Evaluating the therapeutic potential of moxibustion on polycystic ovary syndrome: a rat model study on gut microbiota and metabolite interaction.

Polycystic ovary syndrome (PCOS) is a common systemic disorder related to endocrine disorders, affecting the fertility of women of childbearing age. It is associated with glucose and lipid metabolism disorders, altered gut microbiota, and insulin resistance. Modern treatments like pioglitazone, metformin, and spironolactone target specific symptoms of PCOS, while in Chinese medicine, moxibustion is a common treatment. This study explores moxibustion's impact on PCOS by establishing a dehydroepiandrosterone (DHEA)-induced PCOS rat model. Thirty-six specific pathogen-free female Sprague-Dawley rats were divided into four groups: a normal control group (CTRL), a PCOS model group (PCOS), a moxibustion treatment group (MBT), and a metformin treatment group (MET). The MBT rats received moxibustion, and the MET rats underwent metformin gavage for two weeks. We evaluated ovarian tissue changes, serum testosterone, fasting blood glucose (FBG), and fasting insulin levels. Additionally, we calculated the insulin sensitivity index (ISI) and the homeostasis model assessment of insulin resistance index (HOMA-IR). We used 16S rDNA sequencing for assessing the gut microbiota, 1H NMR spectroscopy for evaluating metabolic changes, and Spearman correlation analysis for investigating the associations between metabolites and gut microbiota composition. The results indicate that moxibustion therapy significantly ameliorated ovarian dysfunction and insulin resistance in DHEA-induced PCOS rats. We observed marked differences in the composition of gut microbiota and the spectrum of fecal metabolic products between CTRL and PCOS rats. Intriguingly, following moxibustion intervention, these differences were largely diminished, demonstrating the regulatory effect of moxibustion on gut microbiota. Specifically, moxibustion altered the gut microbiota by increasing the abundance of UCG-005 and Turicibacter, as well as decreasing the abundance of Desulfovibrio. Concurrently, we also noted that moxibustion promoted an increase in levels of short-chain fatty acids (including acetate, propionate, and butyrate) associated with the gut microbiota of PCOS rats, further emphasizing its positive impact on gut microbes. Additionally, moxibustion also exhibited effects in lowering FBG, testosterone, and fasting insulin levels, which are key biochemical indicators associated with PCOS and insulin resistance. Therefore, these findings suggest that moxibustion could alleviate DHEA-induced PCOS by regulating metabolic levels, restoring balance in gut microbiota, and modulating interactions between gut microbiota and host metabolites.

Associations of schizophrenia with the activities of the HPA and HPG axes and their interactions characterized by hair-based biomarkers.

BACKGROUND: Past studies on schizophrenia (SCZ) and the stress-sensitive neuroendocrine systems have mostly focused on a single system and traditionally utilized acute biomarkers (e.g., biomarkers from blood, urine and saliva) that poorly match the chronic course of schizophrenia in time span. Using eight biomarkers in hair, this study aimed to explore the functional characteristics of SCZ patients in the hypothalamic-pituitary-adrenocortical (HPA) and hypothalamic-pituitary-gonadal (HPG) axes and the interaction between the two axes. METHODS: Hair samples were taken from 137 SCZ patients and 73 controls. The SCZ patients were diagnosed by their attending physician according to the Diagnostic and Statistical Manual of Mental Disorders IV and were clinically stable after treatment. Gender, age, BMI, frequency of hair washing, marital status, education level, family history of mental illness and clozapine dosage were concurrently collected as covariates. The 10-item perceived stress scale (PSS-10) and the social readjustment rating scale were used to assess chronic stress status in SCZ patients. Eight hair biomarkers, cortisol, cortisone, dehydroepiandrosterone (DHEA), testosterone, progesterone, cortisol/cortisone, cortisol/DHEA and cortisol/testosterone, were measured by high performance liquid chromatography tandem mass spectrometer. Among them, cortisol, cortisone, DHEA and cortisol/DHEA reflected the functional activity of the HPA axis, and testosterone and progesterone reflected the functional activity of the HPG axis, and cortisol/cortisone reflected the activity of 11ß-hydroxysteroid dehydrogenase types 2 (11ß-HSD 2), and cortisol/testosterone reflected the HPA-HPG interaction. RESULTS: SCZ patients showed significantly higher cortisone and cortisol/testosterone than controls (p<0.001, η²p=0.180 and p=0.015, η²p=0.031), lower testosterone (p=0.009, η²p=0.034), progesterone (p<0.001, η²p=0.069) and cortisol/cortisone (p=0.001, η²p=0.054). There were significant intergroup differences in male and female progesterone (p=0.003, η²p=0.088 and p=0.030, η²p=0.049) and female testosterone (p=0.028, η²p=0.051). In SCZ patients, cortisol, cortisol/cortisone, cortisol/DHEA and cortisol/testosterone were positively associated with PSS-10 score (ps<0.05, 0.212

Rhythm gene PER1 mediates ferroptosis and lipid metabolism through SREBF2/ALOX15 axis in polycystic ovary syndrome.

OBJECTIVE: This work aimed to investigate the role of rhythm gene PER1 in mediating granulosa cell ferroptosis and lipid metabolism of polycystic ovary syndrome (PCOS). METHODS: We injected dehydroepiandrosterone and Ferrostatin-1 (Fer-1) into mice to explore the mechanism of ferroptosis in PCOS. The effect of PER1 on ferroptosis-like changes in granulosa cells was explored by overexpression of PER1 plasmid transfection and Fer-1 treatment. RESULTS: We found that Fer-1 ameliorated the characteristic polycystic ovary morphology, suppressed ferroptosis in the PCOS mice. PER1 and ALOX15 were highly expressed in PCOS, whereas SREBF2 was lowly expressed. Overexpression of PER1 decreased granulosa cell viability and inhibited proliferation. Meanwhile, overexpression of PER1 increased lipid reactive oxygen species, 4-Hydroxynonenal (4-HNE), Malondialdehyde (MDA), total Fe, and Fe2+ levels in granulosa cells and decreased Glutathione (GSH) content. Fer-1, SREBF2 overexpression, or ALOX15 silencing treatment reversed the effects of PER1 overexpression on granulosa cells. PER1 binds to the SREBF2 promoter and represses SREBF2 transcription. SREBF2 binds to the ALOX15 promoter and represses ALOX15 transcription. Correlation analysis of clinical trials showed that PER1 was positively correlated with total cholesterol, low-density lipoprotein cholesterol, luteinizing hormone, testosterone, 4-HNE, MDA, total Fe, Fe2+, and ALOX15. In contrast, PER1 was negatively correlated with SREBF2, high-density lipoprotein cholesterol, follicle-stimulating hormone, progesterone, and GSH. CONCLUSION: This study demonstrates that the rhythm gene PER1 promotes ferroptosis and dysfunctional lipid metabolism in granulosa cells in PCOS by inhibiting SREBF2/ALOX15 signaling.

New insights into the treatment of polycystic ovary syndrome: HKDC1 promotes the growth of ovarian granulocyte cells by regulating mitochondrial function and glycolysis.

Polycystic ovary syndrome (PCOS) is an endocrine disease, and its pathogenesis and treatment are still unclear. Hexokinase domain component 1 (HKDC1) participates in regulating mitochondrial function and glycolysis. However, its role in PCOS development remains unrevealed. Here, female C57BL/6 mice were intraperitoneally injected with dehydroepiandrosterone (DHEA; 60 mg/kg body weight) to establish an in vivo model of PCOS. In vitro, KGN cells, a human ovarian granular cell line, were used to explore the potential mechanisms. DHEA-treated mice exhibited a disrupted estrus cycle, abnormal hormone levels, and insulin resistance. Dysfunction in mitochondria and glycolysis is the main reason for PCOS-related growth inhibition of ovarian granular cells. Here, we found that the structure of mitochondria was impaired, less ATP was generated and more mitochondrial Reactive Oxygen Species were produced in HKDC1-silenced KGN cells. Moreover, HKDC1 knockdown inhibited glucose consumption and decreased the production of glucose-6-phosphate and lactic acid. Conclusively, HKDC1 protects ovarian granulocyte cells from DHEA-related damage at least partly by preserving mitochondrial function and maintaining glycolysis.

Discovery of novel peptide-dehydroepiandrosterone hybrids inducing endoplasmic reticulum stress with effective in vitro and in vivo anti-melanoma activities.

Steroid hybrids have emerged as a type of advantageous compound as they could offer improved pharmacological and pharmaceutical properties. Here, we report a series of novel peptide-dehydroepiandrosterone hybrids, which would effectively induce endoplasmic reticulum stress (ERS) and lead to apoptosis with outstanding in vitro and in vivo anti-melanoma effects. The lead compound IId among various steroids conjugated with peptides and pyridines showed effective in vivo activity in B16 xenograft mice: in medium- and high-dose treatment groups (60 and 80 mg/kg), compound IId would significantly inhibit the growth of tumours by 98%-99% compared to the control group, with the highest survival rate as well. Further mechanism studies showed that compound IId would damage the endoplasmic reticulum and upregulate the ERS markers C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78), which could further regulate caspase and Bcl-2 family proteins and lead to cell apoptosis. The compound IId was also proven to be effective in inhibiting B16 cell migration and invasion.

Aromatase and steroid sulfatase from human placenta.

Cytochrome P450 aromatase (AROM) and steroid (estrone (E1)/dehydroepiandrosterone (DHEA)) sulfatase (STS) are the two key enzymes responsible for the biosynthesis of estrogens in human, and maintenance of the critical balance between androgens and estrogens. Human AROM, an integral membrane protein of the endoplasmic reticulum, is a member of the Fe-heme containing cytochrome P450 superfamily having a cysteine thiolate as the fifth Fe-coordinating ligand. It is the only enzyme known to catalyze the conversion of androgens with non-aromatic A-rings to estrogens characterized by the aromatic A-ring. Human STS, also an integral membrane protein of the endoplasmic reticulum, is a Ca2+-dependent enzyme that catalyzes the hydrolysis of sulfate esters of E1 and DHEA to yield the respective unconjugated steroids, the precursors of the most potent forms of estrogens and androgens, namely, 17ß-estradiol (E2), 16α,17ß-estriol (E3), testosterone (TST) and dihydrotestosterone (DHT). Expression of these steroidogenic enzymes locally within various organs and tissues of the endocrine, reproductive, and central nervous systems is the key for maintaining high levels of the reproductive steroids. Thus, the enzymes have been drug targets for the prevention and treatment of diseases associated with steroid hormone excesses, especially in breast and prostate malignancies and endometriosis. Both AROM and STS have been the subjects of vigorous research for the past six decades. In this article, we review the procedures of their extraction and purification from human term placenta are described in detail, along with the activity assays.

Approaches to assessing 3ß-hydroxysteroid dehydrogenase-1.

The enzyme 3ß-hydroxysteroid dehydrogenase-1 (3ßHSD1), encoded by the gene HSD3B1, plays an essential role in the peripheral conversion of 3ß-OH, Δ5-steroids to 3-keto, Δ4-steroids. In human physiology, the adrenal produces dehydroepiandrosterone (DHEA) and DHEA-sulfate, which are major precursors for the biosynthesis of potent androgens and estrogens. DHEA is converted by 3ßHSD1 and subsequently is converted by steroid-5α-reductase to potent androgens or by aromatase to estrogens. Assessment of 3ßHSD1 is therefore critical under various conditions. In this chapter, we detail several approaches to assessing 3ßHSD1. First, we describe a genotyping protocol for the identification of a common missense-encoding variation that regulates 3ßHSD1 cellular metabolic activity. This protocol distinguishes between the HSD3B1(1245A) and the HSD3B1(1245C) allele which have lower and higher metabolic activity, respectively. Second, we detail mass spectrometry approaches to determining 3ßHSD1 activity using stable isotope dilution. Third, we describe methods for using tritiated DHEA and high performance liquid chromatography coupled with a beta-RAM to also determine 3ßHSD1 activity. Together, we provide multiple methods of directly assessing 3ßHSD1 activity or anticipated 3ßHSD1 activity.

Biosynthesis of estetrol in human pregnancy: Potential pathways.

Estetrol (E4) has emerged as a novel and highly promising estrogen for therapeutic use. E4 is a weak natural estrogen produced only in pregnancy. Because of its novelty, there is considerable interest by clinicians in how it is produced in pregnancy. Although the fetal liver plays a key role in its production, the placenta is also involved. A current view is that estradiol (E2) formed in the placenta enters the fetal compartment and is then rapidly sulfated. E2 sulfate then undergoes 15α-/16α-hydroxylation in the fetal liver thereby forming E4 sulfate (phenolic pathway). However, another pathway involving 15α,16α-dihydroxy-DHEAS formed in the fetal liver and converted to E4 in the placenta also plays a significant role (neutral pathway). It is not known which pathway predominates, but both pathways appear to be important in E4 biosynthesis. In this commentary, we summarize the well-established pathways in the formation of estrogens in the nonpregnant and pregnant female. We then review what is known about the biosynthesis of E4 and describe the 2 proposed pathways involving the fetus and placenta.

3ßHSD activity saturates at physiological substrate concentrations in intact cells.

BACKGROUND: Conversion of adrenally produced dehydroepiandrosterone (DHEA) to the potent androgen dihydrotestosterone (DHT) is an important mechanism by which prostate cancer reaches castration resistance. At the start of this pathway is a branch point at which DHEA can be converted to Δ4 -androstenedione by the enzyme 3ß-hydroxysteroid dehydrogenase (3ßHSD) or to Δ5 -androstenediol by 17ßHSD. To better understand this process, we studied the kinetics of these reactions in cells. METHODS: Prostate cancer cells (LNCaP cell line) were incubated with steroids (DHEA and Δ5 -androstenediol) over a range of concentrations and the steroid metabolism reaction products were measured by mass spectrometry or by high-performance liquid chromatography to determine reaction kinetics. To confirm the generalizability of results, experiments were also performed in JEG-3 placental choriocarcinoma cells. RESULTS: The two reactions displayed very different saturation profiles, with only the 3ßHSD-catalyzed reaction beginning to saturate within a physiological substrate concentration range. Strikingly, incubating LNCaP cells with low (in the ~10 nM range) concentrations of DHEA resulted in a large majority of the DHEA undergoing 3ßHSD-catalyzed conversion to Δ4 -androstenedione, whereas high concentrations of DHEA (in the 100s of nM range) resulted in most of the DHEA undergoing 17ßHSD-catalyzed conversion to Δ5 -androstenediol. CONCLUSION: Contrary to expectations from previous studies that used purified enzyme, cellular metabolism of DHEA by 3ßHSD begins to saturate in the physiological concentration range, suggesting that fluctuations in DHEA concentrations could be buffered at the downstream active androgen level.

Association of Oxidative Stress with Kidney Injury in a Hyperandrogenemic Female Rat Model.

Background: Polycystic ovary syndrome (PCOS) is the most common reproductive dysfunction in premenopausal women. PCOS is associated with oxidative stress (OS), which is the main risk factor for renal diseases. This study aimed to investigate the mechanisms responsible for renal injury in a hyperandrogenemic female rat model. Methods: This study was conducted from December 2019 to September 2021 at Shiraz Nephro-Urology Research Centre, Shiraz University of Medical Sciences (Shiraz, Iran). Thirty female Sprague-Dawley rats were randomly divided into three groups (n=10), namely control, sham, and dehydroepiandrosterone (DHEA). Plasma total testosterone, plasma creatinine (Cr), and blood urea nitrogen (BUN) levels were measured. In addition, total oxidant status (TOS), total antioxidant capacity (TAC), oxidative stress index (OSI), and histopathological changes in the ovaries and kidneys were determined. Data were analyzed using the GraphPad Prism software, and P<0.05 was considered statistically significant. Results: Plasma total testosterone levels increased by nine-fold in DHEA-treated rats compared to controls (P=0.0001). Administration of DHEA increased Cr and BUN levels and caused severe renal tubular cell injury. In addition, plasma and tissue (kidney and ovary) TAC levels decreased significantly, but TOS levels and OSI values were significantly increased (P=0.019). Significant damage to both glomerular and tubular parts of the kidney and ovarian follicular structure was observed in the DHEA group. Conclusion: Hyperandrogenemia caused systemic abnormalities through OS-related mechanisms and damaged renal and ovarian tissues. DHEA treatment in rat models is recommended to study the mechanisms that mediate PCOS-associated renal injury.

Synthesis of menarandroside A from dehydroepiandrosterone.

Menarandroside A, which bears a 12α-hydroxypregnenolone steroid backbone, was isolated from the plant, Cynanchum menarandrense. Treatment of extracts from this plant containing menarandroside A against secretin tumor cell line (STC-1) intestinal cells, resulted in an increased secretion of glucagon-like peptide 1 (GLP-1), a peptide that plays a role in the regulation of blood sugar levels. Increase in GLP-1 is beneficial for the treatment of type 2 diabetes. We disclose the synthesis of menarandroside A from dehydroepiandrosterone (DHEA). Key features of this synthesis include: (i) Wittig reaction of the C17-ketone of a 12-oxygenated DHEA derivative to introduce the C17-acetyl moiety, and (ii) the stereoselective reduction of a C12-keto intermediate bearing an sp2-center at C17 to yield the C12α-hydroxy group. In addition, an oxidation of a methyl enol ether derivative to an α-hydroxy methyl ester using tetrapropylammonium perruthenate (TPAP) and N-methyl-morpholine-N-oxide (NMO) was discovered.

PDE4 inhibitor Roflumilast modulates inflammation and lipid accumulation in PCOS mice to improve ovarian function and reduce DHEA-induced granulosa cell apoptosis in vitro.

This study was implemented to address the role of Roflumilast in polycystic ovary syndrome (PCOS) as well as to discuss its reaction mechanism in vivo and in vitro. In vivo, mice were administrated with 6 mg dehydroepiandrosterone (DHEA) per 100 g body weight and fed with 60% high fat diet to induce PCOS. The expression of phosphodiesterases 4 (PDE4) was assessed with RT-qPCR. The ovary pathology was observed by hematoxylin and eosin staining and follicles were counted. Enzyme-linked immunosorbent assay was adopted for the estimation of progesterone, testosterone and inflammatory factors and lipid accumulation was observed by Oil Red O staining. With the application of reverse transcription-quantitative PCR (RT-qPCR) and western blot, the messenger RNA (mRNA) and protein expressions of low-density lipoprotein receptor (LDLR) was resolved. In vitro, Cell counting kit-8 and flow cytometry analysis were applied for the assessment of cell proliferation and apoptosis. The proliferation- and apoptosis-related proteins were appraised with western blot. Additionally, the expressions of PDE-4 at both mRNA and protein levels were tested with RT-qPCR and western blot. Here, it was discovered that PDE4 was greatly elevated in PCOS mice and DHEA-induced ovarian granulosa cells (KGN). In PCOS mice, PDE4 was negative correlated with progesterone and had positive correlation with testosterone. Roflumilast could enhanced progesterone expression, increased the number of primary follicles, preantral follicles and antral follicles but reduced testosterone and decreased the number of cystic follicles in PCOS mice. It was also testified that Roflumilast could inhibit the release of inflammatory factors and lipid accumulation in PCOS mice. Besides, the proliferation of DHEA-induced KGN cells was enhanced while the apoptosis was declined by Roflumilast, accompanied by elevated contents of PCNA, Ki67 and antiapoptotic protein Bcl-2. Collectively, Roflumilast inhibited inflammation and lipid accumulation in PCOS mice to improve ovarian function and reduce DHEA-induced granulosa cell apoptosis.

Structure of human placental steroid sulfatase at 2.0 angstrom resolution: Catalysis, quaternary association, and a secondary ligand site.

Human placental estrone (E1)/dehydroepiandrosterone (DHEA) sulfatase (human placental steroid sulfatase; hSTS) is an integral membrane protein of the endoplasmic reticulum. This Ca2+-dependent enzyme catalyzes the hydrolysis of sulfate esters of E1 and DHEA to yield the respective unconjugated steroids, which then act as precursors for the biosynthesis of 17ß-estradiol (E2) and dihydrotestosterone (DHT), respectively, the most potent forms of estrogens and androgens. hSTS is a key enzyme for the local production of E2 and DHT in the breast and the prostate. The enzyme is known to be responsible for maintaining high levels of estrogens in the breast tumor cells. The crystal structure of hSTS purified from human placenta has previously been reported at 2.6 Å resolution. Here we present the structure of hSTS determined at the superior 2.0 Å resolution bringing new clarity to the atomic architecture of the active site. The molecular basis of catalysis and steroid-protein interaction are revisited in light of the new data. We also reexamine the enzyme's quaternary association and its implication on the membrane integration. A secondary ligand binding pocket at the intermolecular interface and adjacent to the active site access channel, buried into the gill of the mushroom-shaped molecule, has been identified. Its role as well as that of a phosphate ion bound to an exposed histidine side chain are examined from the structure-function perspective. Higher resolution data also aids in the tracing of an important loop missing in the previous structure.

Anti-polycystic ovary syndrome effect of electroacupuncture: IMD inhibits ER stress-mediated apoptosis and autophagy in granulosa cells.

Polycystic ovary syndrome (PCOS) is a complicated endocrinopathy affecting women at reproductive age. Increasing evidence has shown the anti-PCOS effect of electroacupuncture (EA), a modified approach of traditional Chinese medical therapy "acupuncture". However, the underlying mechanism of EA-alleviated PCOS waits further explored. In this study, experimental PCOS were induced in rats by dehydroepiandrosterone (DHEA) injection. Testosterone (T)-induced human ovarian granulosa cell (GC) line KGN was used to mimic PCOS in vitro. EA significantly alleviated histological changes and hormone disruption in PCOS rats. Besides, EA inhibited cell apoptosis, autophagy and the activation of endoplasmic reticulum (ER) stress-related PERK/eIF2α/ATF4/CHOP signaling in ovaries of PCOS rats. More interestingly, intermedin (IMD), a member of calcitonin gene-related peptide (CGRP), was evidently up-regulated in ovarian GCs after EA treatment, and its main bioactive form IMD1-53 suppressed cell apoptosis, autophagy and PERK/eIF2α/ATF4/CHOP signaling in T-induced KGN cells. Consistent with IMD1-53, ER stress inhibitor 4-PBA exerted an inhibitory effect on T-induced cell apoptosis and autophagy in KGN cells. Collectively, this study validates the protective effect of EA on DHEA-induced PCOS, and proposes that IMD relieved apoptosis and autophagy in T-induced granulosa cells via inhibiting ER stress.

Follicle isolation methods reveal plasticity of granulosa cell steroidogenic capacity during mouse in vitro follicle growth.

Follicles are the functional unit of the ovary and several methods have been developed to grow follicles ex vivo, which recapitulate key events of oogenesis and folliculogenesis. Enzymatic digestion protocols are often used to increase the yield of follicles from the ovary. However, the impact of these protocols on the outermost theca and granulosa cells, and thereby follicle function, is not well defined. To investigate the impact of enzymatic digestion on follicle function, we collected preantral follicles from CD1 mice either by enzymatic digestion (Enzy-FL) or mechanical isolation (Mech-FL) and compared follicle growth, steroidogenesis and cell differentiation within an encapsulated in vitro follicle growth system which maintains the 3D architecture of the oocyte and its surrounding somatic cells. Follicles were encapsulated in 0.5% alginate and cultured for 8 days. Compared with Enzy-FL, Mech-FL grew more rapidly and produced significantly higher levels of androstenedione, estradiol and progesterone. The expression of theca-interstitial cell marker genes, Cyp17a1, which encodes 17-hydroxylase/17, 20-lyase and catalyzes the hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, and the conversion of these products into dehydroepiandrosterone and androstenedione, and Star, which encodes a transport protein essential for cholesterol entry into mitochondria, were also higher in Mech-FL than in Enzy-FL. Mech-FL maintained an intact theca-interstitial layer on the outer edge of the follicle that phenocopied in vivo patterns as confirmed by alkaline phosphatase staining, whereas theca-interstitial cells were absent from Enzy-FL from the onset of culture. Therefore, preservation of the theca cell layer at the onset of culture better supports follicle growth and function. Interestingly, granulosa cells in the outermost layers of Enzy-FL expressed CYP17A1 by Day 4 of culture while maintaining inhibin α-subunit expression and a cuboidal nucleus. Thus, in the absence of theca-interstitial cells, granulosa cells have the potential to differentiate into androgen-producing cells. This work may have implications for human follicle culture, where enzymatic isolation is required owing to the density of the ovarian cortex.

Serum-Derived Exosomal microRNAs in Lipid Metabolism in Polycystic Ovary Syndrome.

The crosstalk between obesity and insulin resistance (IR) in polycystic ovary syndrome (PCOS) may be related to miRNA regulation secreted by exosomes. However, the underlying mechanism remains to be explored. A model of PCOS with IR was constructed in mice with dehydroepiandrosterone (DHEA) and a high-fat diet (HFD). Serum exosomes were extracted and characterized using transmission electron microscopy (TEM) and western blot analysis (for CD9, CD63, and CD81). The expression of miR-20b-5p and miR-106a-5p in serum exosomes was detected by qRT-PCR. The effects of serum exosomal miR-20b-5p and miR-106a-5p on lipid metabolism and ovary histological structure in PCOS model with IR were also explored. Serum exosomal miR-20b-5p and miR-106a-5p overexpression could inhibit adipocyte differentiation in 3T3-L1 cells with IR and PCOS mice model. Furthermore, the predicted targets of miR-20b-5p and miR-106a-5p were also analyzed with bioinformatics. In DHEA + HFD serum-derived exosomes, the miR-20b-5p and miR-106a-5p levels were markedly decreased. Overexpression of miR-20b-5p and miR-106a-5p alleviated adipocyte differentiation-related genes and triglyceride content in 3T3-L1 cells and liver steatosis in mice. Bioinformatics analysis of miR-20b-5p and miR-106a-5p predicted targets indicated that miR-20b-5p and miR-106a-5p were highly related to lipid metabolism. Serum-derived exosome miR-20b-5p and miR-106a-5p inhibited adipocyte differentiation during the process of PCOS with IR, which might be a novel therapeutic target.

PUFA-Derived N-Acylethanolamide Probes Identify Peroxiredoxins and Small GTPases as Molecular Targets in LPS-Stimulated RAW264.7 Macrophages.

We studied the mechanistic and biological origins of anti-inflammatory poly-unsaturated fatty acid-derived N-acylethanolamines using synthetic bifunctional chemical probes of docosahexaenoyl ethanolamide (DHEA) and arachidonoyl ethanolamide (AEA) in RAW264.7 macrophages stimulated with 1.0 µg mL-1 lipopolysaccharide. Using a photoreactive diazirine, probes were covalently attached to their target proteins, which were further studied by introducing a fluorescent probe or biotin-based affinity purification. Fluorescence confocal microscopy showed DHEA and AEA probes localized in cytosol, specifically in structures that point toward the endoplasmic reticulum and in membrane vesicles. Affinity purification followed by proteomic analysis revealed peroxiredoxin-1 (Prdx1) as the most significant binding interactor of both DHEA and AEA probes. In addition, Prdx4, endosomal related proteins, small GTPase signaling proteins, and prostaglandin synthase 2 (Ptgs2, also known as cyclooxygenase 2 or COX-2) were identified. Lastly, confocal fluorescence microscopy revealed the colocalization of Ptgs2 and Rac1 with DHEA and AEA probes. These data identified new molecular targets suggesting that DHEA and AEA may be involved in reactive oxidation species regulation, cell migration, cytoskeletal remodeling, and endosomal trafficking and support endocytosis as an uptake mechanism.

Inhibitory effect of bushen huoxue formula against dehydroepiandrosterone-induced inflammation in granulosa cells through TLR4/NF-κB signaling pathway.

Androgen exposure may be an important factor in promoting the development of polycystic ovary syndrome (PCOS) and disease progression. Bushen Huoxue Formula (BHF), a traditional Chinese medicine, is prescribed in clinical settings as a PCOS remedy, albeit with unclear pharmacological effects on granulosa cells. The present research explores potentially advantageous BHF impacts and whereby BHF alleviates dehydroepiandrosterone (DHEA)-induced inflammation and endocrine disruption. Six chemical components in BHF were identified and fingerprint analysis showed good reproducibility. Using a human granulosa cell line (KGN), BHF effects on cell viability, secretion of steroidogenic and inflammatory factors were evaluated and TLR4/NF-κB pathway expression was examined. Our results demonstrate that BHF treatment of KGN cells in a DHEA-induced inflammatory state led to increased cell viability, decreased testosterone and estradiol production, and decreased CYP19A1 and HSD3B2 mRNA expression. Further experiments revealed that BHF inhibited the expression of pro-inflammatory cytokines and considerably hindered up-regulation in protein levels of TLR4, MyD88, and TRAF6, while inhibiting the activation of NF-κB and phosphorylation of IκBα. Collectively, BHF administration protected granulosa cells from DHEA-induced injuries through down-regulating pro-inflammatory cytokines and blocking the pathway of TLR4/NF-κB. Therefore, BHF hold promise as a therapeutic formulation for preventing androgen induced PCOS.

Differential proteomics analysis of JEG-3 and JAR placental cell models and the effect of androgen treatment.

The placenta is a vital fetal organ that plays an important role in maintaining fetal sex hormone homeostasis. Xenobiotics can alter placental sex-steroidogenic enzymes and transporters, including enzymes such as aromatase (CYP19A1) and the hydroxysteroid dehydrogenases (HSDs) but studying how compounds disrupt in vivo placental metabolism is complex. Utilizing high-throughput in vitro models is critical to predict the disruption of placental sex-steroidogenic enzymes and transporters, particularly by drug candidates in the early stages of drug discovery. JAR and JEG-3 cells are the most common, simple, and cost-effective placental cell models that are capable of high-throughput screening, but how well they express the sex-steroidogenic enzymes and transporters is not well known. Here, we compared the proteomes of JAR and JEG-3 cells in the presence and absence of physiologically relevant concentrations of dehydroepiandrosterone (DHEA, 8 µM) and testosterone (15 nM) to aid the characterization of sex-steroidogenic enzymes and transporters in these cell models. Global proteomics analysis detected 2931 and 3449 proteins in JAR cells and JEG-3 cells, respectively. However, dramatic differences in sex-steroidogenic enzymes and transporters were observed between these cells. In particular, the basal expression of steroid sulfatase (STS), HSD17B1, and HSD17B7 were unique to JEG-3 cells. JEG-3 cells also showed significantly higher protein levels of aldo-keto reductase (AKR) 1A1 and AKR1B1, while JAR cells showed significantly higher levels of HSD17B4 and HSDB12. Aldehyde dehydrogenase (ALDH) 3A2 and HSD17B11 enzymes as well as the transporters sterol O-acyltransferase (SOAT) 1 and ATP binding cassette subfamily G2 (ABCG2) were comparable between the cell lines, whereas sulfotransferases (SULTs) were uniquely present within JAR cells. Androgen treatments significantly lowered HSD17B11, HSD17B4, HSD17B12, and ALDH3A2 levels in JAR cells. DHEA treatment significantly raised the level of HSD17B1 by 51 % in JEG-3 cells, whereas CYP19A1 was increased to significant levels in both JAR and JEG-3 cells after androgen treatments. The proteomics data were supported by a complementary targeted metabolomics analysis of culture media in the DHEA (8 µM) and testosterone (15 nM) treated groups. This study has indicated that untreated JEG-3 cells express more sex-steroidogenic enzymes and transporters. Nevertheless, JEG-3 and JAR cells are unique and their respective proteomics data can be used to select the best model depending on the hypothesis.

The Assessment of the Hypothalamic-Pituitary-Adrenal Axis After Oncological Treatment in Pediatric Patients with Acute Lymphoblastic Leukemia

Objective: Oncologic treatment can affect the adrenal glands, which in stressful situations may lead to life threatening adrenal crisis. The aim of the study was to assess adrenal function in pediatric acute lymphoblastic leukemia (ALL) survivors and to identify the best markers for this assessment. Methods: Forty-three ALL survivors, mean age 8.5±3.6 years and 45 age and sex-matched healthy controls were recruited to the study. ALL patients were assessed once within five years following oncological treatment completion. Fasting blood samples were collected from all participants to measure: fasting blood glucose (FBG); cortisol; aldosterone; plasma renin activity (PRA); dehydroepiandrostendione-sulfate (DHEA-S); and adrenocorticotropic hormone (ACTH). Moreover, diurnal profile of cortisol levels and 24-hour urinary free cortisol (UFC) were assessed. ALL survivors underwent a test with 1 ug of synthetic ACTH. Results: The study revealed lower level of PRA (1.94±0.98 ng/mL/h vs 3.61±4.85 ng/mL/h, p=0.029) and higher FBG (4.6±0.38 mmol/L vs 4.41±0.39 mmol/L, p=0.018) in the ALL group compared to controls. UFC correlated with evening cortisol (p=0.015, r=0.26), midnight cortisol (p=0.002, r=0.33), and DHEA-S (p=0.004, r=0.32). UFC also correlated with systolic and diastolic blood pressure (p=0.033, r=0.23 and p=0.005, r=0.31, respectively). The ACTH test confirmed impaired adrenal function in 4/43 ALL survivors (9%). Two of the patients who needed permanent hydrocortisone replacement had low UFC, midnight cortisol and DHEA-S levels. Conclusion: These results highlight the importance of reviewing adrenal gland functionality after chemo/radiotherapy in ALL survivors. DHEA-S proved to be a good marker to assess the adrenal glands after oncological therapy. Post-treatment disturbances of the adrenal axis could be associated with metabolic complications.