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Background: Little is known about the effect of postnatal exposure to heavy metals on children's behavior problems. This study aimed to investigate the association between metal exposure during different stages of postnatal life and neurobehavioral outcomes in preschool children., Methods: Urinary concentrations of six metals (arsenic, cadmium, chromium, lead, manganese, and vanadium) were measured using inductively coupled plasma mass spectrometry in 220 participants at two time points: before 1 year and at 5 years of age. Mothers completed the Child Behavior Checklist when the children were 5 years old. Multivariable linear and logistic regression analyses were used to evaluate the association between metal concentrations and behavioral outcomes. We employed Bayesian kernel machine regression (BKMR) to assess possible joint effects and potential interactions between metal mixtures and behavioral outcomes., Results: Concentrations of urinary arsenic (As) in infants were associated with higher scores for anxious/shy behavior problems (β ranging from 0.03 to 0.23). Further analyses showed that As exposure increased the odds of scores falling into the borderline or clinical range on anxious/depressed, affective, and pervasive developmental problems (ORs: 2.45-3.40). Stratification by sex indicated significance in girls but not in boys. BKMR analysis showed that, among the metal mixtures, As displayed a major effect on behavior scores. Concentrations of urinary cadmium in infants were also associated with higher behavioral scores but did not increase the risk of clinical problems. A cross-sectional survey in 5-year-olds did not show a significant association between concurrent metal exposure and behavioral outcome., Conclusion: Our results showed that exposure to As and Cd during infancy was associated with emotional problems in children. The effect of arsenic exposure was more pronounced among female infants. We suggest reducing exposure to toxic metals during early postnatal life to prevent behavioral problems in children.", Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Chronic kidney disease (CKD) is a global health problem characterized by progressive renal fibrosis and excessive extracellular matrix deposition. Oxidative stress and epigenetic regulation, particularly through microRNAs (miRNAs), play crucial roles in the pathogenesis of CKD. In this study, we investigated the role of urinary miR-144-3p, which is upregulated in rats with CKD induced by diabetes and hypertension, in renal fibrosis progression, particularly its regulation of the nuclear factor erythroid-2-related factor 2 (NRF2) pathway. Our findings revealed elevated miR-144-3p levels and reduced NRF2 and target gene levels in kidney tissues of streptozotocin-treated spontaneously hypertensive rats. In vitro experiments demonstrated that miR-144-3p directly binds to the 3'-untranslated region of nrf2, suppressing the NRF2 pathway in renal tubular epithelial cells. Additionally, the profibrogenic factor transforming growth factor (TGF)-β1 increased miR-144-3p expression. TGF-β1-induced NRF2 suppression and reactive oxygen species elevation were found to be mediated through miR-144-3p upregulation. In vivo, cilostazol, an antiplatelet drug with an NRF2-activating effect, ameliorated renal injury in diabetic hypertensive rats by decreasing TGF-β1 and miR-144-3p levels while increasing NRF2 and its target gene levels in the kidneys. These findings highlight the potential therapeutic value of targeting the miR-144-3p/NRF2 pathway to attenuate CKD progression in hypertensive diabetic conditions., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The study was funded by the National Research Foundation of Korea, MSIT (MK Kwak, T Bae), The Catholic University of Korea (MK Kwak), and the Ministry of Education of Korea (SK Kim, GS Bae). The research funding from MSIT and the Ministry of Education of Korea supported meeting attendance and travel (MK Kwak, SK Kim, GS Bae, T Bae)., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Obesity is associated with multiple comorbidities, such as metabolic abnormalities and cognitive dysfunction. Moreover, accumulating evidence indicates that neurodegenerative disorders are associated with chronic neuroinflammation. GLP-1 receptor agonists (RAs) have been extensively studied as a treatment for type 2 diabetes. Emerging evidence has demonstrated a protective effect of GLP-1 RAs on neurodegenerative disease, which is independent of its glucose-lowering effects. In this study, we aimed to examine the effects of a long-acting GLP-1 RA, exenatide, on high-fat diet (HFD)-induced neuroinflammation and related brain function impairment. First, mice treated with exenatide exhibited significantly reduced HFD-increased body weight and blood glucose. In an open field test, exenatide treatment ameliorated the reduction in local motor activity and anxiety in HFD-fed mice. Moreover, HFD induced astrogliosis, microgliosis, and upregulation of IL-1β, IL-6 and TNF-α in hippocampus and cortex. Exenatide treatment reduced HFD-induced astrogliosis and IL-1β and TNF-α expressions. Moreover, exenatide increased phosphor-ERK and M2-type microglia marker arginase-1 expression in the hippocampus and cortex. In addition, we found that scavenger receptor-A4 protein expression was induced by HFD and was subsequently inhibited by exenatide. SR-A4 knockout reversed the locomotor activity impairment but not the anxiety behavior caused by HFD consumption. SR-A4 knockout also reduced HFD-induced neuroinflammation, as shown by the reduced expression of GFAP and IBA-1 compared with that in wild-type control mice. These results demonstrate that exenatide decreases HFD-increased neuroinflammation and promotes anti-inflammatory M2 differentiation. The inhibition of SR-A4 by exenatide exerts anti-inflammatory activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Ferroptosis is a widely known regulator of cell death in connection with the redox state as a consequence of the depletion of glutathione or accumulation of lipid peroxidation. Hepatic stellate cells (HSCs) play a pivotal role in the progression of hepatic fibrosis by increasing the production and secretion of the extracellular matrix. However, the role of ferroptosis in HSC activation and liver fibrogenesis has not been clearly elucidated. The ferroptosis inducer RAS-selective lethal 3 (RSL3) or erastin treatment in HSCs caused cell death. This effect was suppressed only after exposure to ferroptosis inhibitors. We observed induction of ferroptosis by RSL3 treatment in HSCs supported by decreased glutathione peroxidase 4, glutathione deficiency, reactive oxygen species generation, or lipid peroxidation. Interestingly, RSL3 treatment upregulated the expression of plasminogen activator inhibitor-1, a representative fibrogenic marker of HSCs. In addition, treatment with ferroptosis-inducing compounds increased c-JUN phosphorylation and activator protein 1 luciferase activity but did not alter Smad phosphorylation and Smad-binding element luciferase activity. Chronic administration of iron dextran to mice causes ferroptosis of liver in vivo. The expression of fibrosis markers, such as alpha-smooth muscle actin and plasminogen activator inhibitor-1, was increased in the livers of mice with iron accumulation. Hepatic injury accompanying liver fibrosis was observed based on the levels of alanine aminotransferase, aspartate aminotransferase, and hematoxylin and eosin staining. Furthermore, we found that both isolated primary hepatocyte and HSCs undergo ferroptosis. Consistently, cirrhotic liver tissue of patients indicated glutathione peroxidase 4 downregulation in fibrotic region. In conclusion, our results suggest that ferroptosis contribute to HSC activation and the progression of hepatic fibrosis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The significant advances in nano-drug delivery systems (NDDS) for anticancer agents have led to the development of computational techniques, such as machine learning and neural networks to identify the optimal architectural and compositional design in a wide variety of therapeutic nanoformulations. On the other hand, few studies have examined downsized plug-in reaction-ware embodied in an autonomous platform for the instant reconfigurable production of engineered nanomaterials to guide optimal NDDS designs and delivery strategies. This paper describes an on-demand system for an electrically operable, continuously processible material produced by sequential spray pyrolysis and vibrating spray for single-pass NDDS assembly. In particular, a mild chemotherapeutic NDDS consisting of amorphous boron nitride (a-BN; a stable base material for loading), doxorubicin (DOX; an anticancer drug), and folic acid-chitosan conjugate (FACHI; a targeting and antiopsonic agent), called a-BN-DOX@FACHI, was fabricated using the developed system. a-BN-DOX@FACHI was assessed for the pH-responsive release of DOX, targeting of the folate receptor, and its resistance to opsonization and macrophage phagocytosis. a-BN-DOX@FACHI was found to be a mild cancer chemotherapeutic with reasonable biosafety. Integrating a metal ablation device with the developed on-demand system enabled the reconfiguration of NDDS from a-BN-DOX@FACHI to a-BN-Au-DOX@FACHI or a-BN-Pt-cisplatin@bovine serum albumin to add a photothermal effect with a range of architectures and compositions., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Liver fibrosis is caused by repetitive hepatic injury. Regulated in development and DNA damage response 1 (REDD1) gene is induced by various stresses and has been studied in cell proliferation and survival. However, the role of REDD1 in hepatic stellate cell activation and hepatic fibrogenesis has not yet been investigated. In the current study, we examined the effect of REDD1 on hepatic fibrogenesis and the underlying molecular mechanism. REDD1 protein was upregulated in the activated primary hepatic stellate cells and transforming growth factor-β (TGF-β)-treated LX-2 cells. REDD1 mRNA levels were also elevated by TGF-β treatment. TGF-β signaling is primarily transduced via the activation of the Smad transcription factor. However, TGF-β-mediated REDD1 induction was not Smad-dependent. Thus, we investigated the transcription factors that influence the REDD1 expression by TGF-β. We found that c-JUN, a component of AP-1, upregulated the REDD1 expression that was specifically suppressed by p38 inhibitor. In silico analysis of the REDD1 promoter region showed putative AP-1-binding sites; additionally, its deletion mutants demonstrated that the AP-1-binding site between -716 and -587 bp within the REDD1 promoter is critical for TGF-β-mediated REDD1 induction. Moreover, REDD1 overexpression markedly inhibited TGF-β-induced plasminogen activator inhibitor-1 (PAI-1) expression and Smad phosphorylation. REDD1 adenovirus infection inhibited CCl 4 -induced hepatic injury in mice, which was demonstrated by reduced ALT/AST levels and collagen accumulation. In addition, we observed that REDD1 inhibited CCl 4 -induced fibrogenic gene induction and restored GSH and malondialdehyde levels. Our findings implied that REDD1 has the potential to inhibit HSC activation and protect against liver fibrosis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The prevalence of chronic kidney disease is increasing globally; however, effective therapeutic options are limited. In this study, we aimed to identify urinary miRNAs reflecting the effect of therapeutic intervention in rats with comorbid hypertension and diabetes. Additionally, the potential beneficial effects of anti-platelet sarpogrelate and cilostazol were investigated. Nephropathy progression in streptozotocin (STZ)-treated spontaneously hypertensive rats (SHRs), including albuminuria, collagen deposition, and histopathological changes, was alleviated by sarpogrelate and antihypertensive agent telmisartan. Global analysis of urinary miRNAs identified that miR-199a-3p was commonly reduced by sarpogrelate and telmisartan treatment. In vitro analysis suggested CD151 as a target gene of miR-199a-3p: miR-199a-3p overexpression repressed CD151 levels and miR-199a-3p interacted with the 3'-untranslated region of the CD151 gene. In addition, we demonstrated that the miR-199a-3p/CD151 axis is associated with the transforming growth factor-β1 (TGF-β1)-induced fibrogenic pathway. TGF-β1 treatment led to miR-199a-3p elevation and CD151 suppression, and miR-199a-3p overexpression or CD151-silencing enhanced TGF-β1-inducible collagen IV and α-smooth muscle actin (α-SMA) levels. In vivo analysis showed that the decrease in CD151 and the increase in collagen IV and α-SMA in the kidney from STZ-treated SHR were restored by sarpogrelate and telmisartan administration. In an additional animal experiment using cilostazol and telmisartan, there was a correlation between urinary miR-199a-3p reduction and the ameliorating effects of cilostazol or combination with telmisartan. Collectively, these results indicate that urinary miR-199a-3p might be utilized as a marker for nephropathy treatment. We also provide evidence of the benefits of antiplatelet sarpogrelate and cilostazol in nephropathy progression., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Acute and adverse effects of ketamine on cognitive functioning have been documented. No longitudinal study has examined whether cognitive deficits can be reversed following ketamine abstinence although it has been suggested in some cross-sectional studies. This study aimed to investigate the changes in cognitive functioning among ketamine users following a 12-week abstinence from ketamine., Methods: In this longitudinal study, 114 ketamine users completed clinical and cognitive assessments at both baseline and 12-week follow-up with the following instruments: Severity of Dependence Scale, Beck Depression Inventory (BDI), Anxiety Subscale of the Hospital Anxiety Depression Scale (HADSA), and a cognitive battery., Results: BDI (p < 0.001) and HADSA (p = 0.044) scores were significantly reduced at the 12-week follow-up. Significant improvements were found in Wechsler Adult Intelligence Scale (Third edition) immediate recall (p < 0.001) and delayed recall (p < 0.001) on the Rey-Osterrieth Complex Figure Test, and in delayed recall (p < 0.001), and immediate recall (p = 0.001) on the Logical Memory component of the Wechsler Memory Scale (Third Edition) at the 12-week follow-up. Participants completed the Stroop Inference Test significantly faster (p < 0.001); and required fewer number of attempts (p < 0.001) and produced fewer perseverative errors (p < 0.001) on the Wisconsin Card Sorting Test at the 12-week follow-up., Conclusion: Chronic ketamine users' verbal and visual memory and executive functions improved after 12 weeks of ketamine abstinence., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Sleep fragmentation (SF) commonly occurs in several pathologic conditions and is especially associated with impairments of hippocampus-dependent neurocognitive functions. Although the effects of SF on hippocampus in terms of protein or gene levels were examined in several studies, the impact of SF at the metabolite level has not been investigated. Thus, in this study, the differentially expressed large-scale metabolite profiles of hippocampus in a rat model of SF were investigated using untargeted metabolomics approaches. Forty-eight rats were divided into the following 4 groups: 4-day SF group, 4-day exercise control (EC) group, 15-day SF group, and 15-day EC group (n = 12, each). SF was accomplished by forced exercise using a walking wheel system with 30-s on/90-s off cycles, and EC condition was set at 10-min on/30-min off. The metabolite profiles of rat hippocampi in the SF and EC groups were analyzed using liquid chromatography/mass spectrometry. Multivariate analysis revealed distinctive metabolic profiles and marker signals between the SF and corresponding EC groups. Metabolic changes were significant only in the 15-day SF group. In the 15-day SF group, L-tryptophan, myristoylcarnitine, and palmitoylcarnitine were significantly increased, while adenosine monophosphate, hypoxanthine, L-glutamate, L-aspartate, L-methionine, and glycerophosphocholine were decreased compared to the EC group. The alanine, aspartate, and glutamate metabolism pathway was observed as the common key pathway in the 15-day SF groups. The results from this untargeted metabolomics study provide a perspective on metabolic impact of SF on the hippocampus., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Transforming growth factor-β (TGF-β) is a potent pathogenic factor of renal injury through the upregulation of extracellular matrix (ECM) expression and facilitation of renal fibrosis. Nuclear factor erythroid 2-like 2 (Nfe2l2; Nrf2), a master regulator of antioxidant and detoxifying systems, is mainly controlled by the binding with cytosolic protein Kelch-like ECH-associated protein 1 (Keap1) and subsequent proteasomal degradation. The protective effect of Nrf2 on renal injury has been attributed to its antioxidant role, where it aids in coping with oxidative stress-associated progression of renal disease. In this study, we investigated the effect of Nrf2 activation on ECM production and TGF-β/Smad signaling using Keap1-silenced MES-13 cells (a genetic glomerular mesangial cell model with Nrf2 overexpression). The TGF-β1-inducible expression of fibronectin and α-smooth muscle actin (α-Sma) was suppressed and Smad2/3 phosphorylation was blocked in Nrf2-high mesangial cells as compared with that in control cells. Notably, in these Nrf2-high mesangial cells, levels of TGF-β1 receptor 1 (TβR1) were substantially diminished, and the protein levels of Smad7, an inhibitor TGF-β1/Smad signaling, were increased. Nrf2-mediated Smad7 elevation and its anti-fibrotic role in Keap1-silenced cells were confirmed by studies with Nrf2-or Smad7-silencing. As a molecular link for Smad7 elevation in Nrf2-high cells, the reduction of Smad-ubiquitination-regulatory factor 1 (Smurf1), an E3 ubiquitin ligase for Smad7, was notable. Silencing of Smurf1 increased Smad7 in the control mesangial cells; however, forced expression of Smurf1 repressed Smad7 levels in Keap1-silenced cells. Additionally, we demonstrate that bardoxolone (BARD; CDDO-methyl), a pharmacological activator of Nrf2, increased Smad7 levels and attenuated TGF-β/Smad/ECM expression in MES-13. Moreover, in an aristolochic acid (AA)-mediated nephropathy mouse model, the renal expression of Nrf2 and Smad7 was elevated by BARD treatment, and AA-induced tubular necrosis and interstitial fibrosis were substantially ameliorated by BARD. Collectively, these results indicate that the Nrf2-Smad7 axis plays a key role in the protection of TGF-β-induced renal fibrosis, and further suggest a novel molecular mechanism of beneficial effect of BARD on renal disease., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Adherent-invasive E. coli colonization and Toll-like receptor (TLR) expression are increased in the gut of inflammatory bowel disease (IBD) patients. However, the underlying mechanism of such changes has not been determined. In the current study, it was examined whether gut serotonin (5-hydroxytryptamine, 5-HT) can induce adherent-invasive E. coli colonization and increase TLR expression. In a co-culture system, commensal E. coli strain (BW25113, BW) adhered minimally to colon epithelial cells, but this was significantly enhanced by 5-HT to the level of a pathogenic strain (EDL933). Without inducing bacterial virulence, such as, biofilm formation, 5-HT enhanced BW-induced signaling in colon epithelial cells, that is, NADPH oxidase (NOX)-dependent superoxide production, the up-regulations of IL-8, TLR2, TLR4, and ICAM-1, and the down-regulations of E-cadherin and claudin-2. In a manner commensurate with these gene modulations, BW induced an increase in NF-κB and a decrease in GATA reporter signals in colon epithelial cells. However, 5-HT-enhanced BW adhesion and colon epithelial responses were blocked by knock-down of NOX2, TLR2, or TLR4. In normal mice, 5-HT induced the invasion of BW into gut submucosa, and the observed molecular changes were similar to those observed in vitro, except for significant increases in TNFα and IL-1β, and resulted in death. In dextran sulfate sodium-induced colitis mice (an IBD disease model), in which colonic 5-HT levels were markedly elevated, BW administration induced death in along with large amount of BW invasion into colon submucosa, and time to death was negatively related to the amount of BW injected. Taken together, our results demonstrate that 5-HT induces the invasion of commensal E. coli into gut submucosa by amplifying commensal bacteria-induced epithelial signaling (superoxide production and the inductions of NOX2 and TLR2/TLR4). The authors suggest that these changes may constitute the molecular basis for the pathogenesis of IBD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new treatments for human diseases. Inhibition of high mobility group box 1 (HMGB1) and restoration of endothelial integrity are emerging as an attractive therapeutic strategies in the management of severe sepsis or septic shock. Here, we examined the effects of methylthiouracil (MTU), used as antithyroid drug, by monitoring the effects on lipopolysaccharide (LPS)- or cecal ligation and puncture (CLP)-mediated release of HMGB1, and on the modulation of HMGB1-mediated inflammatory responses. The anti-inflammatory activities of MTU were determined by measuring permeability, leukocyte adhesion and migration, and the activation of pro-inflammatory proteins in HMGB1-activated HUVECs and mice. MTU inhibited the release of HMGB1 and downregulated HMGB1-dependent inflammatory responses in human endothelial cells. MTU also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with MTU reduced CLP-induced release of HMGB1 and sepsis-related mortality and pulmonary injury. Our results indicate that MTUs could be candidate therapeutic agents for various severe vascular inflammatory diseases via the inhibition of the HMGB1 signaling pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Hepatic fibrosis is considered integral to the progression of chronic liver diseases, leading to the development of cirrhosis and hepatocellular carcinoma. Activation of hepatic stellate cells (HSCs) is the dominant event in hepatic fibrogenesis. We investigated the ability of isorhamnetin, the 3'-O-methylated metabolite of quercetin, to protect against hepatic fibrosis in vitro and in vivo. Isorhamnetin inhibited transforming growth factor (TGF)-β1-induced expression of α-smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), and collagen in primary murine HSCs and LX-2 cells. The TGF-β1- or Smad-induced luciferase reporter activity of Smad binding elements was significantly decreased by isorhamnetin with a concomitant decrease in Smad2/3 phosphorylation. Isorhamnetin increased the nuclear translocation of Nrf2 in HSCs and increased antioxidant response element reporter gene activity. Furthermore, isorhamnetin blocked TGF-β1-induced reactive oxygen species production. The specific role of Nrf2 in isorhamnetin-mediated suppression of PAI-1 and phosphorylated Smad3 was verified using a siRNA against Nrf2. To examine the anti-fibrotic effect of isorhamnetin in vivo, liver fibrosis was induced by CCl4 in mice. Isorhamnetin significantly prevented CCl4-induced increases in serum alanine transaminase and aspartate transaminase levels, and caused histopathological changes characterized by decreases in hepatic degeneration, inflammatory cell infiltration, and collagen accumulation. Moreover, isorhamnetin markedly decreased the expression of phosphorylated Smad3, TGF-β1, α-SMA, and PAI-1. Isorhamnetin attenuated the CCl4-induced increase in the number of 4-hydroxynonenal and nitrotyrosine-positive cells, and prevented glutathione depletion. We propose that isorhamnetin inhibits the TGF-β/Smad signaling pathway and relieves oxidative stress, thus inhibiting HSC activation and preventing liver fibrosis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Atopic dermatitis (AD) is a common disease with genetic and environmental interactions. We previously reported lifetime exposure to cigarette smoke is associated with adult-onset AD. Aryl hydrocarbon receptor (AhR) is important in regulating environmental exposure to xenobiotics, including benzopyrenes (BP), a major polycyclic aromatic hydrocarbon (PAH) present in cigarette smoke. However, how AhR regulates immune responses in sensitization phase of AD remained elusive., Methods: We investigated how BP affects epicutaneous sensitization response through AhR axis. We compared AhR expression in skin from AD patients and healthy controls. We measured immune responses (Langerhans cell migration and T cell polarization in epicutaneous Ova sensitization in mice with or without AhR defect., Results: We found AhR and ARNT (AhR nuclear translocator) are upregulated in AD skin. BP exposure increases Langerhans cell migration, and increases IL-5, IL-13, and IL-17 levels when lymph node cells were re-challenged with Ova. The increased cytokine levels were attenuated in AhR defected mice. AhR agonists (BP and ITE) decreased E-cadherin expression, while AhR antagonist (CH223191) increased it in human primary keratinocytes., Conclusions: These results suggested AhR interacts with BP to polarize T cell responses, along with Langerhans cell migration. This study revealed a regulatory mechanism how cigarette smoking affects atopic sensitization through the benzopyrene-AhR interaction., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Clematis mandshurica Ruprecht root has been used in Asia as a traditional anti-inflammatory, analgesic, and antitumor agent. Its main active component is hederagenin, a naturally occurring triterpene, and in this study, we examined the anti-inflammatory effects of hederagenin in lipopolysaccharide-stimulated RAW 264.7 cells using an enzyme-linked immunosorbent assay, Western blot, and RT-PCR. In addition, its effects on acute inflammation in vivo were observed using a carrageenan-induced mouse hind paw edema assay. Furthermore, the changes on the histopathology and histomorphometry of hind paw skins were examined using carrageenan-treated mice. Treatment with hederagenin (10, 30 and 100μM) resulted in inhibited levels of protein expression of lipopolysaccharide-stimulated iNOS, COX-2, and NF-κB as well as production of NO, PGE2, TNF-α, IL-1β, and IL-6 induced by lipopolysaccharide. Consistent with these results, hederagenin also dose-dependently reduced the lipopolysaccharide-induced mRNA levels of iNOS and COX-2, and of the above-mentioned cytokines. Interestingly, results of the carrageenan-induced mouse hind paw edema assay showed an anti-edema effect of hederagenin. Furthermore, hederagenin (30mg/kg) inhibited the carrageenan-induced increases in skin thicknesses, infiltrated inflammatory cells, and mast cell degranulation. These results suggest that hederagenin may possess anti-inflammatory activities., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The purpose of this study was to develop novel solid lipid nanoparticle (SLN)-loaded dual-reverse thermosensitive hydrogel (DRTH) for rectal administration of flurbiprofen with improved bioavailability and reduced initial burst effect. The flurbiprofen-loaded SLNs were prepared by hot homogenisation technique, after optimising the amounts of lipid mixture (tricaprin and triethanolamine in 8:2 weight ratio), drug and surfactant. The flurbiprofen-loaded thermosensitive SLN composed of drug, lipid mixture and surfactant at a weight ratio of 10/15/1.3 was a solid at room temperature, and changed to liquid form at physiological temperature due to its melting point of about 32°C. This SLN gave the mean particle size of about 190nm and entrapment efficiency of around 90%. The DRTHs were prepared by adding this flurbiprofen-loaded thermosensitive SLN in various poloxamer solutions. Their rheological characterisation, release and stability were investigated while a morphological and pharmacokinetic study was performed after its rectal administration to rats compared with the drug and hydrogel. Poloxamer 188 and SLN decreased the gelation temperature and gelation time, but increased the viscosity at 25°C, gel strength and mucoadhesive force of DRTHs. In particular, the DRTH composed of [SLN/P 407/P 188 (10%/15%/25%)] with the gelation temperature of about 35°C existed as liquid at room temperature, but gelled at 30-36°C, leading to opposite reversible property of SLN. Thus, it was easy to administer rectally, and it gelled rapidly inside the body. This DRTH gave a significantly increased dissolution rate of the drug as compared to the flurbiprofen, but significantly retarded as compared to the hydrogel, including the initial dissolution rate. Moreover, this DRTH gave significantly higher plasma concentration and 7.5-fold AUC values compared to the drug, and lower initial plasma concentration and Cmax value compared to the hydrogel due to reduced initial burst effect. No damage in rectal mucosa was observed after the application of DRTH. Thus, this DRTH system with improved bioavailability and reduced initial burst effect would be recommended as an alternative for the flurbiprofen-loaded rectal pharmaceutical products., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Sestrins (Sesns) are conserved antioxidant proteins that accumulate in cells in response to various stresses. However, the regulatory roles of Sesn2 in the immune system and in inflammatory responses remain obscure. In the present study, we investigated whether Sesn2 regulates Toll like receptor (TLR)-mediated inflammatory signaling and sought to identify the molecular mechanism responsible. In cells expressing Sesn2, it was found that Sesn2 almost completely inhibited lipopolysaccharide (LPS)-induced NO release and iNOS expression. A gene knockdown experiment confirmed the role of Sesn2 in LPS-activated RAW264.7 cells. Consistently, proinflammatory cytokine (e.g., TNF-α, IL-6, and IL-1β) release and expression were inhibited in Sesn2-expressing cells. Furthermore, Sesn2 prevented LPS-elicited cell death and ROS production via inhibition of NADPH oxidase. NF-κB and AP-1 are redox-sensitive transcription factors that regulate the expressions of diverse inflammatory genes. Surprisingly, Sesn2 specifically inhibited AP-1 luciferase activity and its DNA binding, but not those of NF-κB. AP-1 inhibition by Sesn2 was found to be due to a lack of JNK, p38, and c-Jun phosphorylation. Next, we investigated whether Sesn2 protects galactosamine (Gal)/LPS-induced liver injury in mice infected with a recombinant adenovirus Sesn2 (Ad-Sesn2). Ad-Sesn2 present less severe hepatic injury as supported by decreases in the ALT, AST, and hepatocyte degeneration. Moreover, Ad-Sesn2 attenuated Gal/LPS-induced proinflammatory gene expression in mice. The study shows that Sesn2 inhibits TLR-induced proinflammatory signaling and protects cells by inhibiting JNK- or p38-mediated c-Jun phosphorylation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: We aimed to determine the association between psychomotor vigilance task (PVT) performance and sleep-related factors including sleep duration, daytime sleepiness, poor sleep quality, insomnia, and habitual snoring in a population-based sample., Methods: This was a cross-sectional analysis from the ongoing prospective cohort study, the Korean Genome and Epidemiology Study. We measured PVT performance and documented demographics, sleep-related factors, life style, and medical conditions in community dwelling adults (N = 2499; mean age 57.1 ± 7.3; male 1259). Associations between PVT parameters and sleep-related factors were tested, adjusting for age, gender, smoking, alcohol use, education, body mass index, hypertension, diabetes, depression, and the interval between mid-sleep time and PVT test., Results: High Epworth Sleepiness Scale (ESS, ≥8) was associated with slower mean reciprocal response speed (mean RRT) (3.69 ± 0.02 vs. 3.77 ± 0.01, p < 0.001), higher probability for increased lapses (≥4) (OR 1.48, CI 1.12-1.88, p = 0.001), and more negative RRT slope (-0.036 ± 0.002 vs. -0.030 ± 0.001, p = 0.02). Older age, female gender, low education level, depressive mood, and the interval between mid-sleep and PVT test were also associated with poor performance. Sleep duration, habitual snoring, insomnia, or poor sleep quality (the Pittsburgh Sleep Quality Index score > 5) was not related to PVT parameters., Conclusions: At the population level, our results revealed important modifiers of PVT performance, which included subjective reports of daytime sleepiness., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Sulforaphane (SFN), a natural isothiocyanate present in cruciferous vegetables such as broccoli and cabbage, is effective in preventing carcinogenesis, diabetes, and inflammatory responses. Increasing evidence has demonstrated that beyond its role in the activation of protein C, endothelial cell protein C receptor (EPCR) is also involved in vascular inflammation. EPCR activity is markedly changed by ectodomain cleavage and its release as the soluble EPCR. EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of SFN on EPCR shedding. Our results demonstrated that SFN induced potent inhibition of phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-α-, interleukin (IL)-1β, and cecal ligation and puncture (CLP)-induced EPCR shedding. SFN also inhibited the expression and activity of PMA-induced TACE in endothelial cells. In addition, treatment with SFN resulted in reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results demonstrate the potential of SFN as an anti-sEPCR shedding reagent against PMA and CLP-mediated EPCR shedding., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, are pivotal for the development of inflammatory bowel disease (IBD), and down-regulation of the cytokines and cytokine-induced inflammatory responses therefore constitute pharmacological targets for the development of therapeutic strategies in IBD. In the current study, we found that 7-O-succinyl macrolactin A (SMA), a macrolide, potently inhibited TNF-α-induced adhesion of monocytes to colonic epithelial cells in a concentration-dependent manner, similar to rapamycin, a mTOR inhibitor. In addition, oral administration of SMA resulted in a significant suppression of clinical signs of TNBS-induced rat colitis, including weight loss, colon tissue edema, and myeloperoxidase activity, a marker for inflammatory cell infiltration, as well as microscopic damage score in a histomorphological examination of HE-stained colon tissue. More importantly, SMA was more efficacious in inhibition of intestinal inflammation than 5-aminosalicylic acid (5-ASA), an active metabolite of sulfasalazine, the most commonly prescribed agent for the treatment of IBD. Such anti-inflammatory activity showed correlation with significant suppression of adhesion molecules (ICAM-1 and VCAM-1), T-helper 1-type cytokines (TNF-α, IL-6), and chemokines (MCP-1, IL-8). In addition to inhibition of NF-κB nuclear translocation, SMA also caused significant suppression of TNF-α-induced phosphorylation of PI3K, Akt, mTOR and p70S6 kinase, similar to the effect of rapamycin, an immunosuppressant macrolide. Taken together, the current results suggest that managing both mTOR and NF-κB activation pathways using SMA may be a good therapeutic intervention for the treatment of IBD., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Endothelial cell protein C receptor (EPCR) can be shed from the cell surface, and this process is mediated by tumor necrosis factor-α converting enzyme (TACE), and high levels of soluble EPCR are involved in vascular inflammation. Orientin, one of the C-glycosyl flavonoids, has been known to have anxiolytic and antioxidative activities. However, the effect of orientin on lipopolysaccharide (LPS)-induced inflammatory response has not been studied. Here we investigated the barrier protective effects of orientin against pro-inflammatory responses induced by LPS and the associated signaling pathways. We found that orientin inhibited LPS-induced barrier disruption, expression of cell adhesion molecules (CAMs), and adhesion/transendothelial migration of monocytes to human endothelial cells. Orientin induced potent inhibition of phorbol-12-myristate 13-acetate (PMA) and LPS-induced EPCR shedding. Orientin also suppressed LPS-induced hyperpermeability and leukocyte migration in vivo. Furthermore, orientin suppressed the production of tumor necrosis factor-α (TNF-α) or Interleukin (IL)-6 and the activation of nuclear factor-κB (NF-κB) or extracellular regulated kinases (ERK) 1/2 by LPS. Moreover, treatment with orientin resulted in reduced LPS-induced lethal endotoxemia. These results suggest that orientin protects vascular barrier integrity by inhibiting hyperpermeability, expression of CAMs, and adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: Although there is a growing interest in the independent effect of snoring on carotid atherosclerosis, few studies have observed the relationship between snoring and change in carotid atherosclerosis prospectively. Therefore, the present study aimed to prospectively examine the association of snoring with subclinical changes in carotid atherosclerosis during a four-year period., Methods: Participants in an ongoing prospective cohort study (n=3129) were enrolled. Subclinical changes in carotid atherosclerosis were assessed using: (i) mean and maximum intima-media thickness (IMT) on both common carotid arteries; (ii) prevalence of elevated IMT (maximum IMT > or = 1.0 mm); and (iii) presence of plaque. Measurement was performed using B-mode ultrasonogram at baseline and after two and four years. Subjects were classified into three groups, based on self-reported snoring frequency at baseline: habitual, occasional, and non-snorer., Results: After adjustment for conventional cardiovascular risk factors and self-reported witnessed sleep apnea, the present study found significant cross-sectional differences in mean and maximum IMT between female snorers and non-snorers at baseline only. The changes in IMTs and presence of plaque over four years, however, did not differ by three groups, with different snoring frequency in both genders., Conclusions: Snoring did not accelerate subclinical change in carotid atherosclerosis during a four-year follow-up, although baseline difference in IMT between snorers and non-snorers was significant in women, independent of witnessed sleep apnea. Additional longer-term studies with objective assessment of snoring are needed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Changes in serotonin (5-hydroxytryptamine, 5-HT) content in the gut of patients with inflammatory bowel disease (IBD) and animal models of colitis suggest an important role of 5-HT in the pathogenesis of IBD. In this study, we examined the role and mechanism of action of 5-HT in the inflammatory response of colon epithelial cells in vitro and in vivo. In colon epithelial cells (CCD 841, HT-29, Caco-2), direct application of 5-HT induced production of reactive oxygen species (ROS) and monocyte-epithelial adhesion, an initial event of inflammation, which were blocked not only by 5-HT receptor antagonists (tropisetron, RS39604, and SB269970), antioxidants (ascorbic acid, apocynin), and various inhibitors of NADPH oxidase (DPI), CREB (KG-501), and NF-κB (PDTC), but also by transfection with Nox2 siRNA. Nox2-derived production of ROS corresponded with the rapid and brief activation of Rac. In addition, 5-HT induced Nox2, p67(phox), and Duox2 without altering the level of Nox1 or Duox1 in colon epithelial cells, and silencing of Nox2 suppressed 5-HT-induced Duox2 increase. 5-HT also induced an increase in the expression of MCP-1, IL-8, and ICAM-1 and a decrease in E-cadherin expression. Exogenous application of 5-HT to rat colon through the rectum caused a minimal level of inflammation, which was demonstrated by histological examination, MPO activity, and inflammatory cytokine induction. However, 5-HT combined with a low dose of 2,4,6-trinitrobenzene sulfonic acid (TNBS), the level of which caused a minimal level of colitis, exaggerated colon inflammation accompanied by much more enhanced induction of inflammatory cytokines, IL-6, IL-8, and MCP-1, indicating that colon epithelial cells directly exposed to 5-HT are primed toward inflammation. In the colon at the lesion site, treatment with 5-HT resulted in an increase in the level of epithelial Nox2 but not of constitutively expressed Nox1, which is the opposite result of TNBS treatment. Furthermore, 5-HT treatment of Nox2-knockout mice did not induce colon inflammation, in contrast to 5-HT-treated wild-type mice. The results demonstrate that colon epithelial cells directly exposed to 5-HT are primed for inflammatory reactions, which is an important innate immune response, and the underlying mechanism for the priming is associated with Nox2-activated signaling pathways, including ERK/p38 MAPK, NF-κB, and CREB., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Withaferin A (WFA), an active compound from Withania somnifera, is widely researched for its anti-inflammatory, cardioactive and central nervous system effects. However, antiplatelet, anticoagulant, and profibrinolytic properties of WFA have not been studied. In this study, the anticoagulant activities of WFA were measured by monitoring activated partial thromboplastin-time (aPTT), prothrombin time (PT), fibrin polymerization, platelet aggregation, thrombus formation, and the activities of cell-based thrombin and activated factor X (FXa). The effects of WFA on the expressions of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were also tested in tumor necrosis factor-α (TNF-α) activated human umbilical vein endothelial cells (HUVECs). Our data showed that WFA inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation, FeCl3-induced thrombus formation, prolonged aPTT and PT significantly and inhibited the activities and production of thrombin and FXa. WFA prolonged in vivo and ex vivo bleeding time and inhibited TNF-α induced PAI-1 production. Furthermore, PAI-1/t-PA ratio was significantly decreased by WFA. Collectively, these results indicate that WFA possesses antithrombotic activities and suggest that the current study could provide bases for the development of new anticoagulant agents., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Due to the needs of medical care, the probability of using health care service from heroin users is high. This cross-sectional study investigated the frequency and correlates of health service utilization among heroin users. From June to September 2006, 124 heroin users (110 males and 14 females, mean age: 34.2 ± 8.3 years) who entered two psychiatric hospitals (N = 83) and a detention center (N = 41) in northern Taiwan received a face-to-face interview. Therefore, socio-demographic characteristics, patterns of drug use, psychiatric comorbidities, blood-borne infectious diseases and health service utilization were recorded. The behaviors of health service utilization were classified into the frequency of out-patient department visit and hospitalization, as well as the purchase of over-the-counter drugs. During 12 months prior to interview, 79.8% of the participants attended health care service at least once. The rate of having any event in out-patients service visit, hospitalization, and over-the-counter drugs were 66.1%, 29.8% and 25.8% respectively. The frequency of health service utilization was associated with numerous factors. Among these factors, patients who were recruited from hospital and having a mood disorder were conjoint predictors of out-patient department visit, hospitalization and purchase of over-the-counter drugs. According to the results of this study, social education and routine screening for mood disorders can help heroin users to obtain adequate health care service. The findings of this study are useful references for targeting the heroin users for whom a successful intervention represents the greatest cost benefit., (© 2013 Elsevier Ltd. All rights reserved.)

Persicarin and isorhamnetin were isolated from Oenanthe javanica and their anticoagulant activities were examined by monitoring activated partial thromboplastin time (aPTT), prothrombin time (PT), and the activities of cell-based thrombin and activated factor X (FXa). In addition, the effects of persicarin and isorhamnetin on the expressions of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor-α (TNF-α) activated human umbilical vein endothelial cells (HUVECs). The data obtained showed that persicarin and isorhamnetin both prolonged aPTT and PT significantly and inhibited the activities of thrombin and FXa. In addition, they both inhibited the generations of thrombin and FXa in HUVECs. In accordance with these anticoagulant activities, persicarin and isorhamnetin prolonged in vivo bleeding time and inhibited TNF-α induced PAI-1 production. Furthermore, PAI-1/t-PA ratio was significantly decreased by persicarin. Interestingly, the anticoagulant and profibrinolytic effects of persicarin were greater than those of isorhamnetin, which suggest that the sulfonate group of persicarin positively regulates its anticoagulatory function. Accordingly, our results suggest that persicarin and isorhamnetin possess antithrombotic activities and that they could provide bases for the development of new anticoagulant agents., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Arsenic, still a significant environmental threat in several regions in the world, induces various cancers, including lungs, skin, and bladder. Arsenic-induced Bowen's disease (As-BD) is generally an indolent cutaneous intraepithelial carcinoma in susceptible people. Patients with As-BD have been found to have attenuated contact hypersensitivity. Skin samples collected from these patients have reduced numbers of Langerhans cells (LCs), the major epidermal antigen presenting cells expressing Langerins. This study uses an epicutaneous protein sensitization model to investigate the mechanism through which LCs are decreased in As-BD. It further investigates the possibility that arsenic alters LC migration and polarizes Th responses. To do this, we patch-sensitized Balb/c mice or DT-treated Langerin-DTR mice (conditional depletion of Langerin(+) cells) with OVA or PBS, and fed them water containing 300 ppb arsenic or regular water for 200 μl for five days. Ninety-six hours after OVA sensitization, Langerin(+)EpCAM(+) cells in arsenic-treated WT mice were significantly increased in draining lymph nodes and decreased in epidermis without changes in the dermis. Lymph node cells from arsenic-treated WT mice were found to proliferate more than lymph node cells from control PBS-treated mice after OVA challenge in vitro. They also secreted more IFN-γ and IL-12, but not IL-4, IL-13, or IL-17. However, cell proliferation and the induction of IFN-γ by arsenic were found to be abolished in DT-treated Langerin-DTR mice. The expressions of CCL21 and CXCL12 were also increased in lymph nodes from arsenic-treated WT mice. The administration of a neutralizing antibody against CCL21, but not CXCL12, abolished the increase of LCs in lymph nodes in vivo. The results of this study, the first to study oral arsenic polarization of Th1 responses in epicutaneous protein sensitization through CCL21-mediated LC migration, suggest the chronicity of As-BD without invasion might result from enhanced Th1 responses and altered LC migrations by arsenic., (Copyright © 2012 Elsevier Inc. All rights reserved.)

This study examined the bioactivity of yeast (Saccharomyces cerevisiae)-fermented aged black garlic (FBG) on obese mice supplied a high-fat diet (HFD) and its in vitro antioxidant activity. Aged black garlic (BG) exhibits potent antioxidative effects and has been subjected to extensive research. In addition, the bioactivity of some natural products is increased by fermentation. In a preliminary test, this study found that the antioxidant activity of FBG is stronger than that of BG. Therefore, it was hypothesized that the bioactivity of BG would be increased by yeast fermentation and would be a good candidate as a nutraceutical product for improving the oxidative defense systems in older patients or patients affected by various oxidative stresses, for example, diabetes and diabetic complications. To test this hypothesis, the bioactivities of FBG in diabetic and obese mice as well as the antioxidant activity in vitro were examined. After 91 days of continuous HFD supply, the mice showed marked obesity, hyperglycemia, hyperlipemia, and liver and kidney damages. Black garlic and all 3 different doses of FBG showed favorable hepatoprotective, nephroprotective, hypolipidemic, and antiobesity effects compared with the HFD control, but no hypoglycemic effects. In particular, more favorable bioactivity against all 4 HFD-induced diabetic complications was detected in the FBG-treated groups compared with the group given equivalent doses of BG. These findings suggest that the bioactivities of BG can be improved by yeast fermentation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Epithelial-mesenchymal transition (EMT) is an underlying mechanism of tissue fibrosis, generating myofibroblasts, which serve as the primary source of extracellular matrix production from tissue epithelial cells. Recently, EMT has been implicated in immunosuppressive cyclosporin A (CsA)-induced renal fibrosis. In this study, the potential role of NRF2, which is the master regulator of genes associated with the cellular antioxidant defense system, in CsA-induced EMT renal fibrosis has been investigated. Pretreatment of rat tubular epithelial NRK-52E cells with sulforaphane, an activator of NRF2, could prevent EMT gene changes such as the loss of E-cadherin and the increase in alpha-smooth muscle actin (alpha-SMA) expression. Conversely, genetic inhibition of NRF2 in these cells aggravated changes in CsA-induced EMT markers. These in vitro observations could be confirmed in vivo: CsA treatment resulted in severe renal damage and fibrosis with increased expression of alpha-SMA in NRF2-deficient mice compared to wild-type mice. NRF2-mediated amelioration of CsA-caused EMT changes could be accounted for in part by the regulation of heme oxygenase-1 (HO-1). CsA treatment increased HO-1 expression in an NRF2-dependent manner in NRK cells as well as in murine fibroblasts. Induction of HO-1 by CsA seems to be advantageous in that it counteracts EMT gene changes: specific increase in HO-1 expression caused by cobalt protoporphyrin prevented CsA-mediated alpha-SMA induction, whereas genetic inhibition of HO-1 by siRNA substantially enhanced alpha-SMA induction compared to control cells. Collectively, our results suggest that the NRF2-HO-1 system plays a protective role against CsA-induced renal fibrosis by modulating EMT gene changes., (Copyright 2010 Elsevier Inc. All rights reserved.)

The hepatoprotective properties of Artemisiae capillaris herba (AC) and Picrorrhiza rhizoma (PR) are well known. The aim of this study was to determine the optimal composition of AC and PR mixtures for better complimentary or alternative regimens in reducing the level of hepatic fibrosis. Ten weeks of carbon tetrachloride injections caused subacute hepatic damage, manifested as significantly less body weight gain and hepatic protein content, and a higher liver weight, serum aspartate aminotransferase and alanine aminotransferase levels, hepatic malondialdehyde (an index for lipid peroxidation), and hydroxyproline (an index for collagen synthesis) concentrations. The carbon tetrachloride-induced toxic effects were inhibited by 11 different AC/PR mixtures as well as the single AC or PR treatment. More favorable effects were detected in all mixed-formulation groups compared with the AC or PR single formulations. Moreover, the AC/PR 2:1 formulation showed the most favorable hepatoprotective activity. The AC and PR mixtures showed good synergic hepatoprotective activity that was attributed to increasing free-radical scavenging ability. Among the 11 types of mixed formula tested in this study, the AC/PR 2:1 formulation had the most impressive synergic effects on inhibiting the subacute hepatic damage induced by carbon tetrachloride in rats and showed more favorable effects than with an equal dose of silymarin.

Activating transcription factor 3 (ATF3) is an early-induced gene involved in diverse cellular functions in response to various stresses including viral infection. Here we observed marked reduction of ATF3 by coxsackievirus B3 (CVB3) infection and investigated the regulation and functional role of ATF3 in HeLa cells for the understanding of biological significance of ATF3 downregulation. CVB3 infection markedly reduced ATF3 expression at mRNA and protein levels in parallel with p53 degradation, and preservation of p53 expression rescued CVB3 infection-induced ATF3 downregulation. ATF3 overexpression stimulated apoptotic cell death following CVB3 infection, accompanying with augmentation of CVB3 infection-induced eIF2alpha phosphorylation. However, ATF3 overexpression did not affect viral protein production but promoted virus progeny release. Taken together, our results suggest that ATF3 is under control of p53 in part and that the ATF3 downregulation via p53 degradation may contribute to effective viral production as a modulation mechanism of CVB3 infection-induced cell death.

Objective: We investigated the anti-osteoporotic effects of Polycan, a beta-glucan from Aureobasidium pullulans, in ovariectomized mice at doses of 31.25, 62.5, and 125 mg/kg., Methods: Polycan was administered orally once a day for 28 d to bilateral ovariectomized mice, beginning 4 wk after surgery. Changes in body weight, bone weight, bone mineral content, bone mineral density, failure load, histologic profiles, and histomorphometric analyses were determined, in addition to serum osteocalcin, calcium, and phosphorus levels. Alendronate was used as a reference drug., Results: Polycan significantly and dose-dependently suppressed decreases in bone weight, bone mineral content, failure load, bone mineral density, and serum calcium and phosphorus levels and the increase in serum osteocalcin levels. In addition, Polycan significantly suppressed decreases in histomorphometric parameters such as volume, length, and thickness of trabecular bone and thickness of cortical bone and the increase in osteoclast cells in the femur and tibia., Conclusion: Although the effects of Polycan were generally modest and smaller than those of alendronate, the effects on cortical bone thickness were more favorable for Polycan than for alendronate. In addition, Polycan exhibited favorable effects on ovariectomy-induced osteoporosis. However, more long-term studies are needed to confirm the effects of Polycan on osteoporosis.

Chemically synthesized small interfering RNA (siRNA) has been used as an anti-coxsackievirus B3 (CVB3) agent. Herein, we investigated whether vector-derived short hairpin RNAs (shRNA) targeting CVB3 can exert antiviral activities, prior to their further application to viral vector system for efficient in vivo administration. Employing transient transfection assays to in vivo mouse models as well as to in vitro Cos-7 cell cultures, we directly demonstrated the potential antiviral activity of shRNAs following challenges with infectious CVB3. Of the six shRNAs that we designed, three prevented cell death from CVB3 infection by suppressing viral replication and viral production in Cos-7 cells. These were shRNA 2, which targeted the capsid protein VP1, and shRNAs 4 and 5, which targeted two different regions of the RNA-dependent RNA polymerase 3D. Furthermore, shRNAs 2 and 5 also exerted strong antiviral effects in viral replication in vivo, accompanied by attenuated pancreatic tissue damage. Through this direct evaluation system we addressed the development and application of vector-derived shRNAs as an anti-CVB3 agent, revealing new target sequences.

Coxsackievirus (CVB) 3 induces viral myocarditis and ultimately dilated cardiomyopathy (DCM). However, there is no vaccine in clinical use. We constructed recombinant CVB3 plasmids using a highly effective mammalian expression vector and evaluated their immunogenicity in vivo on the basis of survival rate. Four recombinant plasmids were constructed, which encode CVB3 capsid proteins (VP1 or VP3) or VP1 partial proteins (VP1-1 or VP1-2), and used to immunize BALB/c mice by electroporation. Although VP1, VP3, VP1-1, and VP1-2 induced specific antibodies against the corresponding proteins in mice, neutralizing antibodies were not present in the sera. These recombinant plasmids, except VP1-1 (12.5%), dramatically increased the survival rate in mice at 46 days after challenge (42.9-75.0%, p<0.05). VP3 (75.0%) protected mice against viral infection and the middle regions of VP1 (VP1-2, 50.5%) conferred a protective effect like that conferred by VP1 (42.9%), suggesting that the epitopes in VP3 as well as in the middle of VP1 protect against CVB3 infection in vivo. In conclusion, some recombinant CVB3 plasmids used in this study reduced the destruction of myocytes and improved the survival rates in mice immunized and challenged compared with the control. Thus, pCA-VP3 as well as pCA-VP1 are good candidates for a CVB3 DNA vaccine.