial ovarian cancer (EOC) tissues and benign ovarian tumor tissues. Cyclopamine (Cyp, a well-known inhibitor of SHH signaling pathway) and chloroquine (CQ, the pharmaceutical inhibitor of autophagy) were used in vivo and in vitro (autophagic flux, CCK-8 assay, wound healing assay, transwell assay, tumor xenograft model). The mechanism of action was explored through Quantitative RT-PCR and Western Blot.
RESULTS
We found up-regulation of SHH and accumulation of SQSTM1/P62 in epithelial ovarian cancer. Cyp induced autophagy through the PI3K/AKT signaling pathway. Moreover, low-dose Cyp and chloroquine (CQ) significantly promoted the migratory ability of SKOV3 cells.
CONCLUSIONS
Our findings suggest that inhibition of the SHH pathway and autophagy may be a potential and effective therapy for the treatment of ovarian cancer.
Topics: Animals; Autophagic Cell Death; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Movement; Chloroquine; Female; Hedgehog Proteins; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA-Binding Proteins; Sequestosome-1 Protein; Up-Regulation; Veratrum Alkaloids
PubMed: 34076346
DOI: 10.1002/cam4.4018 -
Harmful Algae Mar 2021Marine biotoxins accumulating in seafood products pose a risk to human health. These toxins are often potent in minute amounts and contained within complex matrices;...
Marine biotoxins accumulating in seafood products pose a risk to human health. These toxins are often potent in minute amounts and contained within complex matrices; requiring sensitive, reliable, and robust methods for their detection. The mouse neuroblastoma (Neuro-2a) cytotoxicity assay (N2a-assay) is a sensitive, high-throughput, in vitro method effective for detecting sodium channel-specific marine biotoxins. The N2a-assay can be conducted to distinguish between specific effects on voltage-gated sodium (Na) channels, caused by toxins that activate (e.g., ciguatoxins (CTXs), brevetoxins (PbTxs)) or block (e.g., tetrodotoxins, saxitoxins) the target Na. The sensitivity and specificity of the assay to compounds activating the Na are achieved through the addition of the pharmaceuticals ouabain (O) and veratridine (V). However, these compounds can be toxic to Neuro-2a cells and their application at insufficient or excessive concentrations can reduce the effectiveness of this assay for marine toxin detection. Therefore, during growth incubation, Neuro-2a cells were exposed to O and V, and surviving cells exhibiting a lower sensitivity to O and V (OV-LS) were propagated. OV-LS Neuro-2a cells were selected for 60-80% survival when exposed to 0.22/0.022 mM O/V during the cytotoxicity assay. At these conditions, OV-LS N2a cells demonstrated a 3.5-fold higher survival rate 71% ± 7.9 SD (n = 232), and lower sensitivity to O/V, compared to the original Neuro-2a cells 20% ± 9.0 SD (n = 16). Additionally, OV-LS N2a cells were 1.3-2.6-fold more sensitive for detecting CTX3C 1.35 pg/ml, CTX1B 2.06 pg/ml, and PbTx-3 3.04 ng/ml compared to Neuro-2a cells using 0.1/0.01 mM O/V. Detection of CTX3C in a complex fish matrix using OV-LS cells was 0.0048 pg CTX3C/mg fish tissue equivalent. This work shows the potential for a significant improvement in sensitivity for CTX3C, CTX1B, and PbTx-3 using the OV-LS N2a-assay.
Topics: Animals; Cell Line, Tumor; Ciguatoxins; Marine Toxins; Neuroblastoma; Ouabain; Oxocins; Veratridine
PubMed: 33980434
DOI: 10.1016/j.hal.2021.101994 -
Bioorganic & Medicinal Chemistry Jun 2021Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and...
Constitutive activation of Hedgehog (Hh) pathway is intimately related with the occurrence and development of several malignancies, such as medulloblastoma (MB) and other tumors. Therefore, small molecular inhibitors of Hh pathway are urgently needed. In this study, three new steroidal alkaloids, ⊿ (20R, 24R) 23-oxo-24-methylsolacongetidine, ⊿ (20S, 24R) 23-oxo-24-methylsolacongetidine and veralinine 3-O-α-l-rhamnopyranosyl-(1 → 2)-β-D-glucopyranoside, together with six known alkaloids, 20-epi-verazine, verazine, protoverine 15-(l)-2'-methylbutyrate, jervine, veramarine and β1-chaconine, were isolated and determined from Veratrum grandiflorum Loes. The dual-luciferase bioassay indicated that all compounds exhibited significant inhibitions of Hh pathway with IC values of 0.72-14.31 μM against Shh-LIGHT 2 cells. To determine whether these Hh pathway inhibitors act with the Smoothened (Smo) protein, which is an important oncoprotein and target for this pathway, BODIPY-cyclopamine (BC) competitive binding assay was preferentially performed. Compared with BC alone, all compounds obviously reduced the fluorescence intensities of BC binding with Smo in Smo-overexpression HEK293T cells through fluorescence microscope and flow cytometer. By directly interacting with Smo, it revealed that they were actually novel natural Smo inhibitors. Then, their anti-tumor effects were investigated against the human MB cell line DAOY, which is a typical pediatric brain tumor cells line with highly expressed Hh pathway. Interestingly, most of compounds had slight proliferation inhibitions on DAOY cells after treatment for 24 h same as vismodegib, while β1-chaconine showed the strongest inhibitory effect on the growth of DAOY with IC value of 5.35 μM. In conclusion, our studies valuably provide several novel natural Smo inhibitors for potential targeting treatment of Hh-dependent tumors.
Topics: Alkaloids; Cell Line, Tumor; Cell Proliferation; HEK293 Cells; Humans; Medulloblastoma; Molecular Structure; Smoothened Receptor; Spectrum Analysis; Steroids; Veratrum
PubMed: 33910157
DOI: 10.1016/j.bmc.2021.116166 -
Neurochemistry International Jun 2021In the adult brain, sonic hedgehog acts on cerebral microvascular endothelial cells to stabilize the blood-brain barrier. The expression of sonic hedgehog by astrocytes...
Down-regulation of astrocytic sonic hedgehog by activation of endothelin ET receptors: Involvement in traumatic brain injury-induced disruption of blood brain barrier in a mouse model.
In the adult brain, sonic hedgehog acts on cerebral microvascular endothelial cells to stabilize the blood-brain barrier. The expression of sonic hedgehog by astrocytes is altered during brain injury, and this change has been shown to affect permeability of blood-brain barrier. However, much remains unknown about the regulation of astrocytic sonic hedgehog production. Our results showed that endothelin-1 reduced sonic hedgehog mRNA expression and extracellular protein release in mouse cerebral cultured astrocytes, but had no effect in bEnd.3, a mouse brain microvascular endothelial-derived cell line. The effect of endothelin-1 on astrocyte sonic hedgehog expression was suppressed by an ET antagonist BQ788, but was unchanged by the ET antagonist FR139317. In cultured astrocytes and bEnd.3, endothelin-1 did not affect the expression of the sonic hedgehog receptor-related molecules, patched-1 and smoothened. In an animal model of traumatic brain injury, fluid percussion injury on the mouse cerebrum increased the expression of sonic hedgehog, patched-1, and smoothened. Repeated administration of BQ788 enhanced sonic hedgehog expression at 5 days after fluid percussion injury. Histochemical examination revealed sonic hedgehog expression in glial fibrillary acidic protein-positive astrocytes in the cerebrum after fluid percussion injury. Administration of exogenous sonic hedgehog and BQ788 suppressed Evans blue extravasation, an indicator of blood vessel permeability, induced by fluid percussion injury. The effects of BQ788 on fluid percussion injury-induced Evans blue extravasation were reduced by the administration of jervine, a sonic hedgehog inhibitor. Altogether, these results suggest that endothelin-1 down-regulates astrocytic sonic hedgehog to promote disruption of the blood-brain barrier during traumatic brain injury.
Topics: Animals; Astrocytes; Blood-Brain Barrier; Brain Injuries, Traumatic; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Down-Regulation; Endothelin-1; Hedgehog Proteins; Male; Mice; Oligopeptides; Piperidines; Receptor, Endothelin B; Veratrum Alkaloids
PubMed: 33838160
DOI: 10.1016/j.neuint.2021.105042 -
Pharmaceutical Biology Dec 2021Peimine and paeoniflorin can be combined for the treatment of cough in paediatrics. The interaction during the co-administration could dramatically affect the...
CONTEXT
Peimine and paeoniflorin can be combined for the treatment of cough in paediatrics. The interaction during the co-administration could dramatically affect the bioavailability of drugs.
OBJECTIVE
The interaction between peimine and paeoniflorin was investigated in this study.
MATERIALS AND METHODS
The pharmacokinetics of paeoniflorin (20 mg/kg) with or without the coadministration of peimine (5 mg/kg for 10 days before paeoniflorin) was orally investigated in Sprague-Dawley rats ( = 6). The group without the peimine was set as the control group. The metabolic stability of paeoniflorin was studied in rat liver with microsomes. The effect of peimine on the absorption of paeoniflorin was investigated with Caco-2 cell monolayers.
RESULTS
The (244.98 ± 10.95 vs. 139.18 ± 15.14 μg/L) and AUC (3295.92 ± 263.02 vs. 139.18 ± 15.14 h·μg/L) of paeoniflorin was increased by peimine. The was prolonged from 5.33 ± 1.65 to 14.21 ± 4.97 h and the clearance was decreased from 15.43 ± 1.75 to 4.12 ± 0.57 L/h/kg. Consistently, peimine increased the metabolic stability of paeoniflorin with rat liver microsomes with the increased (56.78 ± 2.62 vs. 26.33 ± 3.15 min) and the decreased intrinsic clearance (24.42 ± 3.78 vs. 52.64 ± 4.47 μL/min/mg protein). Moreover, the transportation of paeoniflorin was also inhibited by peimine as the efflux ratio decreased from 3.06 to 1.63.
DISCUSSION AND CONCLUSIONS
Peimine increased the systemic exposure of paeoniflorin through inhibiting the activity of CYP3A4 and P-gp. These results provide a reference for further studies in a broader population.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Area Under Curve; Caco-2 Cells; Cevanes; Cytochrome P-450 CYP3A; Drug Interactions; Glucosides; Half-Life; Humans; Male; Microsomes, Liver; Monoterpenes; Rats; Rats, Sprague-Dawley
PubMed: 33721550
DOI: 10.1080/13880209.2021.1875013