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Nanomedicine (London, England) Jun 2016This study aimed to characterize the role of tropoelastin in eliciting a nitric oxide response in endothelial cells.
AIMS
This study aimed to characterize the role of tropoelastin in eliciting a nitric oxide response in endothelial cells.
MATERIALS AND METHODS
Nitric oxide production in cells was quantified following the addition of known nitric oxide synthase pathway inhibitors such as LNAME and 1400W. The effect of eNOS siRNA knockdowns was studied using western blotting and assessed in the presence of PI3K-inhibitor, wortmannin.
RESULTS
Tropoelastin-induced nitric oxide production was LNAME and wortmannin sensitive, while being unaffected by treatment with 1400W.
CONCLUSION
Tropoelastin acts through a PI3K-specific pathway that leads to the phosphorylation of eNOS to enhance nitric oxide production in endothelial cells. This result points to the benefit of the use of tropoelastin in vascular applications, where NO production is a characteristic marker of vascular health.
Topics: Amidines; Androstadienes; Animals; Benzylamines; Cattle; Cells, Cultured; Endothelial Cells; Enzyme Inhibitors; Humans; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase Type III; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; RNA Interference; RNA, Small Interfering; Recombinant Proteins; Tropoelastin; Wortmannin
PubMed: 27175893
DOI: 10.2217/nnm-2016-0052 -
Virology Journal Mar 2019Porcine sapelovirus (PSV), a species of the genus Sapelovirus within the family Picornaviridae, are a significant cause of enteritis, pneumonia, polioencephalomyelitis...
BACKGROUND
Porcine sapelovirus (PSV), a species of the genus Sapelovirus within the family Picornaviridae, are a significant cause of enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. However, the life cycle of PSV on the molecular level is largely unknown.
METHODS
Here, we used chemical inhibitors, RNA interference, and overexpression of dominant negative (DN) mutant plasmids to verify the roles of distinct endocytic pathways involved in PSV entry into porcine small intestinal epithelial cell line (IPEC-J2).
RESULTS
Our experiments indicated that PSV infection was inhibited when cells were pre-treated with NHCl or chloroquine. Inhibitors nystatin, methyl-β-cyclodextrin, dynasore and wortmannin dramatically reduced PSV entry efficiency, whereas the inhibitors chlorpromazine and EIPA had no effect. Furthermore, overexpression caveolin DN mutant and siRNA against caveolin also decreased virus titers and VP1 protein synthesis, whereas overexpression EPS15 DN mutant and siRNA against EPS15 did not reduce virus infection.
CONCLUSIONS
Our findings suggest that PSV entry into IPEC-J2 cells depends on caveolae/lipid raft mediated-endocytosis, that is pH-dependent and requires dynamin and PI3K but is independent of clathrin and macropinocytosis.
Topics: Ammonium Chloride; Animals; Caveolae; Cell Line; Chloroquine; Clathrin; Dynamins; Endocytosis; Epithelial Cells; Hydrazones; Nystatin; Picornaviridae; RNA Interference; RNA, Small Interfering; Swine; Virus Internalization
PubMed: 30909932
DOI: 10.1186/s12985-019-1144-6 -
Journal of Virology Jul 2022Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. MicroRNAs (miRNAs) are small noncoding RNAs that...
Avian reovirus (ARV) causes viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally by silencing or degrading their targets, thus playing important roles in the host response to pathogenic infection. However, the role of miRNAs in host response to ARV infection is still not clear. In this study, we show that ARV infection markedly increased gga-miR-30c-5p expression in DF-1 cells and that transfection of cells with gga-miR-30c-5p inhibited ARV replication while knockdown of endogenous gga-miR-30c-5p enhanced viral growth in cells. Importantly, we identified the autophagy related 5 (ATG5), an important proautophagic protein, as a bona fide target of gga-miR-30c-5p. Transfection of DF-1 cells with gga-miR-30c-5p markedly reduced ATG5 expression accompanied with reduced conversion of ARV-induced-microtubule-associated protein 1 light chain 3 II (LC3-II) from LC3-I, an indicator of autophagy in host cell, while knockdown of endogenous gga-miR-30c-5p enhanced ATG5 expression as well as ARV-induced conversion of LC3-II, facilitating viral growth in cells. Furthermore, knockdown of ATG5 by RNA interference (RNAi) or treatment of cells with autophagy inhibitors (3-MA and wortmannin) markedly reduced ARV-induced LC3-II and syncytium formation, suppressing viral growth in cells, while overexpression of ATG5 increased ARV-induced LC3-II and syncytium formation, promoting viral growth in cells. Thus, gga-miR-30c-5p suppressed viral replication by inhibition of ARV-induced autophagy via targeting ATG5. These findings unraveled the mechanism of how host cells combat against ARV infection by self-encoded small RNA and furthered our understanding of the role of microRNAs in host response to pathogenic infection. Avian reovirus (ARV) is an important poultry pathogen causing viral arthritis, chronic respiratory diseases, and retarded growth, leading to considerable economic losses to the poultry industry across the globe. Elucidation of the pathogenesis of ARV infection is crucial to guiding the development of novel vaccines or drugs for the effective control of these diseases. Here, we investigated the role of miRNAs in host response to ARV infection. We found that infection of host cells by ARV remarkably upregulated gga-miR-30c-5p expression. Importantly, gga-miR-30c-5p suppressed ARV replication by inhibition of ARV-induced autophagy via targeting autophagy related 5 (ATG5) accompanied by suppression of virus-induced syncytium formation, thus serving as an important antivirus factor in host response against ARV infection. These findings will further our understanding of how host cells combat against ARV infection by self-encoded small RNAs and may be used as a potential target for intervening ARV infection.
Topics: Animals; Autophagy; Autophagy-Related Protein 5; Chickens; MicroRNAs; Orthoreovirus, Avian; Reoviridae Infections; Virus Replication
PubMed: 35867570
DOI: 10.1128/jvi.00759-22 -
Ecotoxicology and Environmental Safety Jul 2023The widely used plasticizer bisphenol A (BPA) is known as an endocrine-disrupting chemical (EDC). Many studies have shown that BPA contributes to diseases involving...
The widely used plasticizer bisphenol A (BPA) is known as an endocrine-disrupting chemical (EDC). Many studies have shown that BPA contributes to diseases involving immune system alterations, but the underlying mechanisms have yet to be elucidated. We previously reported that BPA at concentration of 100 μM caused human B cell death in accordance with an increase in nuclear factor (erythroid-derived 2)-like 2(Nrf2) expression. Autophagy is a cellular process that degraded and recycles cytoplasmic constituents. Here, we investigated whether BPA induces autophagy through Nrf2, which is associated with regulation of B cell death using human WiL2-NS lymphoblast B cells. Then, cell viability was assessed by various assays using trypan blue, MTT or Celltiter glo luminescent substrate and DAPI. When WiL2-NS cells were treated with BPA, cell viability was decreased and LC3 autophagy cargo protein/puncta was increased. BPA-induced autophagy was confirmed by the modification of LC3 puncta formation or autophagy flux turnover with the treatment of hydroxychloroquine(HCQ), NHCl and PI3K inhibitors including 3-methyladenine(3-MA), LY294002 and wortmannin. BPA treatment increased the expression of autophagy-related gene(Atg)7 and Beclin1 as well as Nrf2 induced by the production of reactive oxygen species (ROS). The inhibition of autophagy with siAtg7 or siBeclin1 and Nrf2 depletion aggravated BPA-induced cell death. BPA enhanced the bound of Nrf2 to the specific region on Beclin1 and Atg7 promoter. Spleen tyrosine kinase(Syk) activity was enhanced in response to BPA treatment. Bay61-3606, Syk inhibitor, decreased LC3 and the expression of Atg7 and Beclin1, leading to the increase of BPA-induced B cell death. The results suggest that BPA-induced autophagy ameliorates human B cell death through Nrf2-mediated regulation of Atg7 and Beclin1 expression.
Topics: Humans; Beclin-1; NF-E2-Related Factor 2; Phosphatidylinositol 3-Kinases; Autophagy; Cell Death; Autophagy-Related Protein 7
PubMed: 37257343
DOI: 10.1016/j.ecoenv.2023.115061 -
JA Clinical Reports Nov 2022Although chlorhexidine allergy has been shown to be mediated by immunoglobulin (Ig) E, few reports investigated the mechanism of chlorhexidine-induced anaphylaxis using...
BACKGROUND
Although chlorhexidine allergy has been shown to be mediated by immunoglobulin (Ig) E, few reports investigated the mechanism of chlorhexidine-induced anaphylaxis using basophil activation tests (BATs).
CASE PRESENTATION
A 79-year-old man underwent cholecystectomy under general anesthesia. Anaphylaxis was diagnosed based on the clinical symptoms and high serum tryptase and histamine levels. Skin tests showed positive results only for chlorhexidine. Subsequently, BATs demonstrated that the causative agent was likely chlorhexidine. The inhibitory effect of wortmannin, an inhibitor of phosphoinositide 3-kinase, on basophil activation suggested an IgE-dependent mechanism underlying chlorhexidine-induced anaphylaxis. An 89-year-old man underwent inguinal hernioplasty under general anesthesia. Anaphylaxis was diagnosed based on the clinical symptoms and high serum tryptase and histamine levels. Skin tests and BATs with wortmannin were performed, showing similar results to case 1.
CONCLUSIONS
BATs suggested an IgE-dependent mechanism for chlorhexidine-induced anaphylaxis and might be useful for investigating the mechanisms underlying drug-induced anaphylaxis.
PubMed: 36417006
DOI: 10.1186/s40981-022-00581-w -
Biomaterials Jul 2018Most ovarian cancer patients respond well to initial platinum-based chemotherapy. However, within a year, many patients experience disease recurrence with a platinum...
Most ovarian cancer patients respond well to initial platinum-based chemotherapy. However, within a year, many patients experience disease recurrence with a platinum resistant phenotype that responds poorly to second line chemotherapies. As a result, new strategies to address platinum resistant ovarian cancer (PROC) are needed. Herein, we report that NP co-delivery of cisplatin (CP) and wortmannin (Wtmn), a DNA repair inhibitor, synergistically enhances chemoradiotherapy (CRT) and reverses CP resistance in PROC. We encapsulated this regimen in FDA approved poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) NPs to reduce systemic side effects, enhance cellular CP uptake, improve Wtmn stability, and increase therapeutic efficacy. Treatment of platinum-sensitive ovarian cancer (PSOC) and PROC murine models with these dual-drug loaded NPs (DNPs) significantly reduced tumor burden versus treatment with combinations of free drugs or single-drug loaded NPs (SNPs). These results support further investigation of this NP-based, synergistic drug regimen as a means to combat PROC in the clinic.
Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Chemoradiotherapy; Cisplatin; Drug Carriers; Drug Synergism; Female; Humans; Mice; Nanoparticles; Ovarian Neoplasms; Polyesters; Polyethylene Glycols; Wortmannin; Xenograft Model Antitumor Assays
PubMed: 29631163
DOI: 10.1016/j.biomaterials.2018.03.055 -
European Journal of Immunology May 2023Polyphosphates are highly conserved, linear polymers of monophosphates that reside in all living cells. Bacteria produce long chains containing hundreds to thousands of...
Polyphosphates are highly conserved, linear polymers of monophosphates that reside in all living cells. Bacteria produce long chains containing hundreds to thousands of phosphate units, which can interfere with host defense to infection. Here, we report that intratracheal long-chain polyphosphate administration to C57BL/6J mice resulted in the release of proinflammatory cytokines and influx of Ly6G polymorphonuclear neutrophils in the bronchoalveolar lavage fluid causing a disruption of the physiologic endothelial-epithelial small airway barrier and histologic signs of lung injury. Polyphosphate-induced effects were attenuated after neutrophil depletion in mice. In isolated murine neutrophils, long-chain polyphosphates modulated cytokine release induced by lipopolysaccharides (LPS) from Gram-negative bacteria or lipoteichoic acid from Gram-positive bacteria. In addition, long-chain polyphosphates induced immune evasive effects in human neutrophils. In detail, long-chain polyphosphates downregulated CD11b and curtailed the phagocytosis of Escherichia coli particles by neutrophils. Polyphosphates modulated the migration capacity by inducing CD62L shedding resulting in CD62L and CD11b neutrophils. The release of IL-8 induced by LPS was also significantly reduced. Pharmacologic blockade of PI3K with wortmannin antagonized long-chain polyphosphate-induced effects on LPS-induced IL-8 release. In conclusion, polyphosphates govern immunomodulation in murine and human neutrophils, suggesting polyphosphates as a therapeutic target for bacterial infections to restore innate immune defense.
Topics: Humans; Mice; Animals; Neutrophils; Lipopolysaccharides; Polyphosphates; Interleukin-8; Mice, Inbred C57BL; Cytokines; Bronchoalveolar Lavage Fluid; Escherichia coli; Immunomodulation; Lung
PubMed: 36959687
DOI: 10.1002/eji.202250339 -
Journal of Radiation Research Mar 2020Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase...
Wortmannin, a fungal metabolite, is a specific inhibitor of the phosphatidylinositol 3-kinase (PI3K) family, which includes double-stranded DNA dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated kinase (ATM). We investigated the effects of wortmannin on DNA damage in DNA-PK-deficient cells obtained from severe combined immunodeficient mice (SCID cells). Survival of wortmannin-treated cells decreased in a concentration-dependent manner. After treatment with 50 μM wortmannin, survival decreased to 60% of that of untreated cells. We observed that treatment with 20 and 50 μM wortmannin induced DNA damage equivalent to that by 0.37 and 0.69 Gy, respectively, of γ-ray radiation. The accumulation of DNA double-strand breaks (DSBs) in wortmannin-treated SCID cells was assessed using pulsed-field gel electrophoresis. The maximal accumulation was observed 4 h after treatment. Moreover, the presence of DSBs was confirmed by the ability of nuclear extracts from γ-ray-irradiated SCID cells to produce in vitro phosphorylation of histone H2AX. These results suggest that wortmannin induces cellular toxicity by accumulation of spontaneous DSBs through inhibition of ATM.
Topics: Animals; Cell Line; Cell Survival; DNA Breaks, Double-Stranded; Histones; Humans; Phosphatidylinositol 3-Kinase; Phosphorylation; Protein Kinase Inhibitors; Radiation Tolerance; Wortmannin
PubMed: 32052028
DOI: 10.1093/jrr/rrz102 -
Frontiers in Bioscience (Landmark... Nov 2023We explore the effects of endothelial progenitor cell (EPC)-derived exosomes (EPCexos) and of astragaloside IV (ASIV)-stimulated EPCexos (ASIV-EPCexos) on type I...
OBJECTIVE
We explore the effects of endothelial progenitor cell (EPC)-derived exosomes (EPCexos) and of astragaloside IV (ASIV)-stimulated EPCexos (ASIV-EPCexos) on type I diabetic-wound healing, and determine the basic molecular mechanisms of action.
METHODS
EPCs were exposed to different concentrations of ASIV to generate ASIV-EPCexos. A chronic-wound healing model involving streptozotocin-stimulated diabetic rats was established. These rats were treated with EPCexos, ASIV-EPCexos, rapamycin, and wortmannin. Wound healing was evaluated by direct photographic observation, hematoxylin and eosin staining, and Masson's trichrome staining.
RESULTS
ASIV treatment increased the abilities of EPCs (e.g., proliferation), as well as exosome secretion. EPCexo showed a "cup holder" like structure. Treatment with ASIV-EPCexos increased the wound-healing rate, collagen-deposition area, bromodeoxyuridine uptake, VEGF expression, and the number of CD31- and αSMA- positive cells, whereas decreased epidermal thickness and CD45 expression. The expression of the PI3K/AKT/mTOR pathway increased, whereas the expression of inflammatory factor decreased. However, rapamycin and wortmannin reversed these changes.
CONCLUSIONS
ASIV-EPCexos may accelerate type I diabetic-wound healing via the PI3K/AKT/mTOR pathway. This study may lay the foundation for new clinical treatment options for patients with type I diabetic wounds.
Topics: Animals; Rats; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Endothelial Progenitor Cells; Exosomes; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Sirolimus; TOR Serine-Threonine Kinases; Wortmannin; Wound Healing
PubMed: 38062822
DOI: 10.31083/j.fbl2811282 -
Arthritis Research & Therapy Dec 2014Bone erosion in inflammatory arthritis depends on the recruitment and activation of bone resorbing cells, the osteoclasts. Interleukin-23 (IL-23) has been primarily...
INTRODUCTION
Bone erosion in inflammatory arthritis depends on the recruitment and activation of bone resorbing cells, the osteoclasts. Interleukin-23 (IL-23) has been primarily implicated in mediating inflammatory bone loss via the differentiation of Th17 receptor activator of nuclear factor κB ligand (RANKL)-producing cells. In this article, we describe a new role of IL-23 in activating the synthesis and production of leukotriene B4 (LTB4) in innate immune cells.
METHODS
We utilized whole blood-derived human peripheral blood mononuclear cells (PBMCs), differentiated them towards an osteoclast lineage and then performed immunofluorescence and cytochemical staining to detect the expression of LTB4-associated receptors and enzymes such as phospholipase A2, 5-lipoxygenase and leukotriene A4 hydrolase, as well as the presence of tartrate-resistant acid phosphatase (TRAP) and F-actin rings on fully mature osteoclasts. We used enzyme immunoassays to measure LTB4 levels in culture media derived from IL-23-treated human PBMCs. We used real-time calcium imaging to study the effect of leukotrienes and requirements of different calcium sources and signaling proteins in activating intracellular calcium flux using pharmacological inhibitors to phospholipase C (U73122), membrane calcium channels (2-APB) and phosphatidylinositol 3-kinase (Wortmannin) and utilized qPCR for gene expression analysis in macrophages and osteoclasts.
RESULTS
Our data show that LTB4 engagement of BLT1 and BLT2 receptors on osteoclast precursors leads to activation of phospholipase C and calcium release-activated channel-mediated intracellular calcium flux, which can activate further LTB4 autocrine production. IL-23-induced synthesis and secretion of LTB4 resulted in the upregulation of osteoclast-related genes NFATC1, MMP9, ACP5, CTSK and ITGB3 and the formation of giant, multinucleated TRAP+ cells capable of F-actin ring formation. These effects were dependent on Ca2+ signaling and were completely inhibited by BLT1/BLT2 and/or PLC and CRAC inhibitors.
CONCLUSIONS
In conclusion, IL-23 can initiate osteoclast differentiation independently from the RANK-RANKL pathway by utilizing Ca2+ signaling and the LTB4 signaling cascade.
Topics: Calcium; Calcium Signaling; Cell Differentiation; Humans; Intracellular Fluid; Leukocytes, Mononuclear; Leukotriene B4; Osteoclasts
PubMed: 25443625
DOI: 10.1186/s13075-014-0496-y