Structure-activity relationship and docking analysis of nature flavonoids as inhibitors of human and rat gonadal 3ß-hydroxysteroid dehydrogenases for therapeutic purposes.

The potential inhibitory effects of flavonoids on gonadal steroid biosynthesis have gained attention due to their widespread presence in natural plant sources. Specifically, our study focused on evaluating the inhibitory efficacy of these compounds on human 3ß-hydroxysteroid dehydrogenase 2 (h3ß-HSD2) and rat homolog r3ß-HSD1, enzymes responsible for the conversion of pregnenolone to progesterone. Through our investigations, we observed that the potency of flavonoids was silymarin (IC50, 1.31 µM) > luteolin (4.63 µM) > tectorigenin > (5.86 µM), and rutin (44.12 µM) in inhibiting human KGN cell microsomal h3ß-HSD2. Similarly, the potency of flavonoids was silymarin (9.50 µM) > luteolin (11.49 µM) > tectorigenin (14.06 µM), and rutin (145.71 µM) in inhibiting rat testicular r3ß-HSD1. Silymarin, luteolin, and tectorigenin acted as mixed inhibitors of both human and rat 3ß-HSDs. Luteolin and tectorigenin were able to penetrate human KGN cells to inhibit progesterone secretion. Furthermore, docking analysis and structure-activity relationship analysis highlighted the importance of hydrogen bond formation for the inhibitory efficacy of these compounds against h3ß-HSD2 and r3ß-HSD1. Overall, this study demonstrates that silymarin exhibits the most potent inhibition of human and rat gonadal 3ß-HSDs, and significant SAR differences exist among the tested compounds.

Predisposing factors for adrenal crisis in chronic adrenal insufficiency: a case-control study.

OBJECTIVE: This study aims to identify susceptibility markers for adrenal crises (AC) in educated patients with chronic adrenal insufficiency (AI). DESIGN: A case-control study involving 66 patients with AI analyzing the impact of glucocorticoid and mineralocorticoid exposure, adrenomedullary function, inflammatory parameters, and educational status on AC frequency. Patients were categorized into low (n = 32) and high (n = 34) AC frequency groups based on AC occurrence (below or 2 times above the average of the reported AC frequency of 8.3 AC/100 patient-years in a previous prospective study). METHODS: Parameters, including cortisol plasma profile and urinary steroid excretion after administration of the morning glucocorticoid dose, 24-h urinary steroid profiling, salivary cortisol profiling, and hair cortisol, estimated cortisol exposure. Polymorphisms (single nucleotide polymorphism [SNP]) of the glucocorticoid receptor (NR3C1) and mineralocorticoid receptor (NR3C2) associated with individual steroid sensitivity were assessed together with SNPs for 11ß-hydroxysteroid dehydrogenase 1 (HSD11B1) and 11ß-hydroxysteroid dehydrogenase 2 (HSD11B2). Mineralocorticoid replacement was evaluated by serum and urinary electrolytes and osmolality, plasma-renin concentration, and ambulatory blood pressure levels. We additionally measured plasma and urinary catecholamines, serum levels of IL6 and hsCRP, and SNPs of IL6 and TNF-alpha. Patient knowledge of AC prevention was assessed by questionnaires. RESULTS: Frequent AC patients had higher daily glucocorticoid doses and hair cortisol levels, with no significant differences in other parameters investigated. AC frequency is inversely correlated with the frequency of self-reported adjustments of the glucocorticoid replacement. CONCLUSION: Higher glucocorticoid dosages in high-risk patients, despite unaffected cortisol metabolism, may be linked to decreased cortisol sensitivity or impaired glucocorticoid absorption. Proactive dose adjustments show a protective effect against AC, regardless of biological vulnerability.

Resistance to Resveratrol Treatment in Experimental PTSD Is Associated with Abnormalities in Hepatic Metabolism of Glucocorticoids.

Glucocorticoids are metabolized by the CYP3A isoform of cytochrome P450 and by 11-ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD-1). Experimental data suggest that post-traumatic stress disorder (PTSD) is associated with an increase in hepatic 11ß-HSD-1 activity and a concomitant decrease in hepatic CYP3A activity. Trans-resveratrol, a natural polyphenol, has been extensively studied for its antipsychiatric properties. Recently, protective effects of trans-resveratrol were found in relation to PTSD. Treatment of PTSD rats with trans-resveratrol allowed the rats to be divided into two phenotypes. The first phenotype is treatment-sensitive rats (TSR), and the second phenotype is treatment-resistant rats (TRRs). In TSR rats, trans-resveratrol ameliorated anxiety-like behavior and reversed plasma corticosterone concentration abnormalities. In contrast, in TRR rats, trans-resveratrol aggravated anxiety-like behavior and decreased plasma corticosterone concentration. In TSR rats, hepatic 11ß-HSD-1 activity was suppressed, with a concomitant increase in CYP3A activity. In TRR rats, the activities of both enzymes were suppressed. Thus, the resistance of PTSD rats to trans-resveratrol treatment is associated with abnormalities in hepatic metabolism of glucocorticoids. The free energy of binding of resveratrol, cortisol, and corticosterone to the human CYP3A protein was determined using the molecular mechanics Poisson-Boltzmann surface area approach, indicating that resveratrol could affect CYP3A activity.

Direct inhibition of human and rat 11ß-hydroxysteroid dehydrogenase 2 by per- and polyfluoroalkyl substances: Structure-activity relationship and in silico docking analysis.

Per- and polyfluoroalkyl substances (PFAS) are persistent in the environment and may disrupt the endocrine system. Our previous study showed that perfluorooctanoic acid (PFOA, C8) and perfluorooctanesulfonic acid (PFOS, C8S) can inhibit 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) activity leading to an active glucocorticoid accumulation. In this study, we extended investigation for 17 PFAS, including carboxylic and sulfonic acids, with different carbon-chain lengths, to determine their inhibitory potency and structure-activity relationship in human placental and rat renal 11ß-HSD2. C8-C14 PFAS at 100 µM significantly inhibited human 11ß-HSD2 with a potency as C10 (half-maximal inhibitory concentration, IC50, 9.19 µM) > C11 (15.09 µM) > C12 (18.43 µM) > C9 (20.93 µM) > C13 (124 µM) > C14 (147.3 µM) > other C4-C7 carboxylic acids, and C8S > C7S = C10S > other sulfonic acids. For rat 11ß-HSD2, only C9 and C10 and C7S and C8S PFAS exhibited significant inhibitory effects. PFAS are primarily mixed/competitive inhibitors of human 11ß-HSD2. Preincubation and simultaneous incubation with the reducing agent dithiothreitol significantly increased human 11ß-HSD2 but not rat 11ß-HSD2, and preincubation but not simultaneous incubation with dithiothreitol partially reversed C10-mediated inhibition on human 11ß-HSD2. Docking analysis showed that all PFAS bound to the steroid-binding site and carbon-chain length determined the potency of inhibition, with the optimal molecular length (12.6 Å) for potent inhibitors PFDA and PFOS, which is comparable to the molecular length (12.7 Å) of the substrate cortisol. The length between 8.9 and 17.2 Å is the probable threshold molecular length to inhibit human 11ß-HSD2. In conclusion, the carbon-chain length determines the inhibitory effect of PFAS on human and rat 11ß-HSD2, and the inhibitory potency of long-chain PFAS on human and rat 11ß-HSD2 showed V-shaped pattern. Long-chain PFAS may partially act on the cysteine residues of human 11ß-HSD2.

The effect of cold exposure on the levels of glucocorticoids, 11-hydroxysteroid dehydrogenase 2, and placental vascularization in a rat model.

OBJECTIVE: Cold exposure (CE) before birth is one of the initial stressors that may impact mammalian pregnancy, changing placental and fetal development and affecting the health of the offspring. While glucocorticoids (GCs) participate in the body's response to the stress of CE, the specific mechanisms of their action are unclear. This study aims to determine the effect of CE stress on the placenta and to test whether stress, caused by cold exposure in pregnancy impairs fetal development by changing placental angiogenesis via excessive GC expression. MATERIALS AND METHODS: CE rat model was created by exposing 30 SD rats to cold preconception, or during the first, second, and third weeks of pregnancy. Serum cortisol and soluble fms-like tyrosine kinase-1 (sFlt-1) expression levels, physiological index changes (food intake, body weight change and blood pressure), and pregnancy outcomes (fetal rat weight, number of live fetal rats, and placental weight) were collected at baseline and at different time points after the conception. Protein expression levels of 11 ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), glucocorticoid receptor, vascular endothelial growth factor A (VEGF-A), placental growth factor (PIGF), and sFlt-1 in placental tissues were measured by western blotting. Cytokeratin (CK) and laminin (LN) in trophoblasts, and α-actin in vascular smooth muscle of the spiral arteries of pregnant rats after the systemic cold treatment were assessed by immunofluorescence and visualized by fluorescent microscopy. To test the effect of 11ß-HSD2 levels on the placental recasting, human first-trimester extravillous trophoblast cells (HTR8/SVneo) underwent knockdown using specific 11ß-HSD2 siRNA constructs.  Expression levels of 11ß-HSD2 were analyzed by quantitative real-time PCR (qPCR) and into HTR8 cells, and the expression levels of the 11ß-HSD2 gene in each group were measured using qPCR. Cell migration and invasion was assessed by Transwell migration assay, and sFlt-1 levels in HTR8 cells were measured by ELISA. RESULTS: CE pre-conception led to consistently increasing serum corticosterone and sFlt-1 levels throughout pregnancy, and persistently increased diastolic blood pressure (DBP) in rat CE model compared to control animals. CE during the second week of gestation (Gp.3) was associated with significantly lower placental weight (p=0.0003). Cold exposure in the third week (Gp.4) was associated with significantly (p=0.001) lower fetal weight. CE pre-conception was associated with significantly decreased placental levels of 11ß-HSD2, glucocorticoid receptor, VEGF-A, PIGF, and sFlt-1 proteins and α-actin compared to the control group. Silencing 11ß-HSD2 by siRNA led to reduced cell migrations and invasion, and markedly increased expression levels of sFlt-1 in HTR8/SVneo cells (p<0.05). CONCLUSIONS: Pre-conception cold exposure and during early pregnancy leads to increased GCs levels and impaired placental 11ß-HSD2 activity. We suggest that the subsequent 11ß-HSD2-induced increase in the sFlt-1expression during early pregnancy may affect placental vascular remodeling and change placental morphological structure and function.

C11-hydroxy and C11-oxo C19 and C21 Steroids: Pre-Receptor Regulation and Interaction with Androgen and Progesterone Steroid Receptors.

C11-oxy C19 and C11-oxy C21 steroids have been identified as novel steroids but their function remains unclear. This study aimed to investigate the pre-receptor regulation of C11-oxy steroids by 11ß-hydroxysteroid dehydrogenase (11ßHSD) interconversion and potential agonist and antagonist activity associated with the androgen (AR) and progesterone receptors (PRA and PRB). Steroid conversions were investigated in transiently transfected HEK293 cells expressing 11ßHSD1 and 11ßHSD2, while CV1 cells were utilised for agonist and antagonist assays. The conversion of C11-hydroxy steroids to C11-oxo steroids by 11ßHSD2 occurred more readily than the reverse reaction catalysed by 11ßHSD1, while the interconversion of C11-oxy C19 steroids was more efficient than C11-oxy C21 steroids. Furthermore, 11-ketodihydrotestosterone (11KDHT), 11-ketotestosterone (11KT) and 11ß-hydroxydihydrotestosterone (11OHDHT) were AR agonists, while only progestogens, 11ß-hydroxyprogesterone (11ßOHP4), 11ß-hydroxydihydroprogesterone (11ßOHDHP4), 11α-hydroxyprogesterone (11αOHP4), 11α-hydroxydihydroprogesterone (11αOHDHP4), 11-ketoprogesterone (11KP4), 5α-pregnan-17α-diol-3,11,20-trione (11KPdione) and 21-deoxycortisone (21dE) exhibited antagonist activity. C11-hydroxy C21 steroids, 11ßOHP4, 11ßOHDHP4 and 11αOHP4 exhibited PRA and PRB agonistic activity, while only C11-oxo steroids, 11KP4 and 11-ketoandrostanediol (11K3αdiol) demonstrated PRB agonism. While no steroids antagonised the PRA, 11OHA4, 11ß-hydroxytestosterone (11OHT), 11KT and 11KDHT exhibited PRB antagonism. The regulatory role of 11ßHSD isozymes impacting receptor activation is clear-C11-oxo androgens exhibit AR agonist activity; only C11-hydroxy progestogens exhibit PRA and PRB agonist activity. Regulation by the downstream metabolites of active C11-oxy steroids at the receptor level is apparent-C11-hydroxy and C11-oxo metabolites antagonize the AR and PRB, progestogens the former, androgens the latter. The findings highlight the intricate interplay between receptors and active as well as "inactive" C11-oxy steroids, suggesting novel regulatory tiers.

Cucumis melo var. momordica as a Potent Antidiabetic, Antioxidant and Possible Anticovid Alternative: Investigation through Experimental and Computational Methods.

Diabetes mellitus is a typical life threatening of disease, which generate due to the dysfunction of ß cells of pancreas. In 2014, WHO stated that 422 million people were infected with DM. The current pattern of management of diabetes included synthetic or plant based oral hypoglycemic drugs and insulin but drug resentence is become a very big issues in antidiabetic therapy. Thus, it's very earnest to discover now medication for this disease. Now the days, it is well acknowledged that diabetic patients are more prone towards covid and related complications. Thus, medical practitioners reformed the methodology of prescribing medication for covid infected antidiabetic therapy and encouraging the medication contains dual pharmacological properties. It is also well know that polyphenols specifically hold a significant role in oxidative stress and reduced the severity of many inflammatory diseases. Cucumis melo has rich history as ethano-pharmacological use in Indian subcontinent. The fruit and seed are well-known for the treatment of various diseases due to the presence of phenolics. Therefore, in this study, the combined mixture of flower and seeds were used for the extraction of polyphenolic rich extract and tested for antidiabetic activity through the antioxidant and in vivo experiments. The antioxidant potential measurement exhibited that the selected plant extract has the significant competence to down-regulate oxidative stress (DPPH scavenging IC50 at 60.7±1.05 µg/mL, ABTS IC50 at 62.15±0.50 µg/mL). Furthermore, the major polyphenolic phyto-compounds derived from the Cucumis melo were used for in silico anticovid activity, docking, and complementarity studies. The anticovid activity prognosis reflected that selected phyto-compounds amentoflavone and vanillic acid have optimal possibility to interact with 3C-like protease and through this moderate anticovid activity can be exhibit. The docking experiments established that the selected compounds have propensity to interact with protein tyrosine phosphatase 1B, 11ß-Hydroxysteroid dehydrogenase, superoxide dismutase, glutathione peroxidase, and catalase ß-glucuronidase receptor. In vivo experiments showed that 500 mg/kg, Cucumis melo extract ominously amplified body weight, plasma insulin, high-density lipoprotein levels, and biochemical markers. Furthermore, extract significantly downregulate the blood glucose, total cholesterol, triglycerides, low-density lipoprotein, and very low-density lipoprotein.

The role of mineralocorticoids and glucocorticoids under the impact of 11ß-hydroxysteroid dehydrogenase in human breast lesions.

We attempted to explore the possible involvement of the in situ availability of mineralocorticoids and mineralocorticoid receptor (MR) in the pathogenesis of mammary ductal carcinoma. We also explored their individual profiles among different subtypes of invasive ductal carcinomas of no special type (IDC-NST) by evaluating the status of MR, Glucocorticoid receptor (GR), and 11ß hydroxysteroid dehydrogenase (HSD) 1/2 at each stage of the putative cascade of the mammary ductal proliferative disorders. In this study, IDC-NST, ductal carcinoma in situ (DCIS), atypical ductal hyperplasia (ADH), and non-pathological breast tissues were all evaluated by immunohistochemistry. MR was significantly lower in ADH than in DCIS or IDC-NST. 11ßHSD2 was significantly lower in ADH than normal breast tissue and 11ßHSD1 was significantly higher in DCIS than normal, ADH, or IDC-NST. MR in progesterone receptor (PR)-positive IDC-NST cases tended to be associated with the Ki-67 labeling index. Results of the present study demonstrated that the status of MR and GR in conjunction with the 11ßHSDs was correlated with the development of low-grade proliferative disorders in mammary glands. In addition, the potential crosstalk between MR and PR could also influence cell proliferation of breast carcinoma cells but further investigations are required for clarification.

Cortisol induces follicle regression, while FSH prevents cortisol-induced follicle regression in pigs.

During follicular development, a few dominant follicles develop to large antral dominant follicles, whereas the remaining follicles undergo atretic degeneration. Because vascularization on the follicular surface is a morphological feature of dominant follicles, we previously classified these follicles as vascularized follicles (VFs) and non-VFs (NVFs). In NVFs, progesterone producing genes were expressed similarly to that in VFs; however, the progesterone concentration in follicular fluid was low in large NVFs. Therefore, we estimated that progesterone is converted to cortisol, which induces the loss of follicular functions. In this study, we comparative analyzed the expression of genes for progesterone converting enzymes (Cytochrome (CYP)11B1, CYP21A2, Hydroxysteroid (HSD)11B2) and cortisol receptor (NR3C1) in VF and NVF granulosa cells. In NVFs, expression of cortisol producing genes (CYP11B1 and CYP21A2) was higher than in VFs. Expression of the gene for the cortisol metabolizing enzyme HSD11B2 in NVFs was significantly lower than in VFs. In NVFs, accompanied by increasing cortisol concentration in follicular fluid, apoptosis of granulosa and cumulus cells was observed. Cultivation with FSH and metyrapone (a CYP11B1 inhibitor) of NVF cumulus-oocyte complexes inhibited apoptosis of cumulus cells and induced cumulus cell proliferation and oocyte maturation. Cortisol-induced CYP11B1 and CYP21A2 expression, whereas FSH-induced HSD11B2 mRNA expression in VF granulosa cells in the presence of cortisol. Furthermore, an addition of 18ß-glycyrrhetinic acid (18-GA; a HSD17B2 inhibitor) to cortisol and FSH-containing medium increased apoptosis of VF granulosa cells. These results suggested that cortisol is a stimulatory factor that induces follicular atresia; furthermore, inhibition of cortisol production by FSH might increase the number of healthy preovulatory follicles in pigs.

Transcriptome characterization of BPG axis and expression profiles of ovarian steroidogenesis-related genes in the Japanese sardine.

BACKGROUND: The clupeoid fishes are ecologically and commercially important fish species worldwide that exhibit a high level of population fluctuation, accompanied by alteration of reproductive traits. However, knowledge about their reproductive physiology in order to understand mechanisms underlying such population dynamics is limited. The endocrine system along with the brain-pituitary-gonadal (BPG) axis is critical for regulating reproduction. The aims of this study were to provide transcript data and genes related to the BPG axis, and to characterize the expression profiles of ovarian steroidogenesis-related genes in the Japanese sardine (Sardinops melanostictus, Clupeidae). RESULTS: RNA sequencing was performed using the sardine brain, pituitary, and gonad in both sexes. A total of 290,119 contigs were obtained and 115,173 non-redundant ORFs were annotated. The genes differentially expressed between ovary and testis were strongly associated with GO terms related to gamete production. The tissue-specific profile of the abundance of transcripts was characterized for the major regulators in the BPG axis, such as gonadotropin-releasing hormone, gonadotropin, and steroidogenic enzyme. By comparing between ovary and testis, out of eight different 17ß-hydroxysteroid dehydrogenase (Hsd17b) genes identified, higher hsd17b7 expression was found in testis, whereas higher expression of hsd17b8, hsd17b10, hsd17b12a, and hsd17b12b was found in ovary. The cDNAs encoding key endocrine factors in the ovarian steroidogenic pathway were cloned, sequenced, and quantitatively assayed. In the pituitary, follicle-stimulating hormone beta peaked during vitellogenesis, while luteinizing hormone beta peaked at the completion of vitellogenesis. In the ovary, follicle-stimulating hormone receptor and luteinizing hormone receptor were upregulated from mid- to late phase of vitellogenesis. Furthermore, three steroidogenic enzyme genes (cyp11a1, cyp17a1, and cyp19a1a) gradually increased their expression during ovarian development, accompanying a rise in serum estradiol-17ß, while 3ß-hydroxysteroid dehydrogenase and steroidogenic acute regulatory protein did not change significantly. CONCLUSIONS: This is the first report of deep RNA sequencing analysis of Japanese sardine, in which many key genes involved in the BPG axis were identified. Expression profiles of ovarian steroidogenesis-related genes provide a molecular basis of the physiological processes underlying ovarian development in the sardine. Our study will be a valuable resource for clarifying the molecular biology of clupeoid fishes.

The low-expression programming of 11ß-HSD2 mediates osteoporosis susceptibility induced by prenatal caffeine exposure in male offspring rats.

BACKGROUND AND PURPOSE: Prenatal caffeine exposure (PCE) can cause developmental toxicity of long bones in offspring, but the long-term effects and the underlying mechanism have not been fully clarified. Here, we investigated the effects of PCE peak bone mass accumulation and osteoporosis susceptibility in offspring and its intrauterine programming mechanism. EXPERIMENTAL APPROACH: Pregnant Wistar rats were administrated intragastrically with saline or caffeine (120 mg·kg-1 ·day-1 ) on gestational days 9-20. The serum and bone samples were collected from the fetal and postnatal offspring for bone mass, genes expression and corticosterone analysis. Then, rat bone marrow mesenchymal stem cells (BMSCs) were treated with corticosterone in vitro to confirm the molecular mechanism. KEY RESULTS: PCE caused fetal bone dysplasia in male and female offspring. In adulthood, PCE reduced peak bone mass and increased osteoporosis susceptibility in male offspring but not in females. Meanwhile, PCE only decreased the H3K9ac and expression levels of 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2) before and after birth in the male offspring but not in the females. Moreover, the high level of corticosterone induced by PCE down-regulated the H3K9ac and expression levels of 11ß-HSD2 through promoting glucocorticoid receptor (GR; NR3C1) into the nucleus of bone marrow mesenchymal stem cells (BMSCs) and recruiting histone deacetylase 11 (HDAC11) binding to 11ß-HSD2 promoter region, which further enhanced the effect of corticosterone on suppressing osteogenic function of BMSCs. CONCLUSION AND IMPLICATIONS: PCE caused osteoporosis susceptibility in male adult offspring, which attributed to the low-functional programming of 11ß-HSD2 induced by corticosterone via GR/HDAC11 signalling.

Lipopolysaccharide Downregulates 11ß-Hydroxysteroid Dehydrogenase 2 Expression through Inhibiting Peroxisome Proliferator-Activated Receptor-γ in Placental Trophoblasts.

It is increasingly recognized that excessive glucocorticoids induce fetal intrauterine growth restriction (IUGR). Placental 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), a glucocorticoid-catalyzing enzyme, prevents active glucocorticoids from maternal circulation into the fetus, thus protecting against IUGR. Previous studies demonstrated gestational LPS exposure caused fetal IUGR. The aim of the current study was to investigate the effects of LPS on 11ß-HSD2 in mice placentas and human placental trophoblasts. Pregnant ICR(CD-1) mice were i.p. injected with LPS (200 µg/kg) on gestational day 16. As expected, gestational LPS exposure downregulated 11ß-HSD2 in mice placentas. In vitro, LPS downregulated 11ß-HSD2 in human placental trophoblasts. Additional experiment showed that LPS, which activated NF-κB, suppressed rosiglitazone-induced activation of peroxisome proliferator-activated receptor-γ (PPARγ) in mice placentas and human placental trophoblasts. Moreover, NF-κB p65 knockdown and specific NF-κB inhibitor attenuated LPS-induced suppression of PPARγ nuclear translocation in human placental trophoblasts. In addition, NF-κB p65 knockdown attenuated LPS-induced downregulation of 11ß-HSD2 in human placental trophoblasts. Mechanically, LPS promoted physical interaction between NF-κB p65 and PPARγ in the cytoplasm and nucleus of placental trophoblasts. Finally, pretreatment with rosiglitazone, a PPARγ agonist, partially alleviated LPS-induced reduction of fetal weight and crown-rump length. Taken together, these results suggest that LPS downregulates 11ß-HSD2 through suppressing PPARγ in placental trophoblasts. Placental 11ß-HSD2 downregulation may contribute partially to LPS-induced fetal IUGR.

11ß-Hydroxysteroid dehydrogenases control access of 7ß,27-dihydroxycholesterol to retinoid-related orphan receptor γ.

Oxysterols previously were considered intermediates of bile acid and steroid hormone biosynthetic pathways. However, recent research has emphasized the roles of oxysterols in essential physiologic processes and in various diseases. Despite these discoveries, the metabolic pathways leading to the different oxysterols are still largely unknown and the biosynthetic origin of several oxysterols remains unidentified. Earlier studies demonstrated that the glucocorticoid metabolizing enzymes, 11ß-hydroxysteroid dehydrogenase (11ß-HSD) types 1 and 2, interconvert 7-ketocholesterol (7kC) and 7ß-hydroxycholesterol (7ßOHC). We examined the role of 11ß-HSDs in the enzymatic control of the intracellular availability of 7ß,27-dihydroxycholesterol (7ß27OHC), a retinoid-related orphan receptor γ (RORγ) ligand. We used microsomal preparations of cells expressing recombinant 11ß-HSD1 and 11ß-HSD2 to assess whether 7ß27OHC and 7-keto,27-hydroxycholesterol (7k27OHC) are substrates of these enzymes. Binding of 7ß27OHC and 7k27OHC to 11ß-HSDs was studied by molecular modeling. To our knowledge, the stereospecific oxoreduction of 7k27OHC to 7ß27OHC by human 11ß-HSD1 and the reverse oxidation reaction of 7ß27OHC to 7k27OHC by human 11ß-HSD2 were demonstrated for the first time. Apparent enzyme affinities of 11ß-HSDs for these novel substrates were equal to or higher than those of the glucocorticoids. This is supported by the fact that 7k27OHC and 7ß27OHC are potent inhibitors of the 11ß-HSD1-dependent oxoreduction of cortisone and the 11ß-HSD2-dependent oxidation of cortisol, respectively. Furthermore, molecular docking calculations explained stereospecific enzyme activities. Finally, using an inducible RORγ reporter system, we showed that 11ß-HSD1 and 11ß-HSD2 controlled RORγ activity. These findings revealed a novel glucocorticoid-independent prereceptor regulation mechanism by 11ß-HSDs that warrants further investigation.

Endocrine modulation of Brucella abortus-infected osteocytes function and osteoclastogenesis via modulation of RANKL/OPG.

Osteoarticular brucellosis is the most frequent complication of active disease. A large amount of cells in bone are osteocytes. Since bone remodeling process is regulated by hormones we sought to study the effect of cortisol and DHEA in Brucella abortus-infected osteocytes. Cortisol treatment inhibited the expression of IL-6, TNF-α, MMP-2 and RANKL in B. abortus-infected osteocytes. DHEA could reverse the inhibitory effect of cortisol on MMP-2 production. B. abortus infection inhibited connexin 43 (Cx43) expression in osteocytes. This expression was increased when cortisol was incorporated during the infection and DHEA treatment partially reversed the effect of cortisol. Osteocytes-infected with B. abortus induced osteoclast's differentiation. Yet, the presence of cortisol, but not DHEA, during osteocyte infection inhibited osteoclastogenesis. Glucocorticoid receptor (GR) is implicated in the signaling of cortisol. Infection with B. abortus was able to increase GRα/ß ratio. Levels of intracellular cortisol are not only dependent on GR expression but also a result of the activity of the isoenzymes 11ß-hydroxysteroid dehydrogenase (11ß-HSD)-1 (cortisone to cortisol conversion), 11ß-HSD2 (cortisol to cortisone conversion). B. abortus infection increased 11ß-HSD 1/2 ratio and cortisone mimicked the effect of cortisol. Our results indicated that cortisol and DHEA could modulate osteocyte responses during B. abortus infection.

A new tremulane sesquiterpenoid from the fungus Irpex lacteus.

A new tremulane sesquiterpenoid, named irlactam A (1), was isolated from cultures of the fungus Irpex lacteus. The new structure was elucidated by spectroscopic data analysis. The compound was tested for its cytotoxicities on HL-60, SMMC-7721, A-549, MCF-7, and SW480 cells and for its inhibitory activity against isozymes of 11 ß-hydroxysteroid dehydrogenases (11 ß-HSD).

Glucocorticoid-activation system mediated glucocorticoid-insulin-like growth factor 1 (GC-IGF1) axis programming alteration of adrenal dysfunction induced by prenatal caffeine exposure.

Glucocorticoids play a major factor in fetal maturation and fate decision after birth. We have previously demonstrated that prenatal caffeine exposure (PCE) resulted in adrenal dysplasia. However, its molecular mechanism has not been clarified. In the present study, a rat model of intrauterine growth retardation (IUGR) was established by PCE, and offspring were sacrificed. Moreover, NCI-H295 A cells were used to confirm glucocorticoid-related molecular mechanism. Results showed that PCE fetal weight decreased, and the IUGR rate increased, while serum corticosterone levels increased but insulin-like growth factor 1 (IGF1) levels decreased. Fetal adrenals exhibited an activated glucocorticoid-activation system, and the downregulated expression of IGF1 signal pathway and steroidal synthetases. For adult rats, there was no significant change in the glucocorticoid-activation system in the PCE group, the IGF1 signal pathway showed increased trend, and the expression levels of adrenal steroidal synthetases were close to normal. The data in vitro showed that the cortisol of 1200 nM can inhibit the expression of adrenocortical cell steroidal synthetases and IGF1 signal pathway when compared with the control. Meanwhile, the glucocorticoid-activation system was activated while GR inhibitor mifepristone can reverse the effect of cortisol. Furthermore, cortisol can also promote GR into the nucleus after its activation. Based on these findings, we speculated that high concentrations of glucocorticoid in utero led to GR in the nucleus through its activation and then inhibited the IGF1 signaling pathway by activating the glucocorticoid-activation system, which could further downregulate steroid synthesis.

Glucocorticoid activation by 11ß-hydroxysteroid dehydrogenase enzymes in relation to inflammation and glycaemic control in chronic kidney disease: A cross-sectional study.

OBJECTIVE: Patients with chronic kidney disease (CKD) have dysregulated cortisol metabolism secondary to changes in 11ß-hydroxysteroid dehydrogenase (11ß-HSD) enzymes. The determinants of this and its clinical implications are poorly defined. METHODS: We performed a cross-sectional study to characterize shifts in cortisol metabolism in relation to renal function, inflammation and glycaemic control. Systemic activation of cortisol by 11ß-HSD was measured as the metabolite ratio (tetrahydrocortisol [THF]+5α-tetrahydrocortisol [5αTHF])/tetrahydrocortisone (THE) in urine. RESULTS: The cohort included 342 participants with a median age of 63 years, median estimated glomerular filtration rate (eGFR) of 28 mL/min/1.73 m2 and median urine albumin-creatinine ratio of 35.5 mg/mmol. (THF+5αTHF)/THE correlated negatively with eGFR (Spearman's ρ = -0.116, P = 0.032) and positively with C-reactive protein (ρ = 0.208, P < 0.001). In multivariable analysis, C-reactive protein remained a significant independent predictor of (THF+5αTHF)/THE, but eGFR did not. Elevated (THF+5αTHF)/THE was associated with HbA1c (ρ = 0.144, P = 0.008) and diabetes mellitus (odds ratio for high vs low tertile of (THF+5αTHF)/THE 2.57, 95% confidence interval 1.47-4.47). Associations with diabetes mellitus and with HbA1c among the diabetic subgroup were independent of eGFR, C-reactive protein, age, sex and ethnicity. CONCLUSIONS: In summary, glucocorticoid activation by 11ß-HSD in our cohort comprising a spectrum of renal function was associated with inflammation and impaired glucose control.

Harnessing the gatekeepers of glucocorticoids for chemoprevention of non-melanoma skin cancer.

Despite effective surgical methods for non-melanoma skin cancer (NMSC), patients suffer from tissue damage, scarring, or even disfigurement; thus, there is a need for chemopreventive approaches. Because of the complex interplay between glucocorticoids (GCs), inflammation, and cancer, we sought to determine the role of 11ß-hydroxysteroid dehydrogenase 1 and 2 (11ßHSD1 and 2) in regulating GCs during skin cancer development and progression. 11ßHSDs modulate the activation of GCs in a tissue-specific manner and have been reported to play a role in development and progression of other types of cancer, but their role has not yet been reported in NMSC. Here, we found a significant upregulation of 11ßHSD2 protein in skin cancer cells when compared to normal skin cells, suggesting a role for this enzyme in the multifactorial process of skin cancer development. In addition, inhibition of 11ßHSD2 with siRNA resulted in significant reduction in colony formation in vitro. Finally, our in vivo study elucidated that inhibition of 11ßHSD2 with pharmacological inhibitor, Glycyrrhetinic acid (GA) could significantly diminish tumorigenesis in a well-studied in vivo mouse model of NMSC. Overall, these studies highlight for the first time a potential novel role for 11ßHSD2 in NMSC development and may allow for new GC treatment approaches capable of avoiding deactivation by the enzyme. If 11ßHSD2 can be inhibited as we have done here, or circumvented using modified GCs, this may lead to more efficacious outcomes for NMSC patients by preventing deactivation of the GC and minimizing resistance.

Pro-inflammatory Monocyte Phenotype and Cell-Specific Steroid Signaling Alterations in Unmedicated Patients With Major Depressive Disorder.

Several lines of evidence have strongly implicated inflammatory processes in the pathobiology of major depressive disorder (MDD). However, the cellular origin of inflammatory signals and their specificity remain unclear. We examined the phenotype and glucocorticoid signaling in key cell populations of the innate immune system (monocytes) vs. adaptive immunity (T cells) in a sample of 35 well-characterized, antidepressant-free patients with MDD and 35 healthy controls individually matched for age, sex, smoking status and body mass index. Monocyte and T cell phenotype was assessed by flow cytometry. Cell-specific steroid signaling was determined by mRNA expression of pre-receptor regulation (11ß-hydroxysteroid dehydrogenase type 1; 11ß -HSD1), steroid receptor expression [glucocorticoid receptor (GR) and mineralocorticoid receptor (MR)], and the downstream target glucocorticoid-induced leucine-zipper (GILZ). We also collected salivary cortisol samples (8:00 a.m. and 10:00 p.m.) on two consecutive days. Patients showed a shift toward a pro-inflammatory phenotype characterized by higher frequency and higher absolute numbers of non-classical monocytes. No group differences were observed in major T cell subset frequencies and phenotype. Correspondingly, gene expression indicative of steroid resistance (i.e., lower expression of GR and GILZ) in patients with MDD was specific to monocytes and not observed in T cells. Monocyte phenotype and steroid receptor expression was not related to cortisol levels or serum levels of IL-6, IL-1ß, or TNF-α. Our results thus suggest that in MDD, cells of the innate and adaptive immune system are differentially affected with shifts in monocyte subsets and lower expression of steroid signaling related genes.

A new cadinane sesquiterpenoid from cultures of the Basidiomycete Panus conchatus.

A new cadinane sesquiterpenoid, named panutorulon A (1), was isolated from cultures of the basidiomycete Panus conchatus. The new structure was elucidated by means of spectroscopic methods. Compound 1 was tested for its cytotoxicity against five human cancer cell lines and for its inhibitory activity against isozymes of 11ß-hydroxysteroid dehydrogenases (11ß-HSD).