High Concentrations Of Dexamethasone Research Articles

Endothelial Cell-Derived Exosomes Inhibit Osteoblast Apoptosis and Steroid-Induced Necrosis of Femoral Head Progression by Activating the PI3K/Akt/Bcl-2 Pathway

The aim of the study was to investigate the therapeutic potential of exosomes secreted by endothelial cells (EC-exos) on steroid-induced osteonecrosis of femoral head (SNFH). First, we successfully obtained EC-exos through differential centrifugation. Then, the effects of EC-exos on mouse embryo osteoblast precursor (MC3T3-E1) cells under high concentration of dexamethasone (Dex) were analysed in vitro, which included cell migration, viability, and apoptosis. In vivo, a SNFH rat model was successfully established and treated with EC-exos. Micro-computed tomography (micro-CT) and haematoxylin and eosin (H&E) were used to observe femoral trabeculae. Our in vitro results showed that EC-exos improved cell viability and migration of osteoblasts and reduced the apoptotic effect of high concentration of Dex on osteoblasts in vitro. Phosphoinositide 3-kinase (PI3K)/Akt/Bcl-2 signalling pathway was activated in MC3T3-E1 cells under the response to EC-exos. In vivo, increased bone volume per tissue volume (BV/TV) p=0.031, trabecular thickness (Tb.Th) p=0.020, and decreased separation (Tb.Sp) p=0.040 were observed in SNFH rats treated with EC-exos. H&E staining revealed fewer empty lacunae and pyknotic osteocytes in trabeculae. The expression of Bcl-2 and Akt in EC-exos group was significantly increased in trabeculae tissue. Overall, our finding indicated that EC-exos could attenuate SNFH by inhibiting osteoblast apoptosis via the PI3K/Akt/Bcl-2 pathway.

Dexamethasone Is Not Sufficient to Facilitate Tenogenic Differentiation of Dermal Fibroblasts in a 3D Organoid Model

Self-assembling three-dimensional organoids that do not rely on an exogenous scaffold but maintain their native cell-to-cell and cell-to-matrix interactions represent a promising model in the field of tendon tissue engineering. We have identified dermal fibroblasts (DFs) as a potential cell type for generating functional tendon-like tissue. The glucocorticoid dexamethasone (DEX) has been shown to regulate cell proliferation and facilitate differentiation towards other mesenchymal lineages. Therefore, we hypothesized that the administration of DEX could reduce excessive DF proliferation and thus, facilitate the tenogenic differentiation of DFs using a previously established 3D organoid model combined with dose-dependent application of DEX. Interestingly, the results demonstrated that DEX, in all tested concentrations, was not sufficient to notably induce the tenogenic differentiation of human DFs and DEX-treated organoids did not have clear advantages over untreated control organoids. Moreover, high concentrations of DEX exerted a negative impact on the organoid phenotype. Nevertheless, the expression profile of tendon-related genes of untreated and 10 nM DEX-treated DF organoids was largely comparable to organoids formed by tendon-derived cells, which is encouraging for further investigations on utilizing DFs for tendon tissue engineering.

Dose-dependent thresholds of dexamethasone destabilize CAR T-cell treatment efficacy.

Chimeric antigen receptor (CAR) T-cell therapy is potentially an effective targeted immunotherapy for glioblastoma, yet there is presently little known about the efficacy of CAR T-cell treatment when combined with the widely used anti-inflammatory and immunosuppressant glucocorticoid, dexamethasone. Here we present a mathematical model-based analysis of three patient-derived glioblastoma cell lines treated in vitro with CAR T-cells and dexamethasone. Advanced in vitro experimental cell killing assay technologies allow for highly resolved temporal dynamics of tumor cells treated with CAR T-cells and dexamethasone, making this a valuable model system for studying the rich dynamics of nonlinear biological processes with translational applications. We model the system as a nonautonomous, two-species predator-prey interaction of tumor cells and CAR T-cells, with explicit time-dependence in the clearance rate of dexamethasone. Using time as a bifurcation parameter, we show that (1) dexamethasone destabilizes coexistence equilibria between CAR T-cells and tumor cells in a dose-dependent manner and (2) as dexamethasone is cleared from the system, a stable coexistence equilibrium returns in the form of a Hopf bifurcation. With the model fit to experimental data, we demonstrate that high concentrations of dexamethasone antagonizes CAR T-cell efficacy by exhausting, or reducing the activity of CAR T-cells, and by promoting tumor cell growth. Finally, we identify a critical threshold in the ratio of CAR T-cell death to CAR T-cell proliferation rates that predicts eventual treatment success or failure that may be used to guide the dose and timing of CAR T-cell therapy in the presence of dexamethasone in patients.

The role of Serpina3n in the reversal effect of ATRA on dexamethasone-inhibited osteogenic differentiation in mesenchymal stem cells

BackgroundGlucocorticoid-induced osteoporosis (GIOP) is the most common secondary osteoporosis. Patients with GIOP are susceptible to fractures and the subsequent delayed bone union or nonunion. Thus, effective drugs and targets need to be explored. In this regard, the present study aims to reveal the possible mechanism of the anti-GIOP effect of all-trans retinoic acid (ATRA).MethodsBone morphogenetic protein 9 (BMP9)-transfected mesenchymal stem cells (MSCs) were used as an in vitro osteogenic model to deduce the relationship between ATRA and dexamethasone (DEX). The osteogenic markers runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin were detected using real-time quantitative polymerase chain reaction, Western blot, and immunofluorescent staining assay. ALP activities and matrix mineralization were evaluated using ALP staining and Alizarin Red S staining assay, respectively. The novel genes associated with ATRA and DEX were detected using RNA sequencing (RNA-seq). The binding of the protein–DNA complex was validated using chromatin immunoprecipitation (ChIP) assay. Rat GIOP models were constructed using intraperitoneal injection of dexamethasone at a dose of 1 mg/kg, while ATRA intragastric administration was applied to prevent and treat GIOP. These effects were evaluated based on the serum detection of the osteogenic markers osteocalcin and tartrate-resistant acid phosphatase 5b, histological staining, and micro-computed tomography analysis.ResultsATRA enhanced BMP9-induced ALP, RUNX2 expressions, ALP activities, and matrix mineralization in mouse embryonic fibroblasts as well as C3H10T1/2 and C2C12 cells, while a high concentration of DEX attenuated these markers. When DEX was combined with ATRA, the latter reversed DEX-inhibited ALP activities and osteogenic markers. In vivo analysis showed that ATRA reversed DEX-inhibited bone volume, bone trabecular number, and thickness. During the reversal process of ATRA, the expression of retinoic acid receptor beta (RARβ) was elevated. RARβ inhibitor Le135 partly blocked the reversal effect of ATRA. Meanwhile, RNA-seq demonstrated that serine protease inhibitor, clade A, member 3N (Serpina3n) was remarkably upregulated by DEX but downregulated when combined with ATRA. Overexpression of Serpina3n attenuated ATRA-promoted osteogenic differentiation, whereas knockdown of Serpina3n blocked DEX-inhibited osteogenic differentiation. Furthermore, ChIP assay revealed that RARβ can regulate the expression of Serpina3n.ConclusionATRA can reverse DEX-inhibited osteogenic differentiation both in vitro and in vivo, which may be closely related to the downregulation of DEX-promoted Serpina3n. Hence, ATRA may be viewed as a novel therapeutic agent, and Serpina3n may act as a new target for GIOP.

Dexamethasone inhibits BMP7-induced osteogenic differentiation in rat dental follicle cells via the PI3K/AKT/GSK-3β/β-catenin pathway.

Impacted third molars are commonly seen in teenagers and young adults and can cause considerable suffering. Preventing eruption of the third molars can reduce pain at the source. Our previous study has shown that dexamethasone (DEX) at a certain concentration can prevent the eruption of third molars without damaging alveolar bone in Sprague-Dawley (SD) rats, but the relevant molecular mechanisms need to be explored. This study aimed to explore the effects of high concentrations of DEX on osteogenic signaling pathways, including BMP/Smad and Wnt/β-catenin pathways, in rat dental follicle cells (rDFCs) and to elucidate the possible mechanisms. The results showed that BMP7 induced osteogenic differentiation by increasing the activity of ALP and the protein levels of OPN in rDFCs. DEX decreased endogenous BMP7 and phosphorylated Smad1/5/8 expression as well as BMP7-induced osteogenic differentiation. DEX also reduced the mRNA and protein levels of β-catenin by enhancing the expression of GSK-3β. In addition, regardless of DEX intervention, overexpression of BMP7 promoted the expression of β-catenin, while knockdown of BMP7 attenuated it. Further investigation revealed that overexpression of BMP7 attenuated the DEX-mediated inhibition of AKT and GSK-3β phosphorylation, but knockdown of BMP7 exerted the opposite effects. This study suggests that high concentrations of DEX may inhibit the expression of β-catenin via the PI3K/AKT/GSK-3β pathway in a manner mediated by BMP7. The findings further illustrate the possible molecular mechanisms by which DEX prevents tooth development.

Dexamethasone promotes mesenchymal stem cell apoptosis and inhibits osteogenesis by disrupting mitochondrial dynamics.

Long‐term or heavy use of glucocorticoids can cause severe necrosis of the femoral head, but the underlying mechanisms are still unclear. Recent studies have found that mitochondrial dynamics play an important role in femoral head necrosis. Here, we investigated the effect of dexamethasone on the mitochondrial function of mesenchymal stem cells. We observed that high concentrations of dexamethasone (10−6 mol·L−1) decreased cell activity, promoted apoptosis, elevated levels of reactive oxygen species and disrupted mitochondrial dynamics. Furthermore, dexamethasone (10−6 mol·L−1) inhibited osteogenesis of stem cells and promoted adipogenesis. These findings may facilitate greater understanding of the adverse effects of dexamethasone on the femoral head.

Sleep Deprivation Modifies Noise-Induced Cochlear Injury Related to the Stress Hormone and Autophagy in Female Mice.

A lack of sleep is linked with a range of inner ear diseases, including hearing loss and tinnitus. Here, we used a mouse model to investigate the effects of sleep deprivation (SD) on noise vulnerability, and explored the mechanisms that might be involved in vitro, focusing particularly corticosterone levels and autophagic flux in cells. Female BALB/c mice were divided into six groups [control, acoustic trauma (AT)-alone, 1 day (d) SD-alone, 1d SD pre-AT, 5d SD-alone, and 5d SD pre-AT]. Cochlear damage was then assessed by analyzing auditory brainstem response (ABR), and by counting outer hair cells (OHCs) and the synaptic ribbons of inner hair cells (IHCs). In addition, we measured levels of serum corticosterone and autophagy protein expression in the basilar membranes by ELISA kits, and western blotting, respectively. We found that SD-alone temporarily elevated ABR wave I amplitude, but had no permanent effect on hearing level or IHC ribbon numbers. Combined with AT, the number of synaptic ribbons in the 1d SD pre-AT group was significantly higher than that in the AT-alone group, whereas the 5d SD pre-AT group showed more severe synaptopathy, and a greater loss of OHCs after 2 weeks than the other experimental groups exposed to noise. Correspondingly, the levels of corticosterone in the AT-alone group were higher than those of the 1d SD pre-AT group, but lower than those of the 5d SD pre-AT group. The 1d SD pre-AT group showed a marked elevation in the expression of microtubule-associated protein 1 light chain 3B (LC3B), whereas the AT-alone group exhibited only a mild increase. In contrast, the levels of LC3B did not change in the 5d SD pre-AT group. Experiments with HEI-OC-1 cells and cochlear basilar membrane cultures showed that high-concentrations of dexamethasone, and the inhibition of autophagy, aggravated cellular apoptosis induced by oxidative stress. In conclusion, noise-induced synaptopathy and hair cell loss can be mitigated by preceding 1d SD, but will be aggravated by preceding 5d SD. These findings may be attributable to corticosterone levels and the extent of autophagy.

A modular steroid-inducible gene expression system for use in rice

BackgroundChemically inducible systems that provide both spatial and temporal control of gene expression are essential tools, with many applications in plant biology, yet they have not been extensively tested in monocotyledonous species.ResultsUsing Golden Gate modular cloning, we have created a monocot-optimized dexamethasone (DEX)-inducible pOp6/LhGR system and tested its efficacy in rice using the reporter enzyme β-glucuronidase (GUS). The system is tightly regulated and highly sensitive to DEX application, with 6 h of induction sufficient to induce high levels of GUS activity in transgenic callus. In seedlings, GUS activity was detectable in the root after in vitro application of just 0.01 μM DEX. However, transgenic plants manifested severe developmental perturbations when grown on higher concentrations of DEX. The direct cause of these growth defects is not known, but the rice genome contains sequences with high similarity to the LhGR target sequence lacO, suggesting non-specific activation of endogenous genes by DEX induction. These off-target effects can be minimized by quenching with isopropyl β-D-1-thiogalactopyranoside (IPTG).ConclusionsOur results demonstrate that the system is suitable for general use in rice, when the method of DEX application and relevant controls are tailored appropriately for each specific application.

Seasonal changes in acute stressor-mediated plasma glucocorticoid regulation in New World flying squirrels

Southern flying squirrels have higher circulating cortisol levels than most vertebrates. However, regulation of tissue exposure to cortisol by the hormone’s carrier protein, corticosteroid-binding globulin (CBG), appears to be altered due to lower-than-expected CBG expression levels, and a reduced affinity for cortisol. To assess the capacity of flying squirrels to regulate acute stress-mediated cortisol levels, we used the dexamethasone (DEX) suppression test followed by the adrenocorticotropic hormone (ACTH) stimulation test in both the breeding and non-breeding seasons, and quantified resultant changes in plasma cortisol and relative CBG levels. Regulation of cortisol via negative feedback, and the acute stress response appeared to function as they do in other vertebrates during the breeding season, but response to DEX in the non-breeding season showed that the sensitivity of the negative feedback mechanism changed across seasons. The relatively high concentrations of DEX required to induce negative feedback suggests that southern flying squirrels have a reduced sensitivity to cortisol compared with other vertebrates, and that high circulating cortisol levels may be required to compensate for low target tissue responsiveness in this species. Cortisol, but not CBG levels, were higher during the non-breeding than breeding season, and females had higher cortisol and CBG levels than males. Our data suggest that flying squirrel cortisol levels are regulated by negative feedback at a higher set point than in related species. Seasonal changes in cortisol levels, target tissue sensitivity to DEX, and in the capacity to respond to stressors appear to be part of the underlying physiology of southern flying squirrels, and may be required to maximize fitness in the face of tradeoffs between survival and reproduction.

Excessive glucocorticoid-induced muscle MuRF1 overexpression is independent of Akt/FoXO1 pathway.

The ubiquitin-proteasome system (UPS)-dependent proteolysis plays a major role in the muscle catabolic action of glucocorticoids (GCs). Atrogin-1 and muscle-specific RING finger protein 1 (MuRF1), two E3 ubiquitin ligases, are uniquely expressed in muscle. It has been previously demonstrated that GC treatment induced MuRF1 and atrogin-1 overexpression. However, it is yet unclear whether the higher pharmacological dose of GCs induced muscle protein catabolism through MuRF1 and atrogin-1. In the present study, the role of atrogin-1 and MuRF1 in C2C12 cells protein metabolism during excessive dexamethasone (DEX) was studied. The involvement of Akt/forkhead box O1 (FoXO1) signaling pathway and the cross-talk between anabolic regulator mammalian target of rapamycin (mTOR) and catabolic regulator FoXO1 were investigated. High concentration of DEX increased MuRF1 protein level in a time-dependent fashion (P<0.05), while had no detectable effect on atrogin-1 protein (P>0.05). FoXO1/3a (Thr24/32) phosphorylation was enhanced (P<0.05), mTOR phosphorylation was suppressed (P<0.05), while Akt protein expression was not affected (P>0.05) by DEX. RU486 treatment inhibited the DEX-induced increase of FoXO1/3a phosphorylation (P<0.05) and MuRF1 protein; LY294002 (LY) did not restore the stimulative effect of DEX on the FoXO1/3a phosphorylation (P>0.05), but inhibited the activation of MuRF1 protein induced by DEX (P<0.05); rapamycin (RAPA) inhibited the stimulative effect of DEX on the FoXO1/3a phosphorylation and MuRF1 protein (P<0.05).

Effects of pigment epithelium-derived factor on osteo-adipogenic transdifferentiation, and osteoprotegerin/receptor activator for nuclear factor-κB ligand ratio of osteoblast after stimulation of high concentrations of dexamethasone

Objective To explore the protective effect of pigment epithelium-derived factor (PEDF) on osteo-adipogenic transdifferentiation, and osteoprotegerin (OPG)/receptor activator of nuclear factor (NF)-κB ligand (RANKL) ratio of osteoblast after stimulation of high concentrations of dexamethasone (DEX). Methods MC3T3-E1 cells were randomly divided into control group, DEX model group and DEX + PEDF group. According to the concentration of PEDF, the DEX + PEDF group was subdivided into 2, 5, 10 nmol/L groups. The inverted phase contrast microscope was used to observe the cell morphology. After 14 d we used cell staining [alkaline phosphatase (ALP) staining, alizarin red staining] to detect the differentiation of osteoblast. Immunofluorescence and Western blotting were used to observe the expression of OPG and RANKL. Results The MC3T3-E1 cells were positive for ALP and expressed OPG and RANKL. After stimulation of high concentrations of DEX, the osteo-adipogenic transdifferentiation of MC3T3-E1 cells was observed. A large number of osteoblasts transfected into adipocytes. High concentrations of DEX significantly raised the expression of RANKL (P=0.008). The osteo-adipogenic transdifferentiation of MC3T3-E1 cells was significantly inhibited by PEDF with an obvious down-regulation of RANKL and up-regulation of OPG showed by Western blotting testing. Compared with DEX group (0.560±0.012), OPG/RANKL expression level of PEDF intervention groups (0.821±0.025, 1.290±0.018 and 1.405±0.120) appeared significantly upregulated (P=0.003, 0.000, 0.019). Conclusion High concentrations of DEX promote osteo-adipogenic transdifferentiation of MC3T3-E1 cells and reduce the expression of OPG/RANKL, whereas PEDF can effectively reverse this process. PEDF play a important role in protecting the osteoblast function. Key words: Dexamethasone; Pigment epithelium-derived factor; Osteoblast; Receptor activator for nuclear factor-κB ligand; Osteoprotegerin; Osteo-adipogenic transdifferentiation

Use of common carp (Cyprinus carpio) and Aeromonas salmonicida for detection of immunomodulatory effects of chemicals on fish

To develop a test for assessing the immunomodulatory effects of chemical pollutants on fish, we evaluated the effects of dexamethasone on the natural host–pathogen interaction between common carp (Cyprinus carpio) and Aeromonas salmonicida. Carp were exposed to 1mgL−1 dexamethasone for the entire experimental period. One week after the exposure test started, the exposed fish, as well as unexposed fish, were bath-infected with A. salmonicida. One hundred percent mortality was observed in bacteria-infected fish exposed to dexamethasone, whereas no infection-associated mortality was observed in infected fish in the absence of dexamethasone exposure. In a separate experiment, dexamethasone exposure significantly suppressed hemolytic complement activity in bacteria-infected fish. These results clearly indicate that exposure to a high concentration of dexamethasone suppressed the carp immune system and caused subsequent mortality. Thus, this proposed test method is likely to be useful for evaluating the immunomodulatory effects of chemicals in fish.

EFFECT OF MACROPHAGE MIGRATION INHIBITORY FACTOR ON VASCULAR REPAIR OF STEROID-INDUCED AVASCULAR NECROSIS OF FEMORAL HEAD IN VITRO

To interpret the mechanisms of vascular repair disorders in steroid-induced avascular necrosis of the femoral head (SANFH) via detection of the changes of proliferation, migration, and macrophage migration inhibitory factor (MIF)/vascular endothelial growth factor (VEGF) expressions of endothelial cells (ECs) under hypoxia/glucocorticoid. According to culture conditions, human umbilical vein ECs (HUVECs) at passage 3 were divided into group A (normal), group B (1.0×10-6 mol/L dexamethasone), group C (hypoxia), and group D (hypoxia+1.0×10-6 mol/L dexamethasone). The cell activity was detected by AlamarBlue; the number of viable cells was detected in live/dead cell staining; the cell morphology was observed after cytoskeleton staining; cell migration ability was compared by scratch test; and the levels of MIF and VEGF expressions were detected by ELISA. At 24 hours after culture, the cell activity and the number of living cells in group C were significantly higher than those in the other 3 groups, showing significant difference between groups (P<0.05), and group D had the worst cell activity and least living cells. Cytoskeleton staining showed that cells had normal morphology in groups A and B; cells had rich cytoskeleton and secretion granules in group C; cytoskeleton form disorder and nucleus pyknosis were observed in group D. Scratch test showed that the cell migration ability of group C was strongest while cell migration ability of group D was weakest. Accumulated concentration of MIF and VEGF in 4 groups significantly increased with time extending. Accumulated concentration of MIF in group C were significantly higher than that in other 3 groups at each time point (P<0.05). Within 24 hours after intervention, stage concentration of MIF during 1-8 hours was significantly lower than that during 0-1 hour and 8-24 hours in every group (P<0.05). Stage concentration of MIF in group C was significantly higher than other groups during 0-1 hour and 8-24 hours (P<0.05). Within 2 hours after intervention, stage concentration of MIF in 4 groups during 0.5-1 hour was significantly higher than that during other stages (P<0.05). Accumulated concentration of VEGF in group C was significantly higher than that in other groups at 8 and 24 hours (P<0.05). The stage concentration of VEGF in groups C and D during 8-24 hours was significantly higher than that during 0-1 hour and 1-8 hours (P<0.05). There was no significant difference in the stage concentration of VEGF within and among group A, B, C, and D at every stage within 2 hours after intervention (P>0.05). In hypoxia environment, the proliferation and migration of ECs is enhanced, and the secretion of VEGF and MIF is increased. High concentration of dexamethasone will suppress the process above, which induces vascular repair disorders and aggravating SANFH.

Effects of Dexamethasone Concentration and Timing of Exposure on Chondrogenesis of Equine Bone Marrow-Derived Mesenchymal Stem Cells.

ObjectiveDexamethasone is known to support mesenchymal stem cell (MSC) chondrogenesis, although the effects of dose and timing of exposure are not well understood. The objective of this study was to investigate these variables using a laboratory model of MSC chondrogenesis.DesignEquine MSCs were encapsulated in agarose and cultured in chondrogenic medium with 1 or 100 nM dexamethasone, or without dexamethasone, for 15 days. Samples were analyzed for extracellular matrix (ECM) accumulation, prostaglandin E2 and alkaline phosphatase secretion, and gene expression of selected collagens and catabolic enzymes. Timing of exposure was evaluated by ECM accumulation after dexamethasone was withdrawn over the first 6 days, or withheld for up to 3 or 6 days of culture.ResultsECM accumulation was not significantly different between 1 and 100 nM dexamethasone, but was suppressed ~40% in dexamethasone-free cultures. Prostaglandin E2 secretion, and expression of catabolic enzymes, including matrix metalloproteinase 13, and type X collagen was generally lowest in 100 nM dexamethasone and not significantly different between 1 nM and dexamethasone-free cultures. Dexamethasone could be withheld for at least 2 days without affecting ECM accumulation, while withdrawal studies suggested that dexamethasone supports ECM accumulation beyond day 6.ConclusionOne nanomolar dexamethasone supported robust cartilage-like ECM accumulation despite not having an effect on markers of inflammation, although higher concentrations of dexamethasone may be necessary to suppress undesirable hypertrophic differentiation. While early exposure to dexamethasone was not critical, sustained exposure of at least a week appears to be necessary to maximize ECM accumulation.

Dickkopf1 Up-Regulation Induced by a High Concentration of Dexamethasone Promotes Rat Tendon Stem Cells to Differentiate Into Adipocytes

Background/Aims: Dexamethasone (Dex)-induced spontaneous tendon rupture and decreased self-repair capability is very common in clinical practice. The metaplasia of adipose tissue in the ruptured tendon indicates that Dex may induce tendon stem cells (TSCs) to differentiate into adipocytes, but the mechanism remains unclear. In the present study, we used in vitro methods to investigate the effects of Dex on rat TSC differentiation and the molecular mechanisms underlying this process. Methods: First, we used qPCR and Western blotting to detect the expression of the adipogenic differentiation markers aP2 and C/EBPα after treating the TSCs with Dex. Oil red staining was used to confirm that high concentration Dex promoted adipogenic differentiation of rat TSCs. Next, we used qPCR and Western blotting to detect the effect of a high concentration of dexamethasone on molecules related to the canonical WNT/β-catenin pathway in TSCs. Results: Treating rat TSCs with Dex promoted the synthesis of the inhibitory molecule dickkopf1 (DKK1) at the mRNA and protein levels. Western blotting results further showed that Dex downregulated the cellular signaling molecule phosphorylated glycogen synthase kinase-3β (P-GSK-3 β (ser9)), upregulated P-GSK-3β (tyr216), and downregulated the pivotal signaling molecule β-catenin. Furthermore, DKK1 knockdown attenuated Dex-induced inhibition of the canonical WNT/β-catenin pathway and of the adipogenic differentiation of TSCs. Lithium chloride (LiCl, a GSK-3β inhibitor) reduced Dex-induced inhibition of the classical WNT/β-catenin pathway in TSCs and of the differentiation of TSCs to adipocytes. Conclusion: In conclusion, by upregulating DKK1 expression, reducing the level of P-GSK-3β (ser9), and increasing the level of P-GSK-3β (tyr216), Dex causes the degradation of β-catenin, the central molecule of the classical WNT pathway, thereby inducing rat TSCs to differentiate into adipocytes.

The effect of electromagnetic fields on the proliferation and the osteogenic or adipogenic differentiation of mesenchymal stem cells modulated by dexamethasone

Although glucocorticoids provide benefits for inflammation or autoimmune disorders, high-dose and long-term use could cause osteonecrosis or osteoporosis as adverse effect for patients. Electromagnetic field (EMF) treatments have been clinically used for many years to promote fracture healing, but whether EMF can attenuate the deleterious effects of glucocorticoids is not clear. In this study, the effects of different concentrations of dexamethasone (DEX) on proliferation and adipogenic or osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were detected and compared, and the effects of EMF treatment (15 Hz, 1 mT, 4 h/day) on 0.1 µM DEX-modulated BMSCs' proliferation and adipogenic or osteogenic differentiation were investigated. Higher concentrations of DEX (0.1 and 1 µM) inhibited proliferation of BMSCs but promoted expression of adipogenic-related genes, increasing the number of lipid droplets. In the early stage of differentiation, DEX restrained expression of RUNX2 and alkaline phosphatase (ALP), but amplified expression of ALP and osteopontin (OPN) in the late stage. EMF treatment of BMSCs influenced by 0.1 µM DEX inhibited the high expression of adipogenic-related genes, stimulated the expression of RUNX2, ALP, OPN, and osteocalcin, and increased the activity of ALP. EMF exposure augmented the expression of p-ERK, which DEX reduced. After using mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK signaling pathway inhibitor, U0126, the effect of EMF was reduced. In conclusion, EMF exposure accelerates BMSCs proliferation, inhibits adipogenic differentiation, and promotes osteogenic differentiation of BMSCs modulated by DEX, and these effects are mediated at least in part by MEK/ERK signaling pathway.

Inhibition mechanism of hydroxypropyl methylcellulose acetate succinate on drug crystallization in gastrointestinal fluid and drug permeability from a supersaturated solution

The effects of drug-crystallization inhibitor in bile acid/lipid micelles solution on drug permeation was evaluated during the drug crystallization process. Hydroxypropyl methylcellulose acetate succinate (HPMC-AS) was used as a drug-crystallization inhibitor, which efficiently suppressed dexamethasone (DEX) crystallization in a gastrointestinal fluid model containing sodium taurocholate (NaTC) and egg-phosphatidylcholine (egg-PC). Changes of molecular state of supersaturated DEX during the DEX crystallization process was monitored in real time using proton nuclear magnetic resonance (1H NMR). It revealed that DEX distribution to bulk water and micellar phases formed by NaTC and egg-PC was not changed during the DEX crystallization process even in the presence of HPMC-AS. DEX permeation during DEX crystallization was evaluated using dissolution/permeability system. The combination of crystallization inhibition by HPMC-AS and micellar encapsulation by NaTC and egg-PC led to considerably higher DEX concentrations and improvement of DEX permeation at the beginning of the DEX crystallization process. Crystallization inhibition by HPMC-AS can efficiently work even in the micellar solution, where NaTC/egg-PC micelles encapsulates some DEX. It was concluded that a crystallization inhibitor contributed to improvement of permeation of a poorly water-soluble drug in gastrointestinal fluid.

Dexamethasone enhances invasiveness of Aspergillus fumigatus conidia and fibronectin expression in A549 cells

Background The efficacies of current treatments for invasive aspergillus (IA) are unsatisfactory and new therapeutic targets or regimens to treat IA are urgently needed. Previous studies have indicated that the ability of conidia to invade host cells is critical in IA development and fibronectin has a hand in the conidia adherence process. In the clinical setting, many patients who receive glucocorticoid for extended periods are susceptible to Aspergillus fumigatus (A. fumigatus) infection, for this reason we investigated the effect of glucocorticoid on conidia invasiveness by comparing the invasiveness of A. fumigatus conidia in the type II human alveolar cell line (A549) cultured with different concentrations of dexamethasone. We also explored the relationships between dexamethasone and fibronectin expression. Methods Following culture with anti-fibronectin antibodies and/or dexamethasone, type II human alveolar A549 cells were infected with conidia of A. fumigatus. After 4 hours, the extracellular free conidia were washed away and the remaining immobilized conidia were released using Triton-X 100 and quantified by counting the colony-forming units. The invasiveness of conidia was measured by calculating the invasion rate (%). The transcription of the fibronectin gene in cells cultured with different concentrations of dexamethasone for 24 hours was tested by fluorogenic quantitative RT-PCR while the expression of fibronectinin cells cultured for 48 hours was tested by Western blotting and immunocytochemistry. Results A significant reduction in the invasiveness of conidia was seen in the cells cultured with anti-fibronectin antibody ((14.42±1.68)% vs. (19.17±2.53)%, P &lt;0.05), but no significant difference was observed in cells cultured with a combination of anti-fibronectin antibody and dexamethasone (6.37 × 10-5 mol/L). There was no correlation between the dexamethasone concentration and the invasiveness of conidia after dexamethasone pretreatment of cells for 4 hours. In contrast, after pretreated for 24 hours, the invasiveness of conidia in the presence of 6.37 × 10-5 mol/L dexamethasone ((24.66±2.41)%) was higher than for the control ((19.17±2.53)%) and the 0.25 × 10-5 mol/L group ((19.93±3.06)%), and the invasiveness in the 1.27 × 10-5 mol/L group ((22.47±2.46)%) was also higher than in the control, P &lt;0.05. The relative transcripts of the fibronectin gene after exposure to 6.37 × 10-5 mol/L dexamethasone (9.19 × 10-3±1.2 × 10-3) was higher than for the control (4.61 × 10-3±1.54×10-3) and the 0.25×10-5 mol/L group (6.20×10-3±1.93×10-3), and expression in the 1.27×10-5 mol/L group (7.94×10-3±2.24×10-3) was also higher than for the control, P &lt;0.05. High concentrations of dexamethasone promoted fibronectin production after culture for 48 hours. Conclusions Dexamethasone can increase invasiveness of A. fumigatus conidia by promoting fibronectin expression. This may partially explain why patients who are given large doses of glucocorticoids for extended periods are more susceptible to A. fumigatus infection.

Seasonal Changes and Regulation of the Glucocorticoid Receptor in the Testis of the Toad Rhinella arenarum

Several studies indicate that wild free-living vertebrates seasonally regulate plasma glucocorticoids. However, not only glucocorticoids but also the amount of receptors is important in determining biological responses. In this context, seasonal regulation of glucocorticoid receptor (GR) is crucial to modulate the response to glucocorticoids. Rhinella arenarum is an anuran exhibiting seasonal variations in plasma glucocorticoids and also in the number of binding sites (B(max)) of the testicular cytosolic GR. In this work, we evaluated if the annual pattern of GR protein in the testis varies seasonally and, by an in vitro approach, the role of glucocorticoids, androgens, and melatonin in the regulation of the GR B(max) and protein level. For this purpose, testes were treated with two physiological concentrations of melatonin (40 and 200 pg/ml), with or without luzindole (melatonin-receptor antagonist); with testosterone, cyanoketone (inhibitor of steroidogenesis) or casodex (androgen-receptor antagonist); or with dexamethasone or RU486 (GR antagonist). After treatments, B(max) and protein level were determined by the binding of [(3)H]dexamethasone and Western blot, respectively. Results showed that GR protein decreases in the winter. The in vitro treatment with melatonin produced a biphasic effect on the B(max) with the lowest concentration decreasing this parameter by a receptor-mediated mechanism. However, melatonin had no effect on the GR protein level. Conversely, a high concentration of dexamethasone up-regulated the GR protein and androgens neither changed the B(max) nor the protein level. These findings suggest that seasonal changes in plasma melatonin and glucocorticoids modulate the effect of glucocorticoids in the testis of R. arenarum.

Mild versus strong anti-inflammatory therapy during early sepsis in mice: A matter of life and death*

A recent literature-based study suggested that low-dose corticosteroid treatment has a beneficial effect on mortality in septic patients, whereas high-dose corticosteroid treatment has not. This suggests that mild down-regulation of the inflammatory response during early sepsis may be beneficial while extensive reduction of the inflammatory response is not. To investigate this hypothesis, we examined the effect of dexamethasone in varying doses on cecal ligation and puncture-induced inflammation and mortality. Animal study. University research laboratory. Male C57BL/6 mice. Mice were subjected to cecal ligation and puncture, and dexamethasone was administered intravenously at a dosage of 0.05 (L/DEX), 0.25 (M/DEX), or 2.5 (H/DEX) mg/kg body weight 20 mins postoperatively. Mice receiving phosphate-buffered saline served as controls. Survival was recorded up to 21 days and inflammatory markers were determined in plasma, lungs, liver, and kidney at 6 hrs following cecal ligation and puncture as well as bacterial load in blood and peritoneal fluid. L/DEX treatment significantly improved survival compared with control mice, whereas treatment with higher concentrations of dexamethasone (M/DEX and H/DEX) did not. Treatment with either M/DEX or H/DEX was associated with significantly (p < .05) reduced cytokine plasma levels as compared with controls at 6 hrs after cecal ligation and puncture. In addition, M/DEX or H/DEX powerfully reduced cytokine messenger RNA expression in the lung, liver, and kidney. In contrast, treatment with L/DEX was associated with a mild, but nonsignificant, reduction of cytokine plasma levels. In addition, L/DEX moderately reduced cytokine messenger RNA expression in lung, liver, and kidney tissue and reduced the occurrence of bacteremia. A modest down-regulation of the early sepsis-associated inflammatory response improves survival in a murine cecal ligation and puncture model. We propose that the success of anti-inflammatory therapies in a septic setting fundamentally depends on finding a treatment balance that reduces the hyper-inflammation-induced pathology but still allows adequate defense against pathogens.