Cortisol level after dexamethasone suppression test in patients with non-functioning adrenal incidentaloma is positively associated with the duration of reactive hyperemia response on microvascular bed.

PURPOSE: Data on endothelial derangements in patients with non-functioning adrenal incidentaloma (NFAI) are scarce. METHODS: We investigated if NFAI patients present clinical, biochemical and endothelial alterations compared to individuals without an adrenal lesion and also the associations among these variables. Forty-two NFAI and 40 controls were evaluated. NFAI diagnosis and controls were defined according to the current guidelines and based on a normal adrenal imaging exam, respectively. Body composition was evaluated by dual emission X-ray absorptiometry. Endothelial reactivity was assessed by two methods: tonometry (Endo-PAT®) and laser speckle contrast imaging (LSCI). RESULTS: There were no differences between groups regarding age, gender, ethnicity, smoking status, and statin use. The frequency of metabolic syndrome according to the International Diabetes Federation criteria was 69% and 57.9%, respectively in NFAI and controls (p = 0.36), whereas the atherosclerotic cardiovascular disease (ASCVD) risk was 63.4% and 66.7% (p = 0.81). The clinical, laboratory, and anthropometric characteristics, as well as body composition, were similar between the groups. Additionally, any differences between groups were observed on endothelial reactivity tests. Nevertheless, we noted an association between cortisol levels after 1 mg-dexamethosone suppression test (1 mg-DST) and the duration of post-occlusive reactive hyperemia tested on microcirculation (r = 0.30; p = 0.03). NFAI patients require more antihypertensive drugs to achieve blood pressure control (p = 0.04). The number of antihypertensive drugs used to control blood pressure correlated with cortisol levels after 1 mg-DST (r = 0.29; p = 0.03). CONCLUSIONS: Since both groups herein investigated had a high frequency of metabolic syndrome and ASCVD risk, it might explain similarities observed on endothelial reactivity. Nevertheless, prolonged reactive hyperemia response on microcirculation was correlated with cortisol levels under suppression.

Glabridin inhibits dexamethasone-induced muscle atrophy.

Prevention of muscle wasting is known to contribute to improving the quality of life and extending a healthy life. Recently, we have reported that licorice flavonoid oil containing glabridin, which is a prenylated isoflavone, enhances muscle mass in mice. In this study, we investigated the prevention effect of glabridin on dexamethasone-induced muscle atrophy and clarified its mechanism in cultured myotubes and in muscle of mice. Treatment with glabridin to C2C12 myotubes inhibited dexamethasone-induced protein degradation through dexamethasone-induced expression of ubiquitin ligases, MuRF1 and Cbl-b, but not atrogin-1. Mechanistically, glabridin inhibited nuclear translocation of the glucocorticoid receptor. Glabridin directly bound to the glucocorticoid receptor, resulting in the inhibition of binding between dexamethasone and the receptor protein. Glabridin also inhibited dexamethasone-induced phosphorylation of p38 and FoxO3a, as the upstream for the induction of ubiquitin ligases in C2C12 myotubes. Moreover, the glabridin-induced inhibition of protein degradation was eliminated by knockdown of the glucocorticoid receptor, but not by p38 knockdown. These data indicated that the inhibitory mechanism of glabridin against dexamethasone-induced muscle atrophy was mainly mediated by the inhibition of binding between dexamethasone and the glucocorticoid receptor in myotubes. Oral administration of glabridin prevented dexamethasone-induced protein degradation in the tibialis anterior muscle of mice. It was confirmed that glabridin inhibited dexamethasone-induced nuclear translocation of the glucocorticoid receptor and phosphorylation of FoxO3a in the muscle of mice. These findings suggest that glabridin is an effective food ingredient for the prevention of glucocorticoid-induced skeletal muscle atrophy.

Effects of fluvoxamine on nerve growth factor-induced neurite outgrowth inhibition by dexamethasone in PC12 cells.

In the present study, we examined the effects of fluvoxamine on nerve growth factor (NGF)-induced neurite outgrowth inhibition by dexamethasone (DEX) in PC12 cells. Fluvoxamine increased NGF-induced neurite outgrowth. Compared with co-treatment with NGF and fluvoxamine, p-Akt levels were higher than the values without fluvoxamine. The phosphorylated extracellular regulated kinase 1/2 levels were slightly increased by co-treatment with NGF and fluvoxamine. Fluvoxamine concentration-dependently improved NGF-induced neurite outgrowth inhibition by DEX. Fluvoxamine also improved the decrease in the NGF-induced p-Akt level caused by DEX. Interestingly, the sigma-1 receptor antagonist NE-100 blocked the improvement effects of fluvoxamine on NGF-induced neurite outgrowth inhibition by DEX. The selective sigma-1 receptor agonist PRE-084 also improved NGF-induced neurite outgrowth inhibition by DEX, which is blocked by NE-100. These results indicate that the improvement effects of fluvoxamine on NGF-induced neurite outgrowth inhibition by DEX may be attributable to the phosphorylation of Akt and the sigma-1 receptor.

Involvement of the Glucocorticoid Receptor in Pro-inflammatory Transcription Factor Inhibition by Daucane Esters from Laserpitium zernyi.

Species of the genus Laserpitium have been used traditionally to treat inflammation and infection. From the herb of Laserpitium zernyi, six new compounds were isolated and their structures elucidated (using IR, NMR, HRMS data) as derivatives of 8-daucene-2,4,10-triol (1, 2, and 4), 7-daucene-2,4,10-triol (3), a lapiferin derivative featuring a C-2 ester moiety (5), and a daucane featuring an exomethylene group at C-8 (6). Also isolated were the rare daucanes vaginatin (7) and laserpitin (8). In a search for selective glucocorticoid receptor (GR) modulators, the compounds were tested for their capacity to inhibit NF-κB and AP-1 pro-inflammatory factors and for a potential competitive effect on a dexamethasone (Dex)-induced GR-driven glucocorticoid response element (GRE) reporter gene. The new 2ß-angeloyloxy-10α-acetoxy-8-daucene-2,4,10-triol (2) significantly inhibited transactivation of both NF-κB and AP-1, while vaginatin (7) was the most active of the compounds tested in blocking AP-1. Both compounds competitively repressed Dex-induced GRE-driven promoter activities, indicative of a potential role for GR. In addition, a decreased potential to inhibit NF-κB was apparent in GR knockout A549 cells. In line with the transcriptional assays, compounds 2 and 7 also significantly lowered CCL-2 chemokine production, albeit to a lesser extent than Dex. The results suggest that daucanes may be interesting candidates in the search for compounds with GR-modulating activities.

Switching from astrocytic neuroprotection to neurodegeneration by cytokine stimulation.

Astrocytes, the largest cell population in the human brain, are powerful inflammatory effectors. Several studies have examined the interaction of activated astrocytes with neurons, but little is known yet about human neurotoxicity under such situations and about strategies of neuronal rescue. To address this question, immortalized murine astrocytes (IMA) were combined with human LUHMES neurons and stimulated with an inflammatory (TNF, IL-1) cytokine mix (CM). Neurotoxicity was studied both in co-cultures and in monocultures after transfer of conditioned medium from activated IMA. Interventions with >20 drugs were used to profile the model system. Control IMA supported neurons and protected them from neurotoxicants. Inflammatory activation reduced this protection, and prolonged exposure of co-cultures to CM triggered neurotoxicity. Neither the added cytokines nor the release of NO from astrocytes were involved in this neurodegeneration. The neurotoxicity-mediating effect of IMA was faithfully reproduced by human astrocytes. Moreover, glia-dependent toxicity was also observed, when IMA cultures were stimulated with CM, and the culture medium was transferred to neurons. Such neurotoxicity was prevented when astrocytes were treated by p38 kinase inhibitors or dexamethasone, whereas such compounds had no effect when added to neurons. Conversely, treatment of neurons with five different drugs, including resveratrol and CEP1347, prevented toxicity of astrocyte supernatants. Thus, the sequential IMA-LUHMES neuroinflammation model is suitable for separate profiling of both glial-directed and directly neuroprotective strategies. Moreover, direct evaluation in co-cultures of the same cells allows for testing of therapeutic effectiveness in more complex settings, in which astrocytes affect pharmacological properties of neurons.

Activating AMP-activated protein kinase by an α1 selective activator compound 13 attenuates dexamethasone-induced osteoblast cell death.

Excessive glucocorticoid (GC) usage may lead to non-traumatic femoral head osteonecrosis. Dexamethasone (Dex) exerts cytotoxic effect to cultured osteoblasts. Here, we investigated the potential activity of Compound 13 (C13), a novel α1 selective AMP-activated protein kinase (AMPK) activator, against the process. Our data revealed that C13 pretreatment significantly attenuated Dex-induced apoptosis and necrosis in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. AMPK activation mediated C13' cytoprotective effect in osteoblasts. The AMPK inhibitor Compound C, shRNA-mediated knockdown of AMPKα1, or dominant negative mutation of AMPKα1 (T172A) almost abolished C13-induced AMPK activation and its pro-survival effect in osteoblasts. On the other hand, forced AMPK activation by adding AMPK activator A-769662 or exogenous expression a constitutively-active (ca) AMPKα1 (T172D) mimicked C13's actions and inhibited Dex-induced osteoblast cell death. Meanwhile, A-769662 or ca-AMPKα1 almost nullified C13's activity in osteoblast. Further studies showed that C13 activated AMPK-dependent nicotinamide adenine dinucleotide phosphate (NADPH) pathway to inhibit Dex-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary murine osteoblasts. Such effects by C13 were almost reversed by Compound C or AMPKα1 depletion/mutation. Together, these results suggest that C13 alleviates Dex-induced osteoblast cell death via activating AMPK signaling pathway.

Short-Term Curcumin Gavage Sensitizes Insulin Signaling in Dexamethasone-Treated C57BL/6 Mice.

BACKGROUND: Long-term dietary curcumin (>12 wk) improves metabolic homeostasis in obese mice by sensitizing insulin signaling and reducing hepatic gluconeogenesis. Whether these occur only secondary to its chronic anti-inflammatory and antioxidative functions is unknown. OBJECTIVE: In this study, we assessed the insulin sensitization effect of short-term curcumin gavage in a rapid dexamethasone-induced insulin resistance mouse model, in which the chronic anti-inflammatory function is eliminated. METHODS: Six-week-old male C57BL/6 mice received an intraperitoneal injection of dexamethasone (100 mg/kg body weight) or phosphate-buffered saline every day for 5 d, with or without simultaneous curcumin gavage (500 mg/kg body weight). On day 7, insulin tolerance tests were performed. After a booster dexamethasone injection and curcumin gavage on day 8, blood glucose and insulin concentrations were measured. Liver tissues were collected on day 10 for quantitative polymerase chain reaction and Western blotting to assess gluconeogenic gene expression, insulin signaling, and the expression of fibroblast growth factor 21 (FGF21). Primary hepatocytes from separate, untreated C57BL/6 mice were used for testing the in vitro effect of curcumin treatment. RESULTS: Dexamethasone injection impaired insulin tolerance (P < 0.05) and elevated ambient plasma insulin concentrations by ~2.7-fold (P < 0.01). Concomitant curcumin administration improved insulin sensitivity and reduced hepatic gluconeogenic gene expression. The insulin sensitization effect of curcumin was demonstrated by increased stimulation of S473 phosphorylation of protein kinase B (P < 0.01) in the dexamethasone-treated mouse liver, as well as the repression of glucose production in primary hepatocytes (P < 0.001). Finally, curcumin gavage increased FGF21 expression by 2.1-fold in the mouse liver (P < 0.05) and curcumin treatment increased FGF21 expression in primary hepatocytes. CONCLUSION: These observations suggest that the early beneficial effect of curcumin intervention in dexamethasone-treated mice is the sensitization of insulin signaling, involving the stimulation of FGF21 production, a known insulin sensitizer.

Inhibition of adipogenic differentiation of bone marrow mesenchymal stem cells by erythropoietin via activating ERK and P38 MAPK.

We examined whether erythropoietin (EPO) can inhibit adipogenic differentiation of mesenchymal stem cells (MSCs) in the mouse bone marrow and its underlying mechanism. We separated and extracted mouse bone marrow MSCs and induced adipogenic differen-tiation using 3-isobutyl-1-methylxanthine, insulin, and dexamethasone. Different concentrations of EPO were added to the cells and observed by Oil Red O staining on the 20th day to quantitatively analyze the degree of cell differentiation. mRNA expression levels of peroxysome proliferator-activated receptor γ (PPARγ), CCAAT enhancer binding protein α, and adiponectin were analyzed by real-time quantitative polymerase chain reaction, and the activity of PPARγ, extracellular sig-nal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK) were determined by western blotting. EPO significantly inhibited adipogenic differentiation of MSCs after 20 days and reduced absorbance values by Oil Red O staining without affecting proliferation activity. EPO downregulated the mRNA expression of PPARγ, CCAAT enhancer binding protein α, fatty acid binding protein 4, and adiponec-tin during adipogenesis and increased protein phosphorylation of ERK, p38 MAPK, and PPARγ during differentiation. EPO downregulated the mRNA expression of PPARγ, CCAAT enhancer binding protein α, fatty acid binding protein 4, and adiponectin by increasing protein phosphor-ylation of ERK, p38 MAPK, and PPARγ during differentiation, which inhibited adipogenic differentiation of MSCs.

Activation of AMPK participates hydrogen sulfide-induced cyto-protective effect against dexamethasone in osteoblastic MC3T3-E1 cells.

Long-time glucocorticoids (GCs) usage causes osteoporosis. In the present study, we explored the potential role of hydrogen sulfide (H2S) against dexamethasone (Dex)-induced osteoblast cell damage, and focused on the underlying mechanisms. We showed that two H2S-producing enzymes, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), were significantly downregulated in human osteonecrosis tissues as well as in Dex-treated osteoblastic MC3T3-E1 cells. H2S donor NaHS as well as the CBS activator S-adenosyl-l-methionine (SAM) inhibited Dex-induced viability reduction, death and apoptosis in MC3T3-E1 cells. NaHS activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling, which participated its cyto-protective activity. AMPK inhibition by its inhibitor (compound C) or reduction by targeted-shRNA suppressed its pro-survival activity against Dex in MC3T3-E1 cells. Further, we found that NaHS inhibited Dex-mediated reactive oxygen species (ROS) production and ATP depletion. Such effects by NaHS were again inhibited by compound C and AMPKα1-shRNA. In summary, we show that H2S inhibits Dex-induced osteoblast damage through activation of AMPK signaling. H2S signaling might be further investigated as a novel target for anti-osteoporosis treatment.

Corticotropin-releasing hormone exerts direct effects on neuronal progenitor cells: implications for neuroprotection.

Neurogenesis during embryonic and adult life is tightly regulated by a network of transcriptional, growth and hormonal factors. Emerging evidence indicates that activation of the stress response, via the associated glucocorticoid increase, reduces neurogenesis and contributes to the development of adult diseases.As corticotrophin-releasing hormone (CRH) or factor is the major mediator of adaptive response to stressors, we sought to investigate its involvement in this process. Accordingly, we found that CRH could reverse the damaging effects of glucocorticoid on neural stem/progenitor cells (NS/PCs), while its genetic deficiency results in compromised proliferation and enhanced apoptosis during neurogenesis. Analyses in fetal and adult mouse brain revealed significant expression of CRH receptors in proliferating neuronal progenitors. Furthermore, by using primary cultures of NS/PCs, we characterized the molecular mechanisms and identified CRH receptor-1 as the receptor mediating the neuroprotective effects of CRH. Finally, we demonstrate the expression of CRH receptors in human fetal brain from early gestational age, in areas of active neuronal proliferation. These observations raise the intriguing possibility for CRH-mediated pharmacological applications in diseases characterized by altered neuronal homeostasis, including depression, dementia, neurodegenerative diseases, brain traumas and obesity.

Leptin restores adult hippocampal neurogenesis in a chronic unpredictable stress model of depression and reverses glucocorticoid-induced inhibition of GSK-3ß/ß-catenin signaling.

Stress and glucocorticoid stress hormones inhibit neurogenesis, whereas antidepressants increase neurogenesis and block stress-induced decrease in neurogenesis. Our previous studies have shown that leptin, an adipocyte-derived hormone with antidepressant-like properties, promotes baseline neurogenesis in the adult hippocampus. This study aimed to determine whether leptin is able to restore suppression of neurogenesis in a rat chronic unpredictable stress (CUS) model of depression. Chronic treatment with leptin reversed the CUS-induced reduction of hippocampal neurogenesis and depression-like behaviors. Leptin treatment elicited a delayed long-lasting antidepressant-like effect in the forced swim behavioral despair test, and this effect was blocked by ablation of neurogenesis with X-irradiation. The functional isoform of the leptin receptor, LepRb, and the glucocorticoid receptor (GR) were colocalized in hippocampal neural stem/progenitor cells in vivo and in vitro. Leptin treatment reversed the GR agonist dexamethasone (DEX)-induced reduction of proliferation of cultured neural stem/progenitor cells from adult hippocampus. Further mechanistic analysis revealed that leptin and DEX converged on glycogen synthase kinase-3ß (GSK-3ß) and ß-catenin. While DEX decreased Ser9 phosphorylation and increased Tyr216 phosphorylation of GSK-3ß, leptin increased Ser9 phosphorylation and attenuated the effects of DEX at both Ser9 and Tyr216 phosphorylation sites of GSK-3ß. Moreover, leptin increased total level and nuclear translocation of ß-catenin, a primary substrate of GSK-3ß and a key regulator in controlling hippocampal neural progenitor cell proliferation, and reversed the inhibitory effects of DEX on ß-catenin. Taken together, our results suggest that adult neurogenesis is involved in the delayed long-lasting antidepressant-like behavioral effects of leptin, and leptin treatment counteracts chronic stress and glucocorticoid-induced suppression of hippocampal neurogenesis via activating the GSK-3ß/ß-catenin signaling pathway.

Acid sphingomyelinase is required for protection of effector memory T cells against glucocorticoid-induced cell death.

The activity of acid sphingomyelinase (aSMase) was previously reported to be involved in glucocorticoid-induced cell death (GICD) of T lymphocytes. This mechanism in turn is believed to contribute to the therapeutic efficacy of glucocorticoids (GCs) in the treatment of inflammatory diseases. In this study, we reassessed the role of aSMase in GICD by using aSMase knockout mice. The absence of aSMase largely abolished the partial protection that effector memory CD4(+) T cells in wild-type mice possess against GICD. Reduced IL-2 secretion by aSMase-deficient CD4(+) T cells suggested that a lack of this important survival factor might be the cause of these cells' enhanced susceptibility to GICD. Indeed, addition of IL-2 restored the protection against GICD, whereas neutralization of IL-2 abrogated the otherwise protective effect seen in wild-type effector memory CD4(+) T cells. The therapeutic implications of the altered sensitivity of aSMase-deficient T cells to GICD were assessed in models of inflammatory disorders; namely, experimental autoimmune encephalomyelitis and acute graft-versus-host disease. Surprisingly, GC treatment was equally efficient in both models in terms of ameliorating the diseases, regardless of the genotype of the T cells. Thus, our data reveal a hitherto unrecognized contribution of aSMase to the sensitivity of effector memory CD4(+) T cells to GICD and call into question the traditionally attributed importance of GICD of T cells to the treatment of inflammatory diseases by GCs.

[Effects of astragalosides on PC12 cells injury and APP expression induced by dexamethasone and Abeta(25-35)].

OBJECTIVE: To study the effects of Astragaloside (AST) on PC12 cells injury and APP expression induced by dexamethasone (DEX) and beta-amyloid protein 25-35 (Abeta(25-35). METHODS: Logarithmic growth phase of the PC12 cells were seeded in culture plates. DEX 5 micromol/L and Abeta(25-35) 1 micromol/L were used to induce PC12 cells injury. MTT assay was used to detect the PC12 cells activity. RT-PCR was used to detect the APP, alpha-secretase and beta-secretase mRNA level of PC12 cells. Western bloting was used to detect APP protein expression of PC12 cells. RESULTS: MTT results showed that, DEX and Abeta(35-35) co-application could significantly decrease PC12 cells activity (P5 < 0.01). AST (10.20 mg/L) and Ginsenoside Rg1 (16 micromol/L) could increase PC12 cells activity. RT-PCR analysis showed that DEX and Abeta(25-35) co-application could significantly increase the beta-secretase mRNA levels and APP770, lower alpha-secretase mRNA levels. AST (10.20 mg/L) and Rg1 could decrease the elevated APP770, beta-secretase mRNA levels and increase the alpha-secretase mRNA level of PCl2 cells. Western bloting analysis result showed that AST (10.20 mg/L) and Rg1 could decrease the APP expression of PC12 cells induced by DEX and Abeta(25-35). CONCLUSION: AST has protective effects on PC12 cell injury induced by DEX and Abeta(25-35). The mechanism may be associated with decreasing the beta-secretase mRNA levels and APP expression, increasing the alpha-secretase mRNA levels.

Dexamethasone enhances 1alpha,25-dihydroxyvitamin D3 effects by increasing vitamin D receptor transcription.

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner.

HPA axis dysregulation is associated with differential methylation of CpG-sites in related genes.

DNA methylation shifts in Hypothalamic-pituitary-adrenal (HPA) axis related genes is reported in psychiatric disorders including hypersexual disorder. This study, comprising 20 dexamethasone suppression test (DST) non-suppressors and 73 controls, examined the association between the HPA axis dysregulation, shifts in DNA methylation of HPA axis related genes and importantly, gene expression. Individuals with cortisol level ≥ 138 nmol/l, after the low dose (0.5 mg) dexamethasone suppression test (DST) were classified as non-suppressors. Genome-wide methylation pattern, measured in whole blood using the EPIC BeadChip, investigated CpG sites located within 2000 bp of the transcriptional start site of key HPA axis genes, i.e.: CRH, CRHBP, CRHR-1, CRHR-2, FKBP5 and NR3C1. Regression models including DNA methylation M-values and the binary outcome (DST non-suppression status) were performed. Gene transcripts with an abundance of differentially methylated CpG sites were identified with binomial tests. Pearson correlations and robust linear regressions were performed between CpG methylation and gene expression in two independent cohorts. Six of 76 CpG sites were significantly hypermethylated in DST non-suppressors (nominal P < 0.05), associated with genes CRH, CRHR1, CRHR2, FKBP5 and NR3C1. NR3C1 transcript AJ877169 showed statistically significant abundance of probes differentially methylated by DST non-suppression status and correlated with DST cortisol levels. Further, methylation levels of cg07733851 and cg27122725 were positively correlated with gene expression levels of the NR3C1 gene. Methylation levels of cg08636224 (FKBP5) correlated with baseline cortisol and gene expression. Our findings revealed that DNA methylation shifts are involved in the altered mechanism of the HPA axis suggesting that new epigenetic targets should be considered behind psychiatric disorders.

Characterization of a novel non-steroidal glucocorticoid receptor antagonist.

Selective antagonists of the glucocorticoid receptor (GR) are desirable for the treatment of hypercortisolemia associated with Cushing's syndrome, psychic depression, obesity, diabetes, neurodegenerative diseases, and glaucoma. NC3327, a non-steroidal small molecule with potent binding affinity to GR (K(i)=13.2nM), was identified in a high-throughput screening effort. As a full GR antagonist, NC3327 greatly inhibits the dexamethasone (Dex) induction of marker genes involved in hepatic gluconeogenesis, but has a minimal effect on matrix metalloproteinase 9 (MMP-9), a GR responsive pro-inflammatory gene. Interestingly, the compound recruits neither coactivators nor corepressors to the GR complex but competes with glucocorticoids for the interaction between GR and a coactivator peptide. Moreover, NC3327 does not trigger GR nuclear translocation, but significantly blocks Dex-induced GR transportation to the nucleus, and thus appears to be a 'competitive' GR antagonist. Therefore, the non-steroidal compound, NC3327, may represent a new class of GR antagonists as potential therapeutics for a variety of cortisol-related endocrine disorders.

Neonatal dexamethasone treatment in the rat leads to kidney damage in adulthood.

UNLABELLED: Recently, concern has been raised that corticosteroid treatment of preterm neonates might be associated with adverse effects later in life, including early development of hypertension. Here, we investigate the impact of neonatal dexamethasone (Dex) treatment on early renal cell proliferation and nephron number. We analyzed mitotic activity in renal cortex of rat pups neonatally treated with Dex. Nephron number was measured and possible renal damage was quantified by counting inflammatory foci, ED-1 positive cells (macrophages), and the desmin score (activated podocytes). Mitotic activity was 34 and 29% lower on d 2 and 4 in Dex-treated rats compared with saline-treated controls. The number of glomeruli was lower at 4 wk, but nephron size was unchanged after Dex treatment, as calculated from glomerular density and (lower) body- and kidney weight. At wk 50, the glomerular number was significantly lower in Dex-treated rats, whereas body and kidney weight were the same as in Sal controls. Dex rats also showed more kidney damage, manifested by a approximately 3.5-fold increase in inflammation foci/mm and in ED-1 positive cells/mm and a approximately 4.3-fold increased desmin score. Temporary suppression of mitotic activity during neonatal Dex treatment leads to reduction of nephron number and more kidney damage later in life. ABBREVIATIONS: :

Abnormal dexamethasone suppression tests in a rifampicin-treated patient with suspected Cushing's syndrome.

The dexamethasone suppression test is a useful endocrinological test to diagnose Cushing's syndrome. However, its interpretation may be influenced by many factors such as stress, alcohol, failure to ingest the dexamethasone, altered metabolism, drug interaction and obesity. This report illustrates such an instance, whereby the result of the test was erratic due to the anti-tuberculous drug rifampicin. Rifampicin has been found to profoundly attenuate the biological effects of dexamethasone, probably by enhancing its metabolism in the liver. The exact mechanism of the drug interaction remains elusive, though induction of hepatic CYP3A4 enzyme complex is a possible mechanism. In a patient treated with rifampicin, the results of dexamethasone suppression tests thus have no diagnostic value and can be very misleading.

Resveratrol Protects Osteoblasts Against Dexamethasone-Induced Cytotoxicity Through Activation of AMP-Activated Protein Kinase.

PURPOSE: Glucocorticoids are used for the treatment of inflammatory diseases, but glucocorticoid treatment is associated with bone damage. Resveratrol is a phytoalexin found in many plants, and we investigated its protective role on dexamethasone-induced dysfunction in MC3T3-E1 cells and primary osteoblasts. MATERIALS AND METHODS: MC3T3-E1 cells and primary osteoblasts were treated with dexamethasone in the presence/absence of different doses of resveratrol for 24 or 48 h. Then, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium (MTT) and lactate dehydrogenase (LDH) assays were used to evaluate cell viability. Apoptosis was analyzed by a flow cytometry. An alkaline phosphatase (ALP) activity assay and Alizarin Red S staining were used to study osteoblast differentiation. Expression of osteoblast-related genes was measured by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The AMP-activated protein kinase (AMPK) signaling pathway and mitochondrial expression of superoxide dismutase were evaluated by Western blotting. Intracellular reactive oxygen species (ROS), adenosine triphosphate (ATP) content, mitochondrial-complex activity, and mitochondrial DNA content were measured to evaluate mitochondrial function. RESULTS: Resveratrol induced the proliferation and inhibited apoptosis of osteoblasts in the presence of dexamethasone. Resveratrol increased the ALP activity and mineralization of osteoblasts. Resveratrol also attenuated dexamethasone-induced inhibition of mRNA expression of osteogenesis maker genes, including bone morphogenetic protein-2, osteoprotegerin, runt-related transcription factor-2, and bone Gla protein. Resveratrol alleviated dexamethasone-induced mitochondrial dysfunction. Resveratrol strongly stimulated expression of peroxisome proliferator-activated receptor-γ coactivator 1α and sirtuin-3 genes, as well as their downstream target gene superoxide dismutase-2. Resveratrol induced phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). Blockade of AMPK signaling using compound C reversed the protective effects of resveratrol against dexamethasone. CONCLUSION: Resveratrol showed protective effects against dexamethasone-induced dysfunction of osteoblasts by activating AMPK signaling.

Prunella vulgaris L protects glucocorticoids-induced osteogenesis inhibition in bone marrow mesenchymal stem cells through activating the Smad pathway.

OBJECTIVE: To elucidate the role of Prunella vulgaris L (PVL) in protecting glucocorticoids (GC)-induced osteogenesis inhibition, thereafter, protecting the deterioration of osteoporosis (OP). MATERIALS AND METHODS: Cell Counting Kit-8 (CCK-8) assay was conducted to assess the influence of PVL treatment on MSCs viability. Osteogenesis in MSCs was induced by Dexamethasone (DEX) stimulation. Regulatory effects of PVL on osteogenesis-related gene expressions, ALP activity, and mineralization ability in DEX-induced MSCs were determined. At last, protein levels of p-Smad1/5/9 and total-Smad1/5/9 influenced by DEX and PVL were measured by Western blot. RESULTS: PVL treatment did not pose a time- or dose-dependent influence on MSCs viability. DEX induction in MSCs downregulated ALP, RUNX2, Bglap, and Osterix. ALP activity and mineralization in DEX-induced MSCs were suppressed. Downregulated osteogenesis-related genes decreased ALP activity and mineralization in MSCs undergoing DEX stimulation were partially reversed by PVL treatment. Moreover, the downregulated p-Smad1/5/9 level in DEX-induced MSCs was elevated by PVL treatment, while total-Smad1/5/9 was not affected. CONCLUSIONS: PVL alleviated GC-induced suppression in MSCs osteogenesis by activating the Smad pathway, thereafter, protecting the deterioration of OP.