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Basic & Clinical Pharmacology &... Jun 2019Tongxinluo capsule (TXL), a Chinese prescription, has been extensively used for treating ischaemic cerebrovascular diseases in China. Studies have demonstrated that TXL...
Tongxinluo capsule (TXL), a Chinese prescription, has been extensively used for treating ischaemic cerebrovascular diseases in China. Studies have demonstrated that TXL protects the blood-brain barrier (BBB) after cerebral ischaemia. However, the underlying protective mechanisms are not fully elucidated. Enlightened by the critical role of sonic hedgehog (Shh) pathway in promoting BBB integrity through up-regulating tight junction (TJ) proteins, we examined whether the Shh pathway could mediate TXL-induced up-regulation of TJ proteins and subsequent protection against BBB disruption after stroke. Ischaemic stroke was induced in adult male C57BL/6J mice by permanent middle cerebral artery occlusion (pMCAO). The mice were orally administered TXL (3.0 g/kg) at 1, 3 and 21 hours after stroke. Meanwhile, cyclopamine, a specific Shh pathway inhibitor, was intraperitoneally injected at 1 and 21 hours after stroke. The following parameters were measured at 6 and 24 hours after pMCAO: BBB permeability; TJ proteins including occludin, claudin-5 and zonula occludens-1 (ZO-1); and Shh signalling molecules such as Shh, Patched, Smoothened (Smo) and Gli-1. Our results showed that TXL protected against BBB disruption at 6 and 24 hours after pMCAO, and cyclopamine partly reversed the protective effect of TXL on BBB. Meanwhile, cyclopamine blocked the effect of TXL-up-regulated expression of occludin, claudin-5 and ZO-1. Moreover, TXL up-regulated the expression of Shh derived from astrocytes, Patched, Smo and Gli-1, and thus activated the Shh pathway. And cyclopamine inhibited TXL-induced activation of the Shh pathway. Thus, our study demonstrates that the Shh pathway mediates TXL-induced protection against BBB disruption after ischaemic stroke.
Topics: Animals; Blood-Brain Barrier; Brain Ischemia; Claudin-5; Drugs, Chinese Herbal; Endothelial Cells; Glial Fibrillary Acidic Protein; Hedgehog Proteins; Infarction, Middle Cerebral Artery; Mice, Inbred C57BL; Occludin; Patched Receptors; Permeability; Signal Transduction; Smoothened Receptor; Stroke; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zonula Occludens-1 Protein
PubMed: 30548093
DOI: 10.1111/bcpt.13186 -
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 -
Cellular Physiology and Biochemistry :... 2018Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumour in adults, with poor survival rate and high mortality rates....
BACKGROUND/AIMS
Glioblastoma multiforme (GBM) is the most devastating and widespread primary central nervous system tumour in adults, with poor survival rate and high mortality rates. Existing treatments do not provide substantial benefits to patients; therefore, novel treatment strategies are required. Peiminine, a natural bioactive compound extracted from the traditional Chinese medicine Fritillaria thunbergii, has many pharmacological effects, especially anticancer activities. However, its anticancer effects on GBM and the underlying mechanism have not been demonstrated. This study was conducted to investigate the potential antitumour effects of peiminine in human GBM cells and to explore the related molecular signalling mechanisms in vitro and in vivo Methods: Cell viability and proliferation were detected with MTT and colony formation assays. Morphological changes associated with autophagy were assessed by transmission electron microscopy (TEM). The cell cycle rate was measured by flow cytometry. To detect changes in related genes and signalling pathways in vitro and in vivo, RNA-seq, Western blotting and immunohistochemical analyses were employed.
RESULTS
Peiminine significantly inhibited the proliferation and colony formation of GBM cells and resulted in changes in many tumour-related genes and transcriptional products. The potential anti-GBM role of peiminine might involve cell cycle arrest and autophagic flux blocking via changes in expression of the cyclin D1/CDK network, p62 and LC3. Changes in Changes in flow cytometry results and TEM findings were also observed. Molecular alterations included downregulation of the expression of not only phospho-Akt and phospho-GSK3β but also phospho-AMPK and phospho-ULK1. Furthermore, overexpression of AKT and inhibition of AKT reversed and augmented peiminine-induced cell cycle arrest in GBM cells, respectively. The cellular activation of AMPK reversed the changes in the levels of protein markers of autophagic flux. These results demonstrated that peiminine mediates cell cycle arrest by suppressing AktGSk3β signalling and blocks autophagic flux by depressing AMPK-ULK1 signalling in GBM cells. Finally, peiminine inhibited the growth of U251 gliomas in vivo.
CONCLUSION
Peiminine inhibits glioblastoma in vitro and in vivo via arresting the cell cycle and blocking autophagic flux, suggesting new avenues for GBM therapy.
Topics: AMP-Activated Protein Kinases; Animals; Antineoplastic Agents, Phytogenic; Autophagy; Brain Neoplasms; Cell Cycle Checkpoints; Cell Line, Tumor; Cevanes; Female; Fritillaria; Glioblastoma; Humans; Mice, Inbred BALB C; Mice, Nude; Signal Transduction
PubMed: 30497066
DOI: 10.1159/000495646 -
Cellular Physiology and Biochemistry :... 2018Injuries of the brain and spinal cord result in the formation of glial (reactive gliosis) and fibrotic (formed by fibroblasts) scars. Recent studies have shown that the...
BACKGROUND/AIMS
Injuries of the brain and spinal cord result in the formation of glial (reactive gliosis) and fibrotic (formed by fibroblasts) scars. Recent studies have shown that the fibrotic scar was much more important for hindering regeneration after brain or spinal cord injury than the astrocytic scar. However, it has been given much less attention for effects and mechanism of fibroblasts during formation of the fibrotic scar. Resveratrol may be a potential anti-scarring agent in burn-related scarring and keloid fibroblasts. However, it is unclear whether and how resveratrol affects formation of the fibrotic scar after brain or spinal cord injury. Earlier studies have shown that the activated Shh signaling has anti-apoptosis, anti-oxidation, anti-inflammation properties. Moreover, resveratrol can activate the Shh signaling. However, it is unclear how resveratrol activates the Shh signaling. Resveratrol is a activator of Sirt1. It is unknown whether resveratrol activates the Shh signaling via Sirt1.
METHODS
NIH3T3 cells, a fibroblast cell line, were used as model cells and treated with drugs. Cell viability was assessed by Cell Counting Kit 8. The expressions and activity of Shh signaling pathway proteins were evaluated by immunocytochemistry and Western blotting. Transcriptional activity of Gli-1 was detected with Dual-Luciferase Reporter Gene Assay Kit.
RESULTS
Resveratrol, Sirt1 agonist STR1720 and recombinant mouse Shh protein, an activator of hedgehog signaling, enhanced the viability of NIH3T3 cells, promoted Smo to translocated to the primary cilia and Gli-1 entered into the nuclei from cytoplasm, and upregulated expressions of Shh, Ptc-1, Smo, and Gli-1 proteins, which can be reversed by Smo antagonist cyclopamine and Sirt1 antagonist Sirtinol. Additionally, resveratrol increased transcriptional activity of Gli-1.
CONCLUSION
We indicate in the first time that it may be mediated by Sirt1 for resveratrol activating the Shh signaling to enhance viability of NIH3T3 cells, and Sirt1 may be a regulator for upstream of the Shh signaling pathway.This study provides a basis for further investigating effects and mechanism of resveratrol during the formation of fibrous scar after brain or spinal cord injury.
Topics: Animals; Benzamides; Cell Survival; Hedgehog Proteins; Mice; NIH 3T3 Cells; Naphthols; Patched-1 Receptor; Resveratrol; Signal Transduction; Sirtuin 1; Smoothened Receptor; Stilbenes; Transcription, Genetic; Up-Regulation; Veratrum Alkaloids; Zinc Finger Protein GLI1
PubMed: 30355933
DOI: 10.1159/000494593 -
International Journal of Oncology Dec 2018Sonic hedgehog (SHH) signaling is an important promotor of desmoplasia, a critical feature in pancreatic cancer stromal reactions involving the activation of pancreatic...
Sonic hedgehog (SHH) signaling is an important promotor of desmoplasia, a critical feature in pancreatic cancer stromal reactions involving the activation of pancreatic stellate cells (PSCs). Gremlin 1 is widely overexpressed in cancer-associated stromal cells, including activated PSCs. In embryonic development, SHH is a potent regulator of Gremlin 1 through an interaction network. This subtle mechanism in the cancer microenvironment remains to be fully elucidated. The present study investigated the association between Gremlin 1 and SHH, and the effect of Gremlin 1 in pancreatic cancer. The expression of Gremlin 1 in different specimens was measured using immunohistochemistry. The correlations among clinicopathological features and levels of Gremlin 1 were evaluated. Primary human PSCs and pancreatic cancer cell lines were exposed to SHH, cyclopamine, GLI family zinc finger-1 (Gli-1) small interfering RNA (siRNA), and Gremlin 1 siRNA to examine their associations and effects using an MTT assay, reverse transcription-quantitative polymerase chain reaction analysis, western blot analysis, and migration or invasion assays. The results revealed the overexpression of Gremlin 1 in pancreatic cancer tissues, mainly in the stroma. The levels of Gremlin 1 were significantly correlated with survival rate and pT status. In addition, following activation of the PSCs, the expression levels of Gremlin 1 increased substantially. SHH acts as a potent promoter of the expression of Gremlin 1, and cyclopamine and Gli-1 siRNA modulated this effect. In a screen of pancreatic cancer cell lines, AsPC-1 and BxPC-3 cells expressed high levels of Gremlin 1, but only AsPC-1 cells exhibited a high expression level of SHH. The results of the indirect co-culture experiment suggested that paracrine SHH from the AsPC-1 cells induced the expression of Gremlin 1 in the PSCs. Furthermore, Gremlin 1 siRNA negatively regulated the proliferation and migration of PSCs, and the proliferation, invasion and epithelial-mesenchymal transition of AsPC-1 and BxPC-3 cells. Based on the data from the present study, it was concluded that an abnormal expression level of Gremlin 1 in pancreatic cancer was induced by SHH signaling, and that the overexpression of Gremlin 1 enabled pancreatic cancer progression.
Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Intercellular Signaling Peptides and Proteins; Male; Neoplasm Staging; Pancreatic Neoplasms; Prognosis; RNA, Small Interfering; Signal Transduction; Survival Analysis; Up-Regulation; Veratrum Alkaloids; Zinc Finger Protein GLI1
PubMed: 30272371
DOI: 10.3892/ijo.2018.4573 -
ACS Nano Oct 2018Pancreatic ductal adenocarcinoma (PDAC) remains one of the most difficult cancers to treat. It is refractory to most existing therapies, including immunotherapies, due...
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most difficult cancers to treat. It is refractory to most existing therapies, including immunotherapies, due to the presence of an excessive desmoplastic stroma, which restricts penetration of drugs and cytotoxic CD8 T cells. Stromal modulation has shown promising results in the enhancement of immune checkpoint blockade treatment in PDAC. We demonstrate here effective stromal modulation by a polymeric micelle-based nanoformulation to codeliver a sonic hedgehog inhibitor (cyclopamine, abbreviated as CPA) and a cytotoxic chemotherapy drug (paclitaxel, abbreviated as PTX). The formulation, M-CPA/PTX, modulated the PDAC stroma by increasing the intratumoral vasculature density, which then promoted the tumor infiltration by cytotoxic CD8 T cells without depletion of tumor-restraining α-smooth muscle action-positive fibroblasts and type I collage in the stroma. The combination of M-CPA/PTX and the PD-1 checkpoint blockade significantly prolonged animal survival in an orthotopic murine PDAC model as well as a genetically engineered mouse model of PDAC. The superior antitumor efficacy was mediated by enhanced tumor infiltration of CD8 T cells without concomitant infiltration of suppressive regulatory T cells or myeloid-derived suppressor cells and by the coordinated action of PTX and interferon-gamma. Our results demonstrate that stroma-modulating nanoformulations are a promising approach to potentiate immune checkpoint blockade therapy of pancreatic cancer.
Topics: Animals; Antineoplastic Agents; Carcinoma, Pancreatic Ductal; Cell Proliferation; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Female; Humans; Injections, Intravenous; Mice; Mice, Inbred C57BL; Paclitaxel; Pancreatic Neoplasms; Stromal Cells; Veratrum Alkaloids
PubMed: 30231203
DOI: 10.1021/acsnano.8b02481 -
ENeuro 2018Action potentials propagating along axons are often followed by prolonged afterdepolarization (ADP) lasting for several tens of milliseconds. Axonal ADP is thought to be...
Action potentials propagating along axons are often followed by prolonged afterdepolarization (ADP) lasting for several tens of milliseconds. Axonal ADP is thought to be an important factor in modulating the fidelity of spike propagation during repetitive firings. However, the mechanism as well as the functional significance of axonal ADP remain unclear, partly due to inaccessibility to small structures of axon for direct electrophysiological recordings. Here, we examined the ionic and electrical mechanisms underlying axonal ADP using whole-bouton recording from mossy fiber terminals in mice hippocampal slices. ADP following axonal action potentials was strongly enhanced by focal application of veratridine, an inhibitor of Na channel inactivation. In contrast, tetrodotoxin (TTX) partly suppressed ADP, suggesting that a Na channel-dependent component is involved in axonal ADP. The remaining TTX-resistant Na channel-independent component represents slow capacitive discharge reflecting the shape and electrical properties of the axonal membrane. We also addressed the functional impact of axonal ADP on presynaptic function. In paired-pulse stimuli, we found that axonal ADP minimally affected the peak height of subsequent action potentials, although the rising phase of action potentials was slightly slowed, possibly due to steady-state inactivation of Na channels by prolonged depolarization. Voltage clamp analysis of Ca current elicited by action potential waveform commands revealed that axonal ADP assists short-term facilitation of Ca entry into the presynaptic terminals. Taken together, these data show that axonal ADP maintains reliable firing during repetitive stimuli and plays important roles in the fine-tuning of short-term plasticity of transmitter release by modulating Ca entry into presynaptic terminals.
Topics: Action Potentials; Animals; Calcium Signaling; Female; Male; Membrane Transport Modulators; Mice; Mice, Inbred C57BL; Mossy Fibers, Hippocampal; Sodium Channels; Veratridine
PubMed: 30225345
DOI: 10.1523/ENEURO.0254-18.2018 -
International Journal of Molecular... Sep 2018Peiminine, an alkaloid extracted from Fritillaria plants, has been reported to have potent anti-inflammatory properties. However, the anti-inflammatory effect of...
Peiminine, an alkaloid extracted from Fritillaria plants, has been reported to have potent anti-inflammatory properties. However, the anti-inflammatory effect of peiminine on a mouse lipopolysaccharide (LPS)-induced mastitis model remains to be elucidated. The purpose of this experiment was to investigate the effect of peiminine on LPS-induced mastitis in mice. LPS was injected through the canals of the mammary gland to generate the mouse LPS-induced mastitis model. Peiminine was administered intraperitoneally 1 h before and 12 h after the LPS injection. In vitro, mouse mammary epithelial cells (mMECs) were pretreated with different concentrations of peiminine for 1 h and were then stimulated with LPS. The mechanism of peiminine on mastitis was studied by hematoxylin-eosin staining (H&E) staining, western blotting, and enzyme-linked immunosorbent assay (ELISA). The results showed that peiminine significantly decreased the histopathological impairment of the mammary gland in vivo and reduced the production of pro-inflammatory mediators in vivo and in vitro. Furthermore, peiminine inhibited the phosphorylation of the protein kinase B (AKT)/ nuclear factor-κB (NF-κB), extracellular regulated protein kinase (ERK1/2), and p38 signaling pathways both in vivo and in vitro. All the results suggested that peiminine exerted potent anti-inflammatory effects on LPS-induced mastitis in mice. Therefore, peiminine might be a potential therapeutic agent for mastitis.
Topics: Animals; Anti-Inflammatory Agents; Cells, Cultured; Cevanes; Disease Models, Animal; Epithelial Cells; Female; Gene Expression Regulation; Infusions, Parenteral; Lipopolysaccharides; MAP Kinase Signaling System; Mammary Glands, Animal; Mastitis; Mice; NF-kappa B; Phosphorylation; Proto-Oncogene Proteins c-akt
PubMed: 30200569
DOI: 10.3390/ijms19092637 -
Molecules (Basel, Switzerland) Sep 2018is a rich source of steroidal alkaloids such as cyclopamine, a known inhibitor of the Hedgehog (Hh) signaling pathway. Here we provide a detailed analysis of the...
is a rich source of steroidal alkaloids such as cyclopamine, a known inhibitor of the Hedgehog (Hh) signaling pathway. Here we provide a detailed analysis of the alkaloid composition of by plant part through quantitative analysis of cyclopamine, veratramine, muldamine and isorubijervine in the leaf, stem and root/rhizome of the plant. To determine whether additional alkaloids in the extracts contribute to Hh signaling inhibition, the concentrations of these four alkaloids present in extracts were replicated using commercially available standards, followed by comparison of extracts to alkaloid standard mixtures for inhibition of Hh signaling using Shh-Light II cells. Alkaloid combinations enhanced Hh signaling pathway antagonism compared to cyclopamine alone, and significant differences were observed in the Hh pathway inhibition between the stem and root/rhizome extracts and their corresponding alkaloid standard mixtures, indicating that additional alkaloids present in these extracts are capable of inhibiting Hh signaling.
Topics: Alkaloids; Animals; Biomass; Hedgehog Proteins; Mice; NIH 3T3 Cells; Plant Extracts; Plant Leaves; Plant Roots; Plant Stems; Reference Standards; Signal Transduction; Veratrum
PubMed: 30200443
DOI: 10.3390/molecules23092222 -
Cell Proliferation Dec 2018The sonic hedgehog (Shh) signalling pathway has an important role in the maintenance of various stem cells and organogenesis during development. However, the effect of...
OBJECTIVES
The sonic hedgehog (Shh) signalling pathway has an important role in the maintenance of various stem cells and organogenesis during development. However, the effect of Shh in skin-derived precursors (SKPs), which have the capacity for multipotency and self-renewal, is not yet clear. The present study investigated the effects of the Shh signalling pathway on the proliferation and self-renewal of murine SKPs (mSKPs).
METHODS
The Shh signalling pathway was activated by treatment with purmorphamine (Shh agonist) or recombinant Shh in mSKPs. Cyclopamine (Shh antagonist) or GANT-61 (Gli inhibitor) was used to inhibit the pathway. Western blot, qPCR, and immunofluorescence were used to analyse the expression of genes related to self-renewal, stemness, epithelial-mesenchymal transition (EMT) and the Shh signalling pathway. In addition, cell proliferation and apoptosis were examined.
RESULTS
Inhibiting the Shh signalling pathway reduced mSKP proliferation and sphere formation, but increased apoptosis. Activating this signalling pathway produced opposite results. The Shh signalling pathway also controlled the EMT phenotype in mSKPs. Moreover, purmorphamine recovered the self-renewal and proliferation of aged mSKPs.
CONCLUSION
Our results suggest that the Shh signalling pathway has an important role in the proliferation, self-renewal and apoptosis of mSKPs. These findings also provide a better understanding of the cellular mechanisms underlying SKP self-renewal and apoptosis that allow more efficient expansion of SKPs.
Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Proliferation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Morpholines; Neoplastic Stem Cells; Purines; Signal Transduction; Skin; Veratrum Alkaloids
PubMed: 30151845
DOI: 10.1111/cpr.12500