11β-hydroxysteroid Dehydrogenase Research Articles

Engineering a two-enzyme system in Mycolicibacterium neoaurum for efficient biotransformation of phytosterols to dihydrotestosterone.

Dihydrotestosterone (DHT) is a valuable steroid drug with widespread clinical applications, but traditional chemical synthesis is environmentally harmful and requires complex reaction conditions. This study introduces a one-step microbial transformation method for the production of DHT from inexpensive phytosterols (PS) using engineered Mycolicibacterium neoaurum (MNR). The heterologous expression of 5α-reductase (5α-R) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in MNR enabled the efficient conversion of PS to DHT. To further enhance 5α-R activity, semi-rational mutagenesis was employed, which significantly increasing DHT production. Molecular dynamics simulations provided insights into the underlying mechanisms driving this enhancement. Additionally, the incorporation of a PntAB-based NADPH regeneration system further improved DHT yield. Process optimization resulted in a maximum DHT concentration of 1.123 g·L-1, representing the first gram-scale microbial production of DHT. Compared to traditional chemical synthesis, this biotransformation method offers milder reaction conditions, reduced environmental impact, and eliminates the need for toxic catalysts. This work demonstrates a sustainable and efficient microbial method for DHT production, with significant industrial potential for the greener manufacturing of steroid hormone drugs.

Daniellia oliveri as a type 2 diabetes remedy: evidence from in-silico evaluation

ABSTRACT The prevalence of diabetes mellitus is increasing, and well-known conventional medications are associated with harmful effects. Therefore, this study aimed to identify the compounds present in the young leaves of Daniellia oliveri and assess their antidiabetic potential using an in-silico model. The best column chromatographic fraction obtained from ethyl acetate fraction was analyzed using HRLC-MS to identify the prominent compounds. The identified compounds were subjected to ADMET prediction using online servers to confirm their draggability. In addition, the compounds were screened for activity against type 2 diabetes using molecular docking. Eight compounds with prominent peaks were identified, viz; gallic acid, 2,6-dihydroxybenzoic acid, salicylic acid, caffeic acid, phenylacetaldehyde, 2,5-dimethylphenol, 3-(−4-Hydroxyphenyl) propionic acid, and 16-hydroxyhexadecanoic acid. Most of the identified compounds were phenolics and had little toxic effects. The molecular docking results revealed the potentials of the identified compounds in inhibiting the therapeutic targets viz; 11β-hydroxysteroid dehydrogenase type 1, human salivary alpha-amylase, glycogen phosphorylase, human protein tyrosine phosphatase 1B, glutamine fructose-6-phosphate amidotransferase, human sirtuin 6, and dipeptidyl peptidase IV, responsible for the development of type 2 diabetes. This study has successfully revalidated and provided scientific insight into the usage of D. oliveri in managing type 2 diabetes by Nigerians. However, the limitation of this study remains the purification of the lead compounds that can serve as lead for type 2 diabetes treatment in conventional medicinal practices.

Behenic acid protects the testosterone cycle and prevents the sperm apoptosis and protein loss in phthalate exposure by inhibiting oxidative stress and stimulating ATPase activity

BackgroundPlastic products use phthalate to enhance their flexibility, transparency, and stability, while behenic acid is a carboxylic acid with antioxidant activity. ObjectivesThis study evaluates whether behenic acid can protect the testosterone cycle and prevent the sperm apoptosis and protein loss in phthalate-treated male rats. MethodsThere were 36 male albino rats in all, divided into six equal sets of six rats each: control, behenic acid (13g/kg), behenic acid (26g/kg), diethyl phthalate (10mg/kg), behenic acid (13g/kg) + diethyl phthalate (10mg/kg), and behenic acid (26g/kg) + diethyl phthalate (10mg/kg)-treated groups. Measurements were made of serum male hormones, sex hormone-binding globulin, sodium/potassium ATPase, superoxide dismutase, glutathione, glucose-6-phosphate dehydrogenase, 3β-hydroxysteroid dehydrogenase, protein, and cholesterol in the testis, as well as malondialdehyde in the sperm, testis, and hypothalamus. Sperm monoclonal proliferating antibody Ki-67, sperm counts, motility, and abnormalities were measured. ResultsOral administration of diethyl phthalate increased malondialdehyde, serum follicle stimulating hormone, sex hormone binding globulin, luteinizing hormone, glucose-6-phosphate dehydrogenase, 3β-hydroxysteroid dehydrogenase, cholesterol, total protein, sperm abnormality, and the percentage of spermatogonia, first spermatocyte, second spermatocyte, and spermatid in the testis. Superoxide dismutase, glutathione, serum testosterone and dehydroepiandrosterone sulphate, sperm count and motility, and sodium/potassium-ATPase activity were all reduced. Additionally, all of the previously described parameters reverted to near control values after receiving two doses of behenic acid in phthalate-treated rats; a higher dose of behenic acid had a more effective effect than a lower dose. Conclusionbehenic acid can protect the testosterone cycle and prevent the sperm apoptosis and protein loss in phthalate-treated male rats by inhibiting oxidative stress and stimulating ATPase activity.

Prolactin locally mediates follicular atresia in hyperprolactinemic vizcachas (Rodentia, Chinchillidae)

Infertility in hyperprolactinemic females is attributed to the dysregulation of GnRH release, subsequently affecting gonadotropin levels, and ultimately leading to anovulation. However, in addition to the hypothalamus, prolactin receptor (PRLR) is expressed in ovaries as well, suggesting potential local effects of PRL in cases of hyperprolactinemia. We have developed an experimental model of sulpiride (SPD)-induced hyperprolactinemia using a wild rodent, the plains vizcacha, and studied the implications of pharmacological PRL levels on folliculogenesis and steroid production. Ovaries of SPD females showed a striking number of atretic follicles along with a reduction in the collective number of viable follicles leading to a higher atretic/viable follicle ratio compared to that of control females (CTL) (P < 0.05). In terms of sensitivity to the hormonal environment, SPD ovaries substantially changed their potential responsiveness to pituitary PRL, as evidenced by the three-fold increase in PRLR expression alongside heightened expression of both gonadotropic receptors in comparison to CTL ovaries (P < 0.05). Circulating estradiol (E2) values doubled post-treatment in the SPD females, which also showed higher expressions of aromatase and 17β-hydroxysteroid dehydrogenase, along with both E2 receptors, ERα and ERβ than the CTL group (P < 0.05). Our findings strongly suggest that hyperprolactinemia-dependent dysregulation of ovarian function can be explained at least partially, by PRL direct actions facilitated by the heightened expression of PRLR in follicles and corpora lutea. Possibly these PRL actions synergize with those triggered by gonadotropic hormones ultimately leading to alteration of the steroidogenic pathway, folliculogenesis disruption and increased atresia.

Potential antiandrogenic effects of parabens and benzophenone-type UV-filters by inhibition of 3α-hydroxysteroid dehydrogenases

Parabens and UV-filters are frequently used additives in cosmetics and body care products that prolong shelf-life. They are assessed for potential endocrine disrupting properties. Antiandrogenic effects of parabens and benzophenone-type UV-filters by blocking androgen receptor (AR) activity have been reported. Effects on local androgen formation received little attention. Local 5α-dihydrotestosterone (DHT) production with subsequent AR activation is required for male external genitalia formation during embryogenesis. We investigated whether parabens and benzophenone-type UV-filters might cause potential antiandrogenic effects by inhibiting oxidative 3α-hydroxysteroid dehydrogenases (3α-HSDs) involved in the backdoor pathway of DHT formation. Five different 3α-HSDs were assessed for their efficiency to catalyze the 3α-oxidation reaction to form DHT and activate AR. 17β-hydroxysteroid dehydrogenase type 6 (HSD17B6), retinol dehydrogenases type 5 and 16 were further assessed using a radiometric in vitro activity assay to determine the conversion of 5α-androstane-3α-ol-17-one to 5α-androstane-3,17-dione in lysates of overexpressing HEK-293 cells. All parabens tested, except p-hydroxybenzoic acid (a main metabolite) inhibited HSD17B6 activity. Hexyl- and heptylparaben, as well as benzophenone (BP)-1 and BP-2, showed the highest inhibitory potencies, with nanomolar IC50 values. Molecular modeling predicted binding modes for the inhibitory parabens and BPs and provided an explanation for the observed structure-activity-relationship. Our results propose a novel mechanism of antiandrogenic action for commercially used parabens and BP UV-filters by inhibiting HSD17B6 and lowering DHT synthesis. Follow-up studies should assess BP-3 metabolism after topical application and whether the identified inhibitors reach concentrations in liver, testis, or prostate to inhibit HSD17B6, thereby causing antiandrogenic effects.

Boldenone and Testosterone Production from Phytosterol via One-Pot Cascade Biotransformations

Testosterone (TS) and its 1(2)-dehydrogenated derivative boldenone (BD) are widely used in medicine, veterinary science and as precursors in organic synthesis of many therapeutic steroids. Green production of these compounds is possible from androstenedione (AD) enzymatically, or from phytosterol (PS) using fermentation stages. In this study, the ascomycete Curvularia sp. VKM F-3040 was shown to convert androstadienedione (ADD, 4 and 10 g/L) to yield 97% and 78% (mol/mol) of BD, respectively. Based on its high 17β-hydroxysteroid dehydrogenase (17β-HSD) activity, a novel cascade biotransformation of PS was developed for production of TS and BD. At the first stage, the strains of Mycolicibacterium neoaurum VKM Ac-1815D or M. neoaurum VKM Ac-1816D converted PS (5 or 10 g/L) into AD or ADD (each in a concentration of 2.5 or 5 g/L), respectively. At the second stage, mycelium of the fungus under the revealed optimal conditions reduced AD or ADD with more than 90% efficiency to form TS or BD, respectively. Based on transcriptome analysis, six candidate genes that might encode 17β-HSDs in the Curvularia sp. genome were revealed. Along with 17β-HSDs, the fungus possessed inducible P450cur 7-monooxygenase, which led to the accumulation of 7α-hydroxytestosterone (7α-OH-TS) as a major product from AD (up to 83% within 24 h after mycelium addition at the second stage of cascade biotransformation). The presence of protein synthesis inhibitor cycloheximide (CHX) prevented 7α/β-hydroxylation due to inhibition of de novo synthesis of the enzyme in the fungal cells. The results demonstrate the high biotechnological potential of the Curvularia sp. strain and open up prospects for the synthesis of valuable 17β-reduced and 7-hydroxylated steroids by cascade biotransformations.

Congenital Adrenal Hyperplasia (CAH) - Causes, Diagnosis, Symptoms, Treatment

Introduction and Purpose: Congenital adrenal hyperplasia represents a group of genetic disorders characterized by improper adrenal steroidogenesis, resulting in deficiency or absence of cortisol and/or aldosterone, and varying degrees of disturbances in sexual development. The aim of this study is to raise awareness about the disease, enabling early diagnosis and contributing to reducing neonatal mortality, as the first clinical manifestation of CAH can be the life-threatening salt-wasting syndrome. State of Knowledge: Congenital adrenal hyperplasia (CAH), also known as congenital adrenal hyperplasia, encompasses a group of inherited disorders affecting the adrenal glands located above the kidneys. Adrenal glands produce various hormones, including cortisol, aldosterone, and androgens. CAH involves deficiencies in the enzymes necessary for the production of these hormones, leading to hormonal disturbances. Summary: Congenital adrenal hyperplasia (CAH), also referred to as congenital adrenal hyperplasia (CAH), describes a group of genetic diseases causing defects in adrenal hormone synthesis. CAH results in deficiency or absence of cortisol and/or aldosterone. The most common causes are mutations in the CYP21 gene (21-hydroxylase deficiency), CYP11B2 gene (11β-hydroxylase deficiency), and HSD3B2 gene (3β-hydroxysteroid dehydrogenase deficiency). Enzymatic deficiencies lead to compensatory increases in CRH and ACTH secretion, causing adrenal hyperplasia and excessive androgen synthesis.

Unlocking Testosterone Production by Biotransformation: Engineering a Fungal Model of Aspergillus nidulans Strain Deficient in Steroid 11α-Hydroxylase Activity and Expressing 17β-Hydroxysteroid Dehydrogenase Enzyme as Proof of Concept

Testosterone holds significant medical and economic importance, with the global market for testosterone replacement therapies valued at approximately USD 1.9 billion in 2023. This hormone is essential for the development and maintenance of male sexual characteristics as well as bone and muscle health. It plays a key role in conditions such as hypogonadism, muscle disorders, and andropause. However, the industrial production of testosterone often involves complex chemical processes that result in low yields, high costs, and environmental damage. Microbial biotransformation of steroids presents an eco-friendly alternative to traditional chemical synthesis. A knockout strain of Aspergillus nidulans deficient in steroid 11α-hydroxylase activity was developed, rendering it incapable of hydroxylating androstenedione, progesterone, and testosterone. In these strains, two newly identified CYP450 enzymes, CYP68L1 from A. nidulans and CYP68L8 from Aspergillus ochraceus, were expressed to confirm their roles as steroid 11α-hydroxylases of androstenedione, progesterone, and testosterone. The availability of these 11α-hydroxylases represents significant progress toward achieving efficient single-step steroid fermentation. Furthermore, the A. nidulans knockout strain serves as an effective model for studying the conversion of androstenedione to testosterone upon the expression of the enzyme 17β-hydroxysteroid dehydrogenase, due to its inability to hydroxylate testosterone.

African walnut (Plukenetia conophora) oil promotes glucose uptake while improving energy metabolism and steroidogenesis and maintaining surface architecture in rat testes.

African walnut (Plukenetia conophora) oil (AWO) has been reported for its nutritional and medicinal properties and has been employed for the management of metabolic diseases including hyperglycemia-mediated ailments. In the present study, AWO was investigated for its ability to stimulate glucose uptake and its effect on energy metabolism, steroidogenesis, and tissue morphology in isolated testes of Wistar rats. Isolated testes were incubated with AWO (30-240 μg/mL) in the presence of 11.1 mMol glucose at 37°C for 2 h. Control consisted of testes incubated with glucose only, while normal control consisted of testes not incubated with AWO and/or glucose. The standard antidiabetic drug was metformin. Incubation with AWO led to significant increase in glucose uptake, hexokinase, glyoxalase 1, glutathione reductase, glutathione peroxidase, 3β-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase activities, GLUT4, glutathione, and ATP levels while concomitantly suppressing glucose-6-phosphatase, fructose-1,6-biphosphatase, glycogen phosphorylase, aldose reductase, polyol dehydrogenase, E-NTPDase, and ATPase activities. Furthermore, incubation with AWO led to improved testicular morphology while elevating testicular levels of magnesium, sulfur, potassium, calcium, and iron. Fatty acid profiling with GC-MS revealed linoleic acid and linolenic acid as the predominant essential fatty acids in AWO. Molecular docking analysis revealed potent molecular interactions of linoleic acid and linolenic acid with GLUT4. These results suggest the ability of AWO to improve testicular glucose metabolism and steroidogenesis and can be explored in the management of male infertility.

Selenium ameliorates oxidized phospholipid-mediated testicular dysfunction and epididymal sperm abnormalities following Bisphenol A exposure in adult Wistar rats

Bisphenol A (BPA) is an endocrine-disrupting compound extensively utilized in the production of polycarbonate polymers and epoxy resins that, upon exposure, pose a significant threat to male reproductive health because of its estrogenic properties. Accumulating evidence suggests that BPA exposure disrupts the normal process of spermatogenesis, alters testicular morphology and function, and interferes with testicular steroidogenesis and hormonal signaling. However, the precise mechanism by which BPA affects testicular function remains unclear. In this study, we explored the mechanism underlying BPA-induced testicular abnormalities and evaluated the protective effects of Selenium (Se). Thirty-two adult male albino Wistar rats were divided into four groups, and BPA was administered at 50 mg/kg body weight, with or without Se supplementation, for 30 days. Se supplementation (2.5 mg/kg body weight) was initiated 1 week before BPA administration. BPA administration resulted in alterations in testicular architecture, characterized by basement membrane disintegration in the seminiferous tubules, reduced spermatogenic cell counts, and increased interstitial tubule noncellular space. Furthermore, BPA exposure increased the levels of oxidized phospholipids, lipid peroxides, and hydroxyl radicals and decreased the activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase. In addition, BPA significantly reduced the activities of 3β- and 17β-hydroxysteroid dehydrogenases, interfering with testicular steroidogenesis. In rats, coadministration of Se and BPA reduced the levels of oxidized phospholipids and increased the activities of antioxidant enzymes, leading to improved testicular function and epididymal sperm parameters, suggesting that Se plays a critical role in alleviating endocrine disruptor-induced testicular dysfunctions in rats.

HSD3B1, prostate cancer mortality and modifiable outcomes.

Androgen receptor stimulation by testosterone and dihydrotestosterone is crucial for prostate cancer progression. Despite the initial effectiveness of androgen deprivation therapy (ADT), castration-resistant prostate cancer eventually develops in most men. A common germline missense-encoding polymorphism in HSD3B1 increases extra-gonadal androgen biosynthesis from adrenal precursors owing to increased availability of the encoded enzyme 3β-hydroxysteroid dehydrogenase 1 (3βHSD1) - hence, it is called the adrenal-permissive enzyme. This mechanism explains the more rapid progression to castration-resistant prostate cancer in men who inherit this allele than in men without it via sustained androgen receptor activation despite ADT. Multiple clinical studies, including data derived from prospective phase III studies, have linked adrenal-permissive allele inheritance to inferior clinical responses to ADT and increased mortality, but reversal is possible with upfront adrenal androgen blockade. The adrenal-permissive allele exhibits divergent frequencies across various groups worldwide, which could contribute to differences in clinical outcomes among these populations. Large-scale data from the Million Veteran Program have shown homozygous HSD3B1 adrenal-permissive allele inheritance to be an independent biomarker of prostate cancer-specific mortality. Together, these observations support the integration of HSD3B1 into germline testing and clinical trials as it might help to identify groups at increased likelihood of benefiting from early, intensified, AR-targeting interventions. Lastly, 3βHSD1 is a promising target for pharmacological inhibition, which enables new strategies for systemic prostate cancer therapy.

Petunidin attenuates vinclozolin instigated testicular toxicity in albino rats via regulating TLR4/MyD88/TRAF6 and Nrf-2/Keap-1 pathway: A pharmacodynamic and molecular simulation approach

Vinclozolin (VZN) is a widely used fungicide which exerts deleterious impacts on various organs including testis. Petunidin (PDN) is a polyphenolic compound that demonstrates a broad range of pharmacological activities. Thirty-two rats were divided into 4 groups including the control, VZN (100 mg/kg), VZN (100 mg/kg) + PDN (4 mg/kg) and PDN (4 mg/kg) treated group. The activities of antioxidant enzymes were assessed by using previously documented protocols. The gene expressions were determined by using qRT-PCR. The levels of hepatic function and apoptotic markers were evaluated by using standard ELISA technique. The histological analysis was carried out as per the standard protocol of histology. It was revealed that VZN disrupted the Nrf-2/Keap-1 pathway. Moreover, the activities of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), heme-oxygenase-1 (HO-1) and glutathione reductase (GSR) were reduced whereas levels of reactive oxygen species (ROS) & malondialdehyde (MDA) were promoted following the VZN intoxication. Furthermore, VZN intoxication reduced total sperm count, viability, motility as well as luteinizing hormone (LH), follicle stimulating hormone (FSH), and plasma testosterone. Besides, administration of VZN decreased the expressions of 3β-Hydroxysteroid dehydrogenase (3β-HSD), steroidogenic acute regulatory protein (StAR) and 17β-Hydroxysteroid dehydrogenase (17β-HSD). Moreover, VZN exposure escalated the expressions of Bcl-2–associated X protein (Bax) and cysteine–aspartic acid protease-3 (Caspase-3) while reducing the expressions of B-cell lymphoma-2 (Bcl-2). Additionally, VZN administration increased the gene expression of toll-like receptor 4 (TLR4), tumor necrosis factor receptor-associated factor 6 (TRAF-6) and myeloid differentiation primary response 88 (MyD88). The levels of interleukin-6 (IL-6), nuclear factor kappa-B (NF-κB), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and the activity of cyclooxygenase-2 (COX-2) were promoted following the VZN administration. Furthermore, VZN intoxication disrupted the normal histology of testicular tissues. However, VZN + PDN treatment ameliorated testicular damage via regulating aforementioned dysregulations owing to its anti-inflammatory, antioxidative as well as anti-apoptotic potentials. Lastly, molecular docking (MD) was performed to assess the effectiveness of PDN as a curative compound by analyzing its binding affinity with the targeted proteins (Keap1, TLR4 and StAR). Our in-silico evaluations confirmed that PDN possesses the potential to interact with binding pockets of these proteins, emphasizing its capability as a curative compound to mitigate VZN-prompted reproductive damage.

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.

α-Linolenic acid promotes testosterone synthesis by improving mitochondrial function in primary rooster Leydig cells

The present study aimed to investigate the direct effects of α-Linolenic acid (ALA) on the in vitro production of testosterone and the expression of key enzymes and proteins related to steroidogenesis in Leydig cells of roosters. MethodsPurified primary Leydig cells isolated from 65-week-old roosters were purified and treated with different concentrations of ALA treatments: (0 μm/L [control], solvent control group (DMSO), 20 μM/L, 40 μM/L, and 80 μM/L) and cell counting-8 (CCK-8) for cell viability assay, Enzyme-linked immunosorbent assay (ELISA) kit for the determination of testosterone in cell supernatants, quantitative (real-time) PCR, and analysis of activities of antioxidants catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA), evaluation of mitochondrial membrane potential, pro- and anti-apoptotic proteins/genes Bcl-2, Bcl-2-associated X protein (Bax), apoptosis-inducing factor (AIF) were done respectively. ResultsOur results showed that ALA significantly increased testosterone secretion in primary rooster Leydig cells (P < 0.05), and 40 μM/L is the optimal dose. Leydig cells supplemented with ALA (20, 40, 80 μM) increased the expression of key enzymes and proteins 3β-hydroxysteroid dehydrogenase (3β-HSD), steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc) concerning steroidogenesis, enhanced antioxidant capability, improved mitochondrial biogenesis, and markedly improved the mitochondrial membrane potential (P < 0.05). Furthermore, the expression of the apoptosis-suppressive gene Bcl-2 was significantly increased, but Bax and AIF expression was decreased in the ALA group compared to that in the control group (P < 0.05). ConclusionALA promoted testosterone production, enhanced steroidogenic enzyme expression, improved mitochondrial function, and antioxidant capacity, and reduced apoptosis in primary rooster Leydig cells, with 40 μM/L identified as the optimal concentration.

Oleanolic acid purified from the stem bark of Olax subscorpioidea Oliv. inhibits the function and catalysis of human 17β-hydroxysteroid dehydrogenase 1

Cancer is a leading cause of global death. Medicinal plants have gained increasing attention in cancer drug discovery. In this study, the stem bark extract of Olax subscorpioidea, which is used in ethnomedicine to treat cancer, was subjected to phytochemical investigation leading to the isolation of oleanolic acid (OA). The structure was elucidated by 1-dimensional and 2-dimensional nuclear magnetic resonance spectroscopic (NMR) data, and by comparing its data with previously reported data. Molecular docking was used to investigate the interactions of OA with nine selected cancer-related protein targets. OA docked well with human 17β-hydroxysteroid dehydrogenase type-1 (17βHSD1), caspase-3, and epidermal growth factor receptor tyrosine kinase (binding affinities: −9.8, −9.3, and −9.1 kcal/mol, respectively). OA is a triterpenoid compound with structural similarity to steroids. This similarity with the substrates of 17βHSD1 gives the inhibitor candidate an excellent opportunity to bind to 17βHSD1. The structural and functional dynamics of OA-17βHSD1 were investigated by molecular dynamics simulations at 240 ns. Molecular mechanics/Poisson-Boltzmann surface area (MMPBSA) studies showed that OA had a binding free energy that is comparable with that of vincristine (–52.76, and −63.56 kcal/mol, respectively). The average C-α root mean square of deviation (RMSD) value of OA (1.69 Å) compared with the unbound protein (2.01 Å) indicated its high stability at the protein’s active site. The binding energy and stability at the active site of 17βHSD1 recorded in this study indicate that OA exhibited profound inhibitory potential. OA could be a good scaffold for developing new anti-breast cancer drugs.

Promising Directions for Regulating Signaling Pathways Involved in the Type 2 Diabetes Mellitus Development

Many studies confirm that substances of natural origin have a pronounced affinity for type 2 diabetes mellitus (T2DM) therapeutic targets. At the moment, there is growing interest in bioactive peptides, phytochemicals, and drugs from other natural sources as highly effective, safe and promising antidiabetic agents. Natural sources are a promising resource for regulating several pathological pathways in T2DM. The review describes ways to mitigate insulin resistance and tissue sensitivity to glucose through PTP1β (protein tyrosine phosphatase 1β), GLP-1R (glucagon-like peptide receptor), DPP-4 (dipeptidyl peptidase-4), AMPK (adenosine monophosphate activated protein kinase), MAPK (mitogen-activated protein kinase). Regulation of obesity and oxidative stress development through CCN3 (nephroblastoma overexpressed gene), PPAR-γ (peroxisome proliferator-activated receptor γ), Nrf2 (nuclear factor erythroid-related factor 2), FFAR (free fatty acid receptors), 11β-HSD1 (11β-hydroxysteroid dehydrogenase). Regulation of hyperglycemia through alpha-amylase inhibitors, regulation of glucose metabolism through GFAT (glutamine fructose-6-phosphate aminotransferase), FOXO1 (forkhead box protein O1), GLUT4 (glucose transporter type 4), PGC-1α (receptor gamma coactivator 1α activating peroxisome proliferator). The review examines the use of natural sources, from which low-molecular-weight and peptide compounds are used as T2DM targets modulators.

Dysregulation of the 3β-hydroxysteroid dehydrogenase type 2 enzyme and steroid hormone biosynthesis in chronic kidney disease.

Chronic kidney disease (CKD) presents a critical global health challenge, marked by the progressive decline of renal function. This study explores the role of the 3β-hydroxysteroid dehydrogenase type 2 enzyme (HSD3B2) and the steroid hormone biosynthesis pathway in CKD pathogenesis and progression. Using an adenine-induced CKD mouse model, we conducted an untargeted metabolomic analysis of plasma samples to identify key metabolite alterations associated with CKD. Immunohistochemistry, Western blotting, and qPCR analyses were performed to confirm HSD3B2 expression in both human and mouse tissues. Additionally, Nephroseq and Human Protein Atlas data were utilized to assess the correlation between HSD3B2 and kidney function. Functional studies were conducted on HK2 cells with HSD3B2 knockdown to evaluate the impact on cell proliferation and apoptosis. Metabolic characteristics revealed significant shifts in CKD, with 61 metabolites increased and 65 metabolites decreased, highlighting the disruption in steroid hormone biosynthesis pathways influenced by HSD3B2. A detailed examination of seven key metabolites underscored the enzyme's central role. HSD3B2 exhibited a strong correlation with kidney function, supported by data from Nephroseq and the Human Protein Atlas. Immunohistochemistry, Western blotting, and qPCR analyses confirmed a drastic reduction in HSD3B2 expression in CKD-affected kidneys. Suppressed proliferation and increased apoptosis rates in HSD3B2 knocked down HK2 cells further demonstrated the enzyme's significance in regulating renal pathophysiology. These findings underscore the potential of HSD3B2 as a clinical diagnostic and therapeutic target in CKD. While further studies are warranted to fully elucidate the mechanisms, our results provide valuable insights into the intricate interplay between steroid hormone biosynthesis and CKD. This offers a promising avenue for precision medicine approaches and personalized treatment strategies.

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.

Taste receptor T1R3 regulates testosterone synthesis via the cAMP-PKA-SP1 pathway in testicular Leydig cells

Taste receptor type 1 subunit 3 (T1R3) is a G protein-coupled receptor encoded by the TAS1R3 gene that can be specifically activated by certain sweeteners or umami agents for sweet/umami recognition. T1R3 is a potential target for regulating male reproduction. However, studies on the impact of non-nutritive sweeteners on reproduction are limited. In the present study, we evaluated the impact of the non-nutritive sweeteners (saccharin sodium, sucralose and acesulfame-K) on testosterone synthesis in testicular Leydig cells of Xiang pigs by comparing the relative abundance of mRNA transcripts and protein expression of T1R3, steroidogenic related factors, and intracellular cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), as well as testosterone levels using Western blotting, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). To clarify the specific mechanism, a dual luciferase assay was used to uncover the relationship between the transcription factors and steroidogenic enzyme. The acute intratesticular injection of a typical non-nutritive sweeteners was conducted to verify this impact in mouse. The results showed that saccharin sodium not only enhanced T1R3 expression in Leydig cells of Xiang pigs, but also caused significant increases in testosterone, cAMP, PKA, phosphorylation of specificity protein 1 (p-SP1), total protein of specificity protein 1 (SP1), steroidogenic acute regulatory protein (StAR), and 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) (P < 0.05). Similarly, treatment of Leydig cells with sucralose and acesulfame-K also increased testosterone level, protein expression of T1R3, 17-α-hydroxylase/17, 20-lyase (CYP17A1), and 3β-HSD1 (P < 0.05). Treatment with SQ22536 (an adenylate cyclas inhibitor) or H89 (a PKA inhibitor) significantly reduced saccharin sodium-induced protein levels of p-SP1, StAR, CYP17A1, and 3β-HSD1 (P < 0.05). In addition, a dual luciferase assay further demonstrated that SP1 significantly increased the promoter activity of CYP17A1 (P < 0.05). When mouse testes were injected with saccharin sodium, T1R3, p-SP1, CYP17A1, and 3β-HSD1 were upregulated, leading to a significant testicular increase in testosterone and cAMP levels (P < 0.05). These results suggest a mechanism by which the taste receptor T1R3 regulates testosterone production, and this mechanism may be linked to the cAMP-PKA pathway. Understanding the interrelationship between T1R3 and the cAMP-PKA-SP1 pathway contributes to clarify the regulatory mechanisms of male reproduction.

Molecular mechanism of high-altitude hypoxia-induced lipid metabolism disorder in mouse spleen tissue

To investigate the molecular mechanism of lipid metabolism disorder in mouse spleen tissues due to high-altitude hypoxia. Ten C57BL/6 male mice were randomly divided into normoxia group (maintained at an altitude of 400 m) and high-altitude hypoxia group (maintained at 4200 m) for 30 days (n=5). Lipidomics and metabolomics analyses of the spleen tissue of the mice were conducted using liquid chromatography-mass spectrometry (LC-MS) to identify the differential metabolites, which were further analyzed by KEGG enrichment and pathway analyses, and the differential genes were screened through transcriptome sequencing. Bioinformatics analysis was conducted to identify the upstream target genes of the differential metabolites in specific metabolic pathways. RT-qPCR and Western blotting were used to detect mRNA expressions of 11β-hydroxysteroid dehydrogenase 1 (HSD11B1), steroid 5α reductase 1 (SRD5A1), prostaglandin-endoperoxide synthase 1 (PTGS1), hematopoietic prostaglandin D synthetase (HPGDS), xanthine dehydrogenase (XDH), purine nucleoside phosphorylase (PNP), hypoxanthine guanine-phosphoribosyltransferase (HPRT) and extracellular 5'-nucleotidase (NT5E) and protein expressions of HSD11B1, SRD5A1, XDH, PNP and HPRT in the mouse spleens. We identified a total of 41 differential lipid metabolites in the mouse spleens, and these metabolites and the differential genes were enriched in steroid hormone biosynthesis, arachidonic acid metabolism, and purine metabolism pathways. Compared to the mice kept in normoxic conditions, the mice exposed to high-altitude hypoxia showed significantly upregulated expressions of adrenosterone, androsterone, prostaglandin D2, prostaglandin J2, xanthine, xanthosine, and uric acid in the spleen with also changes in the expression levels of HSD11B1, SRD5A1, PTGS1, HPGDS, XDH, PNP, HPRT, and NT5E. High-altitude hypoxia can result in lipid metabolism disorder in mouse spleen tissue by affecting steroid hormone biosynthesis, arachidonic acid metabolism, and purine metabolism pathways.