11β-HSD1 Activity Research Articles

Glycyrrhizic acid and patchouli alcohol in Huoxiang Zhengqi attenuate intestinal inflammation and barrier injury via regulating endogenous corticosterone metabolism mediated by 11β-HSD1

Ethnopharmacological relevanceUlcerative colitis (UC), a chronic inflammatory bowel disease, has become a significant public health challenge due to the limited effectiveness of available therapies. Huoxiang Zhengqi (HXZQ), a well-established traditional Chinese formula, shows potential in managing UC, as suggested by clinical and pharmacological studies. However, the active components and mechanisms responsible for its effects remain unclear. Aim of studyThis study aimed to identify the bioactive components of HXZQ responsible for its therapeutic effects on UC and to elucidate their underlying mechanisms. Materials and methodsThe effect of HXZQ against dextran sodium sulfate (DSS)-induced colitis was investigated. Ingredients in HXZQ were characterized and analyzed in colitic mice using liquid chromatography–mass spectrometry (LC-MS) and gas chromatography–mass spectrometry (GC-MS). In vitro, biological activity of compounds was assessed using lipopolysaccharide (LPS)-induced Ana-1 cells and bone marrow-derived macrophages (BMDMs), tumor necrosis factor-alpha (TNF-α)-induced Caco-2 cells, and isolated intestinal crypts from colitic mice. These results were confirmed in vivo. The targets of the components were identified through bioinformatics analysis and validated via molecular docking, enzyme inhibition assays, and in vivo experiments. Hematoxylin and eosin (HE) staining, periodic acid-Schiff (PAS) staining, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative real-time polymerase chain reaction (qPCR) were employed to confirm the pharmaceutical effects. ResultsA clinical equivalent dose of HXZQ (2.5 mL/kg) effectively treated DSS-induced colitis. A total of 113 compounds were identified in HXZQ, with 35 compounds detected in colitic mice. Glycyrrhizic acid (GA) and patchouli alcohol (PA) emerged as key contributors to the anti-colitic effects of HXZQ. Further investigation revealed that HXZQ and its active components decreased the levels of pro-inflammatory cytokines TNF-α, interleukin-1β (IL-1β), and interleukin-6 (IL-6) in colon, likely by inhibiting nuclear factor kappa-B (NF-κB) signaling pathway. This inhibition indirectly activated the intestinal farnesoid X receptor (FXR) signaling pathway, correcting bile acid imbalances caused by colitis. Additionally, these components significantly enhanced the expression of tight junction proteins ZO-1 and Occludin, as well as the adhesion protein E-cadherin, and reduced goblet cell loss, thereby repairing intestinal barrier injury. Mechanistically, GA and PA were found to inhibit 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) activity, leading to increased local active corticosterone levels in the intestine to exert anti-inflammatory effects. Notably, the inhibition of 11β-HSD1 with the selective inhibitor BVT2733 (BVT) ameliorated colitis in mice. ConclusionsHXZQ exhibits therapeutic effects on UC, primarily through GA and PA inhibiting 11β-HSD1. This suggests new natural therapy approaches for UC and positions 11β-HSD1 as a potential target for colitis treatment.

Structural determinants of parabens in inhibiting human and rat gonadal 3β-hydroxysteroid dehydrogenase

This study delved into the impacts of 10 parabens on the activity of human and rat gonadal 3β-hydroxysteroid dehydrogenase (3β-HSD) within human KGN cell and rat testicular microsomes, as well as on the secretion of progesterone in KGN cells and the inhibitory potency was compared between human and rats. Intriguingly, the outcomes revealed that ethyl, propyl, butyl, hexyl, heptyl, nonyl, phenyl, and benzyl parabens displayed varying IC50 values for human 3β-HSD2, from 4.15 to 139.96 μM, demonstrating characteristics of mixed inhibitors. Notably, within KGN cells, all examined parabens, excluding nonyl and phenyl parabens, significantly inhibited progesterone secretion at 5–50 μM. In the case of rats, the IC50 values for these parabens on gonadal 3β-HSD1 fluctuated between 7.15 and 110.76 μM, likewise functioning as mixed inhibitors. Through docking analysis, it was proposed that most parabens effectively bind to NAD+ and/or steroid binding site. Moreover, bivariate correlation analysis unveiled an inverse correlation between IC50 values and structural characteristics such as LogP, molecular weight, heavy atom number, and carbon number within the alcohol moiety of parabens. 3D-QSAR elucidated pivotal regions, comprising hydrogen bond donor, hydrogen bond acceptor, and hydrophobic region, with the most potent inhibitor nonyl paraben engaging with all regions, while the weakest inhibitor ethyl paraben interacted with the regions except for the hydrophobic region. In conclusion, this investigation underscored the inhibitory effects imparted by several parabens on both human and rat gonadal 3β-HSD activity, with their inhibitory potency being modulated by aspects of hydrophobicity and carbon chain length.

Impaired 11β-hydroxysteroid dehydrogenase type 2 activity in kidney disease disrupts 11-oxygenated androgen biosynthesis.

11-oxygenated androgens are a group of adrenal-derived steroids that require peripheral activation. In vitro data highlight a putative role for 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) in 11-oxygenated androgen biosynthesis, converting 11β-hydroxyandrostenedione (11OHA4) to 11-ketoandrostenedione (11KA4), the direct precursor of the potent androgen 11-ketotestosterone (11KT). As the kidney is the major site of HSD11B2 expression, we hypothesized that patients with chronic kidney disease (CKD) would have reduced 11-oxygenated androgen biosynthesis due to impaired HSD11B2 activity. To determine the role of HSD11B2 in 11-oxygenated androgen biosynthesis using a human CKD cohort alongside complementary cell culture and computational modeling approaches. Cross-sectional observational study of patients with CKD (n=85) and healthy controls (n=46) measuring serum and urinary concentrations of glucocorticoids, classic and 11-oxygenated androgens by liquid chromatography-tandem mass spectrometry. A computational model of peripheral 11-oxygenated androgen biosynthesis was fitted to the serum data to calculate relative HSD11B2 expression levels for each participant. HSD11B2 activity declined with eGFR, evidenced by higher cortisol (F)/cortisone (E) ratios in CKD patients compared to controls (p<0.0001). Serum concentrations of E, 11KA4, 11KT and 11β-hydroxytestosterone were lower in patients with CKD compared to controls (p<0.0001 for each). A computational model based on enzyme kinetic parameters of HSD11B2, 11β-hydroxysteroid dehydrogenase type 1, 17β-hydroxysteroid dehydrogenase type 2 and aldo-keto reductase 1C3 confirmed HSD11B2 as the key enzyme responsible for reduced 11-oxygenated androgen biosynthesis in CKD. Predicted HSD11B2 expression correlated with eGFR. This is the first in vivo study to confirm a central role for renal HSD11B2 in 11-oxygenated androgen biosynthesis. Determining the clinical implications of this observation for patients with CKD requires further research.

7990 Reduced Alpha-1 Antitrypsin Activity Is Associated with Higher Plasma Free Cortisol and Increased Tissue Glucocorticoid Exposure in Humans in vivo

Abstract Disclosure: L.D. Boyle: None. M. Nixon: None. C.M. Underhill: None. L.A. Hill: None. N.Z. Homer: None. M. Magennis: None. R. Andrew: None. G.L. Hammond: None. J.G. Lewis: None. R.H. Stimson: None. B.R. Walker: None. Background: Corticosteroid Binding Globulin (CBG) binds &amp;gt;85% of plasma cortisol, modulating its biological activity. Proteolytic cleavage by neutrophil elastase (NE) reduces CBG binding capacity, proposed to increase free cortisol availability to inflamed tissues. Genetic variation at the locus spanning SERPINA1 (encoding alpha-1 antitrypsin, AAT, the endogenous inhibitor of NE) and SERPINA6 (CBG) altered morning total plasma cortisol. We hypothesised that AAT deficiency increased CBG cleavage and hence free plasma cortisol, resulting in amplified tissue cortisol delivery and increased HPA axis negative feedback. We tested this in recall-by-genotype studies of people who are heterozygous for inactivating mutations in SERPINA1. Methods: 16 asymptomatic carriers of deleterious SERPINA1 single nucleotide polymorphisms (rs17580 &amp; rs28929474) and 16 age-, gender- and BMI-matched controls were recruited from the Generation Scotland Biobank. To quantify in vivo whole body glucocorticoid appearance and clearance rates, and estimate tissue cortisol uptake, we performed arterio-venous plasma sampling across abdominal adipose and skeletal muscle during steady-state 9,11,12,12-[2H]4-cortisol tracer infusion. To assess endogenous negative feedback, participants underwent combined receptor antagonist stimulation of the HPA axis (‘CRASH’) testing using RU486 and spironolactone, or placebo in a double blind randomised crossover design. Total cortisol (LC-MS/MS), free cortisol (isotopic dilution &amp; ultrafiltration), CBG binding capacity (radioligand displacement), CBG concentration and AAT (ELISA) were measured in plasma. Tissue cortisol (LC-MS/MS) and glucocorticoid-dependent transcripts (qPCR) were measured in adipose biopsy samples collected at end of study. Results: AAT was ∼30% lower in AAT+/- (p=0.0002 vs control). Plasma CBG concentration, binding capacity and total cortisol were similar between groups. However, plasma free cortisol fraction was higher in AAT+/- subjects (16.1 +/- 0.2 vs 13.9 +/- 0.04 %, p&amp;lt;0.0001). Adipose tissue from AAT+/- subjects displayed increased cortisol levels and increased glucocorticoid-responsive transcripts PER1 and LPL compared to controls. Consistent with release from CBG, total and free cortisol release across skeletal muscle was increased in AAT+/- subjects vs controls. The rate of appearance of 9,12,12-[2H]3-cortisol was reduced in AAT+/- (p=0.03 vs control), consistent with decreased whole body 11β-HSD1 activity. CRASH testing did not reveal additional differences between groups. Discussion: Subclinical AAT deficiency is associated with a higher free cortisol fraction and increased tissue glucocorticoid exposure. Although consistent with the hypothesis of local tissue-mediated control of glucocorticoid exposure via the NE/AAT/CBG axis, these findings were not accompanied by measurable changes in CBG. Presentation: 6/3/2024

9225 Reduced Alpha-1 Antitrypsin Activity Is Associated with Higher Plasma Free Cortisol and Increased Tissue Glucocorticoid Exposure in Humans in vivo

Abstract Disclosure: L.D. Boyle: None. M. Nixon: None. C.M. Underhill: None. L.A. Hill: None. N.Z. Homer: None. M. Magennis: None. R. Andrew: None. G.L. Hammond: None. J.G. Lewis: None. R.H. Stimson: None. B.R. Walker: None. Background Corticosteroid Binding Globulin (CBG) binds &amp;gt;85% of plasma cortisol, modulating its biological activity. Proteolytic cleavage by neutrophil elastase (NE) reduces CBG binding capacity, proposed to increase free cortisol availability to inflamed tissues. Genetic variation at the locus spanning SERPINA1 (encoding alpha-1 antitrypsin, AAT, the endogenous inhibitor of NE) and SERPINA6 (CBG) altered morning total plasma cortisol. We hypothesised that AAT deficiency increased CBG cleavage and hence free plasma cortisol, resulting in amplified tissue cortisol delivery and increased HPA axis negative feedback. We tested this in recall-by-genotype studies of people who are heterozygous for inactivating mutations in SERPINA1. Methods 16 asymptomatic carriers of deleterious SERPINA1 single nucleotide polymorphisms (rs17580 &amp; rs28929474) and 16 age-, gender- and BMI-matched controls were recruited from the Generation Scotland Biobank. To quantify in vivo whole body glucocorticoid appearance and clearance rates, and estimate tissue cortisol uptake, we performed arterio-venous plasma sampling across abdominal adipose and skeletal muscle during steady-state 9,11,12,12-[2H]4-cortisol tracer infusion. To assess endogenous negative feedback, participants underwent combined receptor antagonist stimulation of the HPA axis (‘CRASH’) testing using RU486 and spironolactone, or placebo in a double blind randomised crossover design. Total cortisol (LC-MS/MS), free cortisol (isotopic dilution &amp; ultrafiltration), CBG binding capacity (radioligand displacement), CBG concentration and AAT (ELISA) were measured in plasma. Tissue cortisol (LC-MS/MS) and glucocorticoid-dependent transcripts (qPCR) were measured in adipose biopsy samples collected at end of study. Results AAT was ∼30% lower in AAT+/- (p=0.0002 vs control). Plasma CBG concentration, binding capacity and total cortisol were similar between groups. However, plasma free cortisol fraction was higher in AAT+/- subjects (16.1 +/- 0.2 vs 13.9 +/- 0.04 %, p&amp;lt;0.0001). Adipose tissue from AAT+/- subjects displayed increased cortisol levels and increased glucocorticoid-responsive transcripts PER1 and LPL compared to controls. Consistent with release from CBG, total and free cortisol release across skeletal muscle was increased in AAT+/- subjects vs controls. The rate of appearance of 9,12,12-[2H]3-cortisol was reduced in AAT+/- (p=0.03 vs control), consistent with decreased whole body 11β-HSD1 activity. CRASH testing did not reveal additional differences between groups. Discussion Subclinical AAT deficiency is associated with a higher free cortisol fraction and increased tissue glucocorticoid exposure. Although consistent with the hypothesis of local tissue-mediated control of glucocorticoid exposure via the NE/AAT/CBG axis, these findings were not accompanied by measurable changes in CBG. Presentation: 6/3/2024

Progressive 11β-Hydroxysteroid Dehydrogenase Type 2 Insufficiency as Kidney Function Declines.

It has been postulated that chronic kidney disease (CKD) is a state of relative 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) insufficiency, resulting in increased cortisol-mediated mineralocorticoid receptor (MR) activation. We hypothesized that relative 11βHSD2 insufficiency manifests across a wide spectrum of progressively declining kidney function, including within the normal range. Adult participants were recruited at two academic centers. A discovery cohort (n=500) enrolled individuals with estimated glomerular filtration rate (eGFR) ranging from normal to CKD stage 5, in whom serum cortisol-to-cortisone (F/E) was measured as a biomarker of 11βHSD2 activity. A validation cohort (n=101) enrolled only individuals with normal kidney function (eGFR ≥ 60 mL/min/1.73 m2) in whom 11βHSD2 activity was assessed via serum F/E and 11-hydroxy-to-11-keto androgen (11OH/K) ratios following multiple maneuvers: oral sodium suppression test (OSST), dexamethasone suppression test (DST), and ACTH-stimulation test (ACTHstim). In the discovery cohort, lower eGFR was associated with higher F/E (P-trend<0.001). Similarly, in the validation cohort, with normal eGFR, an inverse association between eGFR and both F/E and 11OH/K ratios was observed (P-trend<0.01), which persisted following DST (P-trend<0.001) and ACTHstim (P-trend< 0.05). The fractional excretion of potassium, a marker of renal MR activity, was higher with higher F/E (P-trend < 0.01) and with lower eGFR (P-trend<0.0001). A continuum of declining 11βHSD2 activity was observed with progressively lower eGFR in individuals spanning a wide spectrum of kidney function, including those with apparently normal kidney function. These findings implicate cortisol-mediated MR activation in the pathophysiology of hypertension and cardiovascular disease in CKD.

Impact of arsenic on male and female reproductive function: a review of the pathophysiology and potential therapeutic strategies.

Arsenic is a ubiquitous metalloid and heavy metal that contributes to the global decline in human fertility. Humans are constantly exposed to arsenic through biotic and abiotic sources, especially ingestion of arsenic-contaminated food and water. Its exposure is associated with several adverse health challenges, including reproductive toxicity. In spite of its reported adverse effects, arsenic exposure remains a global challenge. Hence, this study provides a comprehensive review of the literature on the impact and mechanism of arsenic on male and female reproductive function. Additionally, a review of the potential therapeutic strategies is presented. Evidence from the literature reveals that arsenic upregulates reactive oxygen species (ROS) generation which mediates arsenic-induced suppression of the hypothalamic-pituitary-gonadal axis and inactivation of 3β-HSD and 17β-HSD activities, leading to reduced gonadal steroidogenesis. Through several oxidative stress-dependent signaling, arsenic induces the apoptosis of the germ cells, thus contributing to the development of infertility. At the moment, there is no specific treatment for arsenic-induced reproductive toxicity. However, increasing data form the scientific literature reveals the benefits of antioxidants in ameliorating arsenic-induced reproductive toxicity. These molecules suppress ROS generation and maintain optimal activities of the hypothalamic-pituitary-gonadal axis, leading to optimal steroidogenesis and gametogenesis as well as improved germ cells. Overall, this study revealed the impact and associated mechanism of arsenic-induced reproductive toxicity. It also provides evidence from the literature demonstrating potential therapeutic measures in managing arsenic-induced reproductive toxicity.

Fast and reliable quantification of aldosterone, cortisol and cortisone via LC-MS/MS to study 11β-hydroxysteroid dehydrogenase activities in primary cell cultures

Cell culture experiments can support characterization of enzymatic activities in healthy and tumorous human tissues. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enables simultaneous measurement of several steroids from a single sample, facilitating analysis of molecular pathways involved in steroid biosynthesis. We developed a reliable but fast method for quantification of cortisol, cortisone and aldosterone in cell culture supernatant. Validation, including investigation of matrix-matched calibration, was performed for two different cell types. Utility of the method was demonstrated in the study of 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) activity under conditions of glucocorticoid and mineralocorticoid excess in different cell types. Aldosterone, cortisol and cortisone were extracted by liquid-liquid extraction (LLE) with methyl tert-butyl ether from 1 mL of cell culture supernatant. Steroids were separated on a Kinetex biphenyl column (50 ×2.1 mm, 2.6 µm) with gradient elution of water and methanol containing 2 mM ammonium format and analysed in multiple reaction monitoring mode after positive electrospray ionization. Application of the method included cell culture experiments with two different primary cell types, human coronary artery smooth muscle cells (HCSMC) and human coronary artery endothelial cells (EC). Cells were treated with different concentrations of cortisol, aldosterone and mifepristone, a glucocorticoid receptor antagonist and quantitative PCR was performed. The method exhibits high precision (CV ≤ 6 %) and accuracy (deviation from nominal concentration ≤ 6 %) for concentrations above the limit of quantification (LoQ) which is 0.11, 0.56 and 0.69 nmol/L for aldosterone, cortisone and cortisol, respectively. Calibration curves did not differ when prepared in media or solvent. The method enabled us to confirm activity of HSD11B2 and concentration dependent conversion of cortisol to cortisone in HCSMC (median conversion ratio at 140 nM cortisol = 1.46 %). In contrast we did not observe any HSD11B2 activity in EC. Neither addition of high aldosterone, nor addition of 1 µM mifepristone had impact on glucocorticoid concentrations. Quantitative PCR revealed expression of HSD11B1 and HSD11B2 in HCSMC but not in EC. We present a fast and reliable method for quantification of cortisol, cortisone and aldosterone in cell culture supernatants. The method enabled us to study HSD11B2 activity in two different cell types and will support future experiments investigating mechanisms of target organ damage in conditions of glucocorticoid and mineralocorticoid excess.

Involvement of porcine and human carbonyl reductases in the metabolism of epiandrosterone, 11-oxygenated steroids, neurosteroids, and corticosteroids

Porcine carbonyl reductases (pCBR1 and pCBR-N1) and aldo-keto reductases (pAKR1C1 and pAKR1C4) exhibit hydroxysteroid dehydrogenase (HSD) activity. However, their roles in the metabolism of porcine-specific androgens (19-nortestosterone and epiandrosterone), 11-oxygenated androgens, neurosteroids, and corticosteroids remain unclear. Here, we compared the steroid specificity of the four recombinant enzymes by kinetic and product analyses. In C18/C19-steroids,11-keto- and 11β-hydroxy-5α-androstane-3,17-diones were reduced by all the enzymes, whereas 5α-dihydronandrolone (19-nortestosterone metabolite) and 11-ketodihydrotestosterone were reduced by pCBR1, pCBR-N1, and pAKR1C1, of which pCBR1 exhibited the lowest (submicromolar) Km values. Product analysis showed that pCBR1 and pCBR-N1 function as 3α/β-HSDs, in contrast to pAKR1C1 and pAKR1C4 (acting as 3β-HSD and 3α-HSD, respectively). Additionally, 17β-HSD activity was observed in pCBR1 and pCBR-N1 (toward epiandrosterone and its 11-oxygenated derivatives) and in pAKR1C1 (toward androsterone, 4-androstene-3,17-dione and their 11-oxygenated derivatives). The four enzymes also showed different substrate specificity for 3-keto-5α/β-dihydro-C21-steroids, including GABAergic neurosteroid precursors and corticosteroid metabolites. 5β-Dihydroprogesterone was reduced by all the enzymes, whereas 5α-dihydroprogesterone was reduced only by pCBR1, and 5α/β-dihydrodeoxycorticosterones by pCBR1 and pCBR-N1. The two pCBRs also reduced the 5α/β-dihydro-metabolites of cortisol, 11-deoxycortisol, cortisone, and corticosterone. pCBR1 exhibited lower Km values (0.3–2.9 μM) for the 3-keto-C21-steroids than pCBR-N1 (Km=10–36 μM). The reduced products of the 3-keto-C21-steroids by pCBR1 and pCBR-N1 were their 3α-hydroxy-metabolites. Finally, we found that human CBR1 has similar substrate specificity for the C18/C19/C21-steroids to pCBR-N1. Based on these results, it was concluded that porcine and human CBRs can be involved in the metabolism of the aforementioned steroids as 3α/β,17β-HSDs.

Methanol extract of Abrus precatorius seeds on reproduction indices, hepatorenal profiles in female albino rats

BackgroundAbrus precatorious is a member of the leguminous family with characteristic red and black seeds. The roots, leaves and seeds of this plant are used for different medicinal purpose. It principally carries flavonoids, triterpene glycosides, abrin and alkaloids. The plant had been mentioned for neuromuscular effects, neuro-protective, abortifacient, antiepileptic, anti-viral, antimalarial, antifertility, nephroprotective, immunomodulator, immunostimulatory properties, antiinflammatory activity, antidiabetic, and many others. The aim of this study was to investigate the anti-fertility activity of the methanol extract of Abrus precatorius seeds (APS) in female albino rats administered 45 and 50 mg/kg BW through oral gavage for 30 days. An Abrus precatorius seed (APS) was extracted (1:1 w/v) using methanol to obtain the crude extract. Liquid-liquid fractionation was performed on the crude methanol extract using solvents of different polarity.MethodsEighteen female rats were divided into three groups each containing six animals was used for this experiment. Group one (control), received 1 ml of saline solution, group two received 45 mg/kg and group three received 50 mg/kg body weight by oral gavage daily for a period of 30 days. At the end of the study, Body weight, Organ Weight, Estrous cycle, Hormonal Concentrations, Oxidative Stress Markers, Enzymatic Activities, Biochemical indices (Liver and Kidney panel) and Hematological parameters were also evaluated.ResultsConsequent upon the administration of A.precatorius seed methanol extract 45 and 50 mg/kg BW into the female rats resulted in changes pertaining to Body weight, Estrous cycle, Hormonal Concentrations, Antioxidant Enzymes, Serum Biochemical indices and Hematological indices significantly (p < 0.05) compared to control rats. Initial body weights are significantly (p < 0.05) indicated, whereas final weights were decreased significantly (p < 0.05). Organ weight changes i.e. Ovary, Liver and Kidney weights were found to be significantly (p < 0.05) decreased in APS administered rats compared to control. Estrous cycle study i.e. time duration of Proestrus, Estrus, Metestrus and Diestrus (in days) was significantly prolonged. Hormonal Concentrations represented by FSH, LH, Prolactin, Estradiol were significantly (p < 0.05) decreased, whereas Progesterone levels were significantly (p < 0.05) increased in APS administered female rats compared to control group of rats. All the antioxidant enzyme assayed CAT, SOD, GPx, GR and GST were found to be significantly (p < 0.05) decreased, whereas MDA concentration was found to be increased significantly (p < 0.05). Enzymatic activities and biomolecules includes Cholesterol and Ascorbic acid contents were significantly increased (p < 0.05), but G6PDH and Δ5 − 3β-HSD activities were found to be significantly (p < 0.05) decreased in APS administered group of female rats compared to control group. Biochemical indices of liver tissue presented by Bilirubin, SGPT, SGOT, Albumin and Globulin were found to be significantly (p < 0.05) increased whereas Alkaline Phosphate and Total Protein contents were found to be significantly (p < 0.05) decreased. The biochemical indices of kidney, i.e. Urea and Creatinine were significantly (p < 0.05) increased and Uric acid and Calcium contents were found to be significantly (p < 0.05) decreased in APS administered female rats compared to control group of rats. Hematological parameters including RBC, Hb, ESR and Clotting Time were found to be significantly (p < 0.05) increased but WBC levels were significantly (p < 0.05) increased in APS administered female rats compared to control group of rats.ConclusionA. Precatorius seeds have anti-fertility effect on female rats.

Inflammation dynamically regulates steroid hormone metabolism and action within macrophages in rheumatoid arthritis

RationaleIn inflammatory diseases such as rheumatoid arthritis (RA), steroid metabolism is a central component mediating the actions of immuno-modulatory glucocorticoids and sex steroids. However, the regulation and function of cellular steroid metabolism within key leukocyte populations such as macrophages remain poorly defined. In this study, the inflammatory regulation of global steroid metabolism was assessed in RA macrophages. MethodsBulk RNA-seq data from RA synovial macrophages was used to assess transcripts encoding key enzymes in steroid metabolism and signalling. Changes in metabolism were assessed in synovial fluids, correlated to measures of disease activity and functionally validated in primary macrophage cultures. ResultsRNA-seq revealed a unique pattern of differentially expressed genes, including changes in genes encoding the enzymes 11β-HSD1, SRD5A1, AKR1C2 and AKR1C3. These correlated with disease activity, favouring increased glucocorticoid and androgen levels. Synovial fluid 11β-HSD1 activity correlated with local inflammatory mediators (TNFα, IL-6, IL-17), whilst 11β-HSD1, SRD5A1 and AKR1C3 activity correlated with systemic measures of disease and patient pain (ESR, DAS28 ESR, global disease activity). Changes in enzyme activity were evident in inflammatory activated macrophages in vitro and revealed a novel androgen activating role for 11β-HSD1. Together, increased glucocorticoids and androgens were able to suppress inflammation in macrophages and fibroblast-like-synoviocytes. ConclusionsThis study underscores the significant increase in androgen and glucocorticoid activation within inflammatory polarized macrophages of the synovium, contributing to local suppression of inflammation. The diminished profile of inactive steroid precursors in postmenopausal women may contribute to disturbances in this process, leading to increased disease incidence and severity.

Current Challenges and Future Directions in the Assessment of Glucocorticoid Status.

Glucocorticoid hormones (GC) are secreted in a circadian and ultradian rhythm and play a critical role in maintaining physiological homeostasis, with both excess and insufficient GC associated with adverse effects on health. Current assessment of GC status is primarily clinical, often in conjunction with serum cortisol values, which may be stimulated or suppressed depending on the GC disturbance being assessed. In the setting of extreme perturbations in cortisol levels i.e. markedly low or high levels, symptoms and signs of GC dysfunction may be overt. However, when disturbances in cortisol GC status values are less extreme, such as when assessing optimization of a GC replacement regimen, signs and symptoms can be more subtle or non-specific. Current tools for assessing GC status, are best suited to identifying profound disturbances but may lack sensitivity for confirming optimal GC status. Moreover, single cortisol values do not necessarily reflect an individual's GC status, as they are subject to inter- and intra-individual variation, do not take into account the pulsatile nature of cortisol secretion, variation in binding proteins, or local tissue concentrations as dictated by 11βeta-hydroxysteroid dehydrogenase (11β-HSD) activity, as well as GC receptor sensitivity. In the present review, we evaluate possible alternative methods for the assessment of GC status that do not solely rely on measurement of circulating cortisol levels. We discuss the potential of changes in metabolomic profiles, miRNA, gene expression, epigenetic, and other novel biomarkers such as GDF-15 and osteocalcin, that could in future aid in the objective classification of GC status.

The metabolic activation of pentachlorophenol to chloranil as a potent inhibitor of human and rat placental 3β-hydroxysteroid dehydrogenases

Pentachlorophenol (PCP) is a widely used pesticide. However, whether PCP and its metabolite chloranil have endocrine-disrupting effects by inhibiting placental 3β-hydroxysteroid dehydrogenase 1 (3β-HSD1) remains unclear. The study used in vitro assays with human and rat placental microsomes to measure 3β-HSD activity as well as human JAr cells to evaluate progesterone production. The results showed that PCP exhibited moderate inhibition of human 3β-HSD1, with an IC50 value of 29.83 μM and displayed mixed inhibition in terms of mode of action. Conversely, chloranil proved to be a potent inhibitor, demonstrating an IC50 value of 147 nM, and displaying a mixed mode of action. PCP significantly decreased progesterone production by JAr cells at 50 μM, while chloranil markedly reduced progesterone production at ≥1 μM. Interestingly, PCP and chloranil moderately inhibited rat placental homolog 3β-HSD4, with IC50 values of 27.94 and 23.42 μM, respectively. Dithiothreitol (DTT) alone significantly increased human 3β-HSD1 activity. Chloranil not PCP mediated inhibition of human 3β-HSD1 activity was completely reversed by DTT and that of rat 3β-HSD4 was partially reversed by DTT. Docking analysis revealed that both PCP and chloranil can bind to the catalytic domain of 3β-HSDs. The difference in the amino acid residue Cys83 in human 3β-HSD1 may explain why chloranil is a potent inhibitor through its interaction with the cysteine residue of human 3β-HSD1. In conclusion, PCP is metabolically activated to chloranil as a potent inhibitor of human 3β-HSD1.

Abstract 3590: Tumor intrinsic production of glucocorticoids in ovarian carcinoma

Abstract Background: Cortisol is a glucocorticoid (GC) known to be produced by the hypothalamic-pituitary-adrenal (HPA) axis. Dysregulation of the HPA axis is prevalent in patients with ovarian cancer. These patients have elevated nighttime levels of cortisol and increased tumoral expression of the glucocorticoid receptor (GR); both of which correlate with shortened overall survival (OS). We hypothesize that outside the HPA axis, ovarian cancer may have tumor-intrinsic production of GC. In this study, we examined whether 11 beta-hydroxysteroid dehydrogenase 1 (11β-HSD1; enzyme which activates cortisone to cortisol) has steroidogenic activity in the ovarian cancer tumor microenvironment (TME). Methods: We used the Genotype-Tissue Expression (GTex) Portal and the Cancer Cell Line Encyclopedia (CCLE) to determine expression of steroidogenic enzymes in the ovary and ovarian cancer cell lines. We further performed UMAP analysis of a single-cell RNA-seq dataset of tumor samples from patients with ovarian carcinoma to cluster for cell type. ELISAs were performed to quantify cortisol production and secretion by cells stimulated with metabolite, cortisone. Subcellular fractionation and Western blotting were performed to measure protein expression of 11β-HSD1, GR, and phospho-Ser 211 GR. Results: HSD11B1 mRNA (which encodes 11β-HSD1) is detectable in bulk ovarian tissue at a median expression of 78.19 transcripts per million (TPM) as measured in the GTex data. In vitro, primary human ovarian fibroblasts express functional 11β-HSD1 and produce and secrete cortisol upon cortisone stimulation in a time-dependent manner, with a mean total secretion of 17.91 ± 0.21 ng/ml at 12 hours. Analysis of scRNA-seq data identifies that cancer-associated fibroblasts (CAFs) may be a source of HSD11B1 in the ovarian TME, with roughly 9% of CAFs expressing HSD11B1. In HeyA8 ovarian cancer cells (positive for 11β-HSD1), cortisone stimulation in vitro results in cortisol production with a mean total secretion of 22.04 ± 5.46 ng/ml at 12 hours. Moreover, this cortisol results in GR activation in an 11β-HSD1-dependent manner. Discussion: We identify 11β-HSD1 as a potential mechanism of GC production within ovarian cancer, with CAFs being a putative source. We further show that 11β-HSD1 activity can lead to activation of the GR within cancer cells. Citation Format: Pahul Hanjra, Elaine Stur, Steven Cole, Susan Lutgendorf, Anil Sood. Tumor intrinsic production of glucocorticoids in ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3590.

Spermatogenic potential of Chlorophytum borivilianum Santapau & R.R.Fern. in rats

Traditional applications of the roots of Chlorophytum borivilianum Santapau & R.R.Fern. include spermatogenic potential. Using rats as test subjects, the potential of alcoholic extract (AE-SM) and n-Butanol fraction (NB-SM), which is rich in saponin, to alter the levels of testicular testosterone and cholesterol as well as mRNA expression of the 3β-Hydroxy-∆5- steroid dehydrogenase (3β-HSD) and Acute Steroid Regulatory Protein (StAR) were determined. Histological analyses of testicular tissues were performed, and the quantity of sperm in the seminal fluid was measured. The testosterone concentration increased in AE-SM and NB-SM in a dose-dependent and statistically significant manner. In-vivo mRNA expression of the StAR and 3β-HSD enzymes was also enhanced by the test samples. When compared to control animals, AE-SM and NB-SM increased testicular testosterone by 11.46 and 12.98 fold, respectively, while the extract and fractions revealed a 5.72 and 7.70 fold alteration in StAR mRNA expression. In testicular tissues, AE-SM and NB-SM increased 3β-HSD activity by 4.98 and 9.05 fold, respectively, while changing the expression of the 3β-HSD mRNA by 6.46 and 15.54 factor, respectively. The fluid sampled from the caudal region of the AE-SM and NB-SM indicated an increased sperm concentration, and histological examinations revealed dense Sertoli cell cytoplasm and an increase in the number of sperms in the luminal part of the seminiferous tubules. The components of AE-SM and NB-SM might stimulated testosterone biosynthesis in-vivo by modifying mRNA expression of selected steroidogenic enzymes. By modifying the mRNA expression of the StAR and 3β-HSD, AE-SM and NB-SM can raise the level of testosterone in testicular tissues. As evidenced by an elevated sperm concentration discovered within the seminiferous fluid and histological studies, overexpression of steroidogenic enzymes increased testicular testosterone levels and may have stimulated the spermatogenesis process.Saponin enriched n-butanol fraction might have interacted with CREB and additionally stimulated LH receptors to elevate the StAR and 3β-HSD mRNA expression.

Features of bone remodeling and osteoreparation processes in modeling femoral fracture in genetically modified mice with impaired enzymatic regulation of steroid hormone metabolism

Introduction: The aim of this study was to evaluate the processes of bone remodeling and osteoreparation in modeling femoral fracture in mice with zero expression of 11β-HSD2 (11β-HSD2-/-) or both 11β-HSD2 and apolipoprotein e (11β-HSD2-/-/ApoE-/-).&#x0D; Materials and Methods: The experimental study was conducted on 60 male mice weighing 24-30 g. The study used male mice that lacked the expression of 11β-HSD2 (knockout mice with the Hsd2-/- genotype) and male mice that lacked the expression of 11β-HSD2 and apolipoprotein E (double knockout mice with the Hsd2-/-/ApoE-/- genotype). The control group includes wild type C57bl/6 animals. Modeling of a fracture of the proximal metaphysis of the femur was performed in animals at the age of 6 months using a closed technique. Fracture fusion and bone remodeling and osteoreparation processes were evaluated 6 weeks after fracture modeling.&#x0D; Results and Discussion: It has been shown that a violation of the regulation of steroid hormone metabolism in groups of animals with the Hsd2-/- and Hsd2-/-/ApoE-/- genotypes leads to an increase in the number of ungrown fractures by 3 and 3.5 times, respectively, in comparison with wild-type animals. It was found that the microcirculation level of the proximal metaphysis of the left femur in the area of the formed bone callus in the group of animals with the genotype 11β-HSD2-/- significantly decreased from 92.075±4.33 perfusion units (PE) in the group of wild-type animals to 82.67±3.54 PE (p=0.0002) in the group of animals with the genotype HSD2-/- and up to 75.85±5.64 (p&lt;0.0001) in the group of mice with the Hsd2-/-/ApoE-/- genotype. When calculating the coefficient of endothelial dysfunction, an increase in the coefficient of endothelial dysfunction was found from 1.297±0.19 in intact animals to 2.115±0.45 (p&lt;0.00001) in the group of mice with the Hsd2-/- genotype and to 2.41±0.04 (p&lt;0.00001) in the group of mice with the Hsd2-/-/ApoE-/- genotype. In animals with impaired cortisol metabolism, there was a slowdown in the formation of bone tissue in the fracture area, bone trabeculae had a smaller width, large amounts of fibrous and connective tissue were observed in the lumen between bone fragments, and an increase in intertrabecular spaces was noted&#x0D; Conclusion: The close relationship between the metabolism of 11β-HSD2 and NO, confirmed in this study, can be considered as a promising pharmacotherapeutic target. It is obvious that approaches to changing the activity of 11β-HSD have significant therapeutic potential in the treatment of osteoporosis, bone remodeling disorders and osteoreparation in fractures against the background of formed osteoporotic changes in the violation of steroid hormone metabolism.

Visceral fat sympathectomy ameliorates systemic and local stress response related to chronic sleep restriction.

Disturbance of sleep homeostasis encompasses health issues, including metabolic disorders like obesity, diabetes, and augmented stress vulnerability. Sleep and stress interact bidirectionally to influence the central nervous system and metabolism. Murine models demonstrate that decreased sleep time is associated with an increased systemic stress response, characterized by endocrinal imbalance, including the elevated activity of hypothalamic-pituitary-adrenal axis, augmented insulin, and reduced adiponectin, affecting peripheral organs physiology, mainly the white adipose tissue (WAT). Within peripheral organs, a local stress response can also be activated by promoting the formation of corticosterone. This local amplifying glucocorticoid signaling is favored through the activation of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). In WAT, 11β-HSD1 activity is upregulated by the sympathetic nervous system, suggesting a link between sleep loss, augmented stress response, and a potential WAT metabolic disturbance. To gain more understanding about this relationship, metabolic and stress responses of WAT-sympathectomized rats were analyzed to identify the contribution of the autonomic nervous system to stress response-related metabolic disorders during chronic sleep restriction. Male Wistar rats under sleep restriction were allowed just 6 h of daily sleep over eight weeks. Results showed that rats under sleep restriction presented higher serum corticosterone, increased adipose tissue 11β-HSD1 activity, weight loss, decreased visceral fat, augmented adiponectin, lower leptin levels, glucose tolerance impairment, and mildly decreased daily body temperature. In contrast, sympathectomized rats under sleep restriction exhibited decreased stress response (lower serum corticosterone and 11β-HSD1 activity). In addition, they maintained weight loss, explained by a reduced visceral fat pad, leptin, and adiponectin, improved glucose management, and persisting decline in body temperature. These results suggest autonomic nervous system is partially responsible for the WAT-exacerbated stress response and its metabolic and physiological disturbances.

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

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. 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 β-hydroxysteroid dehydrogenase 2 (11β-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 11β-HSD2 levels on the placental recasting, human first-trimester extravillous trophoblast cells (HTR8/SVneo) underwent knockdown using specific 11β-HSD2 siRNA constructs. Expression levels of 11β-HSD2 were analyzed by quantitative real-time PCR (qPCR) and into HTR8 cells, and the expression levels of the 11β-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. 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 11β-HSD2, glucocorticoid receptor, VEGF-A, PIGF, and sFlt-1 proteins and α-actin compared to the control group. Silencing 11β-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). Pre-conception cold exposure and during early pregnancy leads to increased GCs levels and impaired placental 11β-HSD2 activity. We suggest that the subsequent 11β-HSD2-induced increase in the sFlt-1expression during early pregnancy may affect placental vascular remodeling and change placental morphological structure and function.

Erythritol attenuates testicular dysfunction in diabetic rat via suppression of oxidative stress, inflammation and apoptosis

Hyperglycemia –induced oxidative stress and inflammation have been closely associated with diabetes complications including testicular dysfunction. Conversely, reducing blood glucose and/or use of antioxidant have been associated with reduced diabetes complications. The present study investigated the effect of erythritol (which has both antioxidant and blood glucose lowering function) on diabetes –induced testicular dysfunction in rats. Thirty male Wistar rats (170–200g) were randomly divided into 5 groups: 1) control; 2) erythritol; 3) diabetic; 4) diabetic + erythritol 1000 mg/kg; and 5) diabetic + metformin 300 mg/kg. After 8 weeks of treatment period, blood sample, testes and epididymis were collected for reproductive hormones, biochemical and histological examinations, and sperm analysis respectively. There was a significant (p < 0.05) decrease in sperm count, sperm motility, sperm morphology and serum reproductive hormones (Follicle stimulating hormone (FSH), Leutinizing hormone (LH), testosterone and gonadotropin releasing hormone (GnRH)) of diabetes rat compared to control. Also, diabetes rat showed increase in sperm and testicular malonaldehyde (MDA) and decrease in sperm and testicular superoxide dismutase (SOD) activity and glutathione (GSH) level. Further, diabetes rat showed reduced testicular weight, decreased testicular 17β-HSD and 3β-HSD activity and testicular histo-architectural alteration which were accompanied by decrease testicular vascular endothelial growth factor (VEGF) and concomitant increase in testicular myeloperoxidase activity and level of caspase 3. The present results indicates that induction of diabetes in rat causes reduction in the level of reproductive hormones (Testosterone, LH and FSH) as well as sperm and testicular oxidative stress causing abnormal sperm parameters, and biochemical and histo-architectural alterations in the testes of rats. In addition, the present results suggest that erythritol administration reduced blood glucose and ameliorated hyperglycemia –induced oxidative stress –mediated alterations in both sperm and testes of diabetes rat. Further, the present study suggests that erythritol improved testicular oxidative stress, inflammation and apoptosis by up-regulating VEGF.

Melatonin as a promising therapeutic intervention for restoring ovarian function in letrozole-induced polycystic ovary syndrome rats

Polycystic ovary syndrome (PCOS) is a common hormonal disorder that affects women of reproductive age and is characterized by multiple ovarian cysts, irregular menstrual cycles, and excessive androgen hormone production. The present study aimed to investigate the therapeutic efficacy of melatonin in alleviating PCOS-induced alterations in female Wistar rats. PCOS was induced in female albino rats by administering letrozole at a dose of 1 mg/kg for 21 days. A total of 24 rats were randomly selected and divided into four groups: group I (normal control), group II (melatonin treatment), group III (letrozole treatment), and group IV (melatonin therapy for PCOS rats). Physical parameters (body and uterus weight), hormone profile (LH and FSH), and steroidogenic enzyme activities and an oral glucose test were assessed using standard methods. Histological analysis was performed using hematoxylin and eosin staining. The results demonstrated that exogenous melatonin administration significantly improved PCOS symptoms in rats, including reduced body weight gain, changes in organ weight/body weight index, blood glucose level, percentage diestrus phase, testosterone, estradiol, progesterone, and LH/FSH ratio, as well as 3β-HSD and 17β-HSD enzyme activity. Histopathological findings revealed well-developed follicles, decreased cystic follicles, and increased antral follicles, Graafian follicles, and corpus luteum in PCOS rats treated with melatonin. These positive outcomes suggest that exogenous melatonin may hold promise as a valuable remedy for PCOS conditions in female rats. Further research is warranted to fully elucidate the underlying mechanisms and potential clinical applications of melatonin in the context of PCOS.