Dihydrotestosterone Augments the Angiogenic and Migratory Potential of Human Endothelial Progenitor Cells by an Androgen Receptor-Dependent Mechanism.

Endothelial progenitor cells (EPCs) play a critical role in cardiovascular regeneration. Enhancement of their native properties would be highly beneficial to ensuring the proper functioning of the cardiovascular system. As androgens have a positive effect on the cardiovascular system, we hypothesized that dihydrotestosterone (DHT) could also influence EPC-mediated repair processes. To evaluate this hypothesis, we investigated the effects of DHT on cultured human EPCs' proliferation, viability, morphology, migration, angiogenesis, gene and protein expression, and ability to integrate into cardiac tissue. The results showed that DHT at different concentrations had no cytotoxic effect on EPCs, significantly enhanced the cell proliferation and viability and induces fast, androgen-receptor-dependent formation of capillary-like structures. DHT treatment of EPCs regulated gene expression of androgen receptors and the genes and proteins involved in cell migration and angiogenesis. Importantly, DHT stimulation promoted EPC migration and the cells' ability to adhere and integrate into murine cardiac slices, suggesting it has a role in promoting tissue regeneration. Mass spectrometry analysis further highlighted the impact of DHT on EPCs' functioning. In conclusion, DHT increases the proliferation, migration, and androgen-receptor-dependent angiogenesis of EPCs; enhances the cells' secretion of key factors involved in angiogenesis; and significantly potentiates cellular integration into heart tissue. The data offer support for potential therapeutic applications of DHT in cardiovascular regeneration and repair processes.

Ellagic acid inhibits dihydrotestosterone-induced ferroptosis and promotes hair regeneration by activating the wnt/ß-catenin signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Androgenic alopecia (AGA) is the most prevalent form of hair loss in clinical practice and affects the physical and psychological well-being of adolescents. Paeonia lactiflora Pallas (PL), which is widely used in traditional Chinese medicine, enhances blood function and promotes hair growth, and ellagic acid (EA), a polyphenol in PL extract, shows strong antioxidant, anti-aging, and anti-inflammatory properties and also plays a role in the treatment of various skin conditions. However, its role and mechanism of action in AGA remain unclear. AIM OF THE STUDY: To determine whether EA can rescue slow hair regeneration by regulating dihydrotestosterone (DHT)-induced ferroptosis in AGA mice and clarify the effect of EA on DHT-induced ferroptosis in dermal papilla cells (DPCs). MATERIALS AND METHODS: Male C57BL/6 mice were used to establish a DHT-induced AGA mouse model, whereas DPCs were used to establish a DHT-induced cellular model. Thereafter, we investigated the therapeutic mechanism of action of EA via immunofluorescence, western blot analysis, immunohistochemistry, electron microscopy, and molecular docking. RESULTS: EA stimulated hair regeneration in mice and reversed DHT-induced increases in iron content, lipid peroxidation, and DHT-induced mitochondrial dysfunction by activating the Wnt/ß-catenin signaling pathway. Further, ß-catenin knockdown suppressed the inhibitory effect of EA on DHT-induced ferroptosis in DPCs. CONCLUSION: EA inhibits DHT-induced ferroptosis and promotes hair regrowth in mice by activating the Wnt/ß-catenin signaling pathway. Thus, it has potential for use as a treatment option for AGA.

Effects of Taraxaci Herba (Dandelion) on Testosterone Propionate-Induced Benign Prostatic Hyperplasia in Rats.

Benign prostatic hyperplasia (BPH) is the non-malignant enlargement of the prostate, associated with lower urinary tract symptoms (LUTSs). Taraxaci Herba (TH), commonly known as dandelion, has traditionally been utilized in East Asia to treat symptoms related to LUTSs. Based on this traditional use, our study aimed to explore the inhibitory effects of TH on BPH progression using a testosterone propionate-induced rat model. To induce BPH, male Sprague Dawley rats were castrated and injected subcutaneously with testosterone propionate (3 mg/kg/day) for 28 days. Concurrently, TH extract was administered orally at doses of 100 and 300 mg/kg/day throughout the four-week period of testosterone propionate injections. The TH extract significantly reduced both the absolute and relative weights of the prostate, along with histopathological changes in the gland. Moreover, it lowered serum levels of testosterone and dihydrotestosterone and reduced the expression of the androgen receptor in the prostate. Additionally, the TH extract modulated the protein expressions of Bax and Bcl-2, which are key regulators of apoptosis in prostate cells. Collectively, our findings suggest that TH inhibits BPH development partially by modulating androgen signaling and inducing apoptosis within the prostate.

[Natural 5α-reductase inhibitors in treatment of benign prostatic hyperplasia].

Benign prostatic hyperplasia(BPH) is a common disease of the male urinary system, and its incidence rate in China is increasing. However, the mechanism underlying the pathogenesis of BPH remains unclear. Some studies demonstrated that the incidence of BPH was related to the change in the levels of steroid hormones. Too high content of dihydrotestosterone(DHT) in the body may cause BPH and other related diseases. Testosterone(T) is converted to DHT by 5α-reductase(SRD5A). By inhibiting the activity of this enzyme, the production of DHT can be reduced, and then the incidence of BPH can be lowered. Therefore, it has drawn great attention to screen and discover safer and more effective 5α-reductase inhibitors from natural medicines to treat prostatic hyperplasia without affecting the physiological function of men. This review summarizes the characteristics and tissue distribution of 5α-reductase, the discovery of 5α-reductase inhibitors in traditional Chinese medicine and natural medicines, 5α-reductase inhibitors commonly used in clinical practice and their side effects, as well as the animal models of prostatic hyperplasia and common detection indicators, aiming to provide a reference for more in-depth understanding and research about BPH and development of drugs.

Synergistic therapeutic effect of ginsenoside Rg3 modified minoxidil transfersomes (MXD-Rg3@TFs) on androgenic alopecia in C57BL/6 mice.

Inflammation in hair follicles will reduce the effectiveness of minoxidil (MXD) in the treatment of androgen alopecia (AGA) caused by elevated androgen levels. To target multiple physiological and pathological processes in AGA, a novel natural bioactive compound modified transfersomes (MXD-Rg3@TFs) was prepared to replace cholesterol that may disrupt hair growth, with ginsenosides Rg3 (Rg3) that have anti-inflammatory effects on AGA. The effects of MXD, Rg3 and their combination on AGA were evaluated using dihydrotestosterone (DHT) induced human dermal papilla cells (DPCs), and the results showed that the combination of MXD and Rg3 can significantly promote the proliferation, reduce the level of intracellular ROS and inflammatory factors, and inhibit the aging of DHT induced DPCs. Compared with cholesterol membrane transfersomes (MXD-Ch@TFs), MXD-Rg3@TFs has similar deformability, smaller particle size and better stability. MXD-Rg3@TFs has also significant advantages in shortening telogen phase and prolonging the growth period of hair follicles in C57BL/6 mice than MXD-Ch@TFs and commercial MXD tincture. The prominent ability of MXD-Rg3@TFs to inhibit the conversion of testosterone to DHT and reduce the level of inflammatory factors suggested that Rg3 and MXD in MXD-Rg3@TFs have synergistic effect on AGA therapy. MXD-Ch@TFs with no irritation to C57BL/6 mice skin is expected to reduce the dose of MXD and shorten the treatment time, which would undoubtedly provide a promising therapeutic option for treatment of AGA.

Dual deficiency of melatonin and dihydrotestosterone promotes stromal cell damage and mediates prostatitis via the cGAS-STING pathway in sleep-deprived mice.

PURPOSE: Prostatitis is a highly prevalent condition that seriously affects men's physical and mental health. Although epidemiological investigations have provided evidence of a correlation between insufficient sleep and prostatitis, the pathogenesis of prostatitis remains unclear. We sought to identify the underlying mechanism involved and identify a promising therapeutic target. METHODS: Sleep deprivation (SD) was utilized to establish a mouse model of insufficient sleep in a special device. Prostatitis was observed at different time points post-SD. The degree of prostatitis was evaluated by pathological section and behavioural tests. Using immunofluorescence, western blot, and proteomic analyses, the underlying mechanism of SD-related prostatitis was investigated, and the development and therapeutic target of prostatitis were elucidated. RESULTS: SD, as an initial pathological trigger, resulted in a reduction in dihydrotestosterone and melatonin levels. Proteomic analysis revealed that the cGAS-STING pathway may play a significant role in inducing prostatitis. The subsequent results illustrated that the dual reduction in dihydrotestosterone and melatonin led to an accumulation of reactive oxygen species and the release of mitochondrial DNA (mt-DNA). The accumulation of mt-DNA activated the cGAS-STING pathway, which recruited inflammatory cells into the prostatic stroma through the secretion of interferon-ß. Consequently, an inflammatory microenvironment was formed, ultimately promoting the development of prostatitis. Notably, mice with SD-induced prostatitis gradually recovered to a normal state within 7 days of recovery sleep. However, after being subjected to SD again, these mice tended to have a more pronounced manifestation of prostatitis within a shorter timeframe, which suggested that prostatitis is prone to relapse. CONCLUSIONS: The cGAS-STING pathway activated by dual deficiency of dihydrotestosterone and melatonin plays a comprehensive inflammatory role in SD-related prostatitis. This research provides valuable insights into the pathogenesis, therapeutic targets, and prevention strategies of prostatitis.

Deciphering the mechanisms and interactions of the endocrine disruptor bisphenol A and its analogs with the androgen receptor.

Bisphenol A (BPA) and its various forms used as BPA alternatives in industries are recognized toxic compounds and antiandrogenic endocrine disruptors. These chemicals are widespread in the environment and frequently detected in biological samples. Concerns exist about their impact on hormones, disrupting natural biological processes in humans, together with their negative impacts on the environment and biotic life. This study aims to characterize the interaction between BPA analogs and the androgen receptor (AR) and the effect on the receptor's normal activity. To achieve this goal, molecular docking was conducted with BPA and its analogs and dihydrotestosterone (DHT) as a reference ligand. Four BPA analogs exhibited higher affinity (-10.2 to -8.7 kcal/mol) for AR compared to BPA (-8.6 kcal/mol), displaying distinct interaction patterns. Interestingly, DHT (-11.0 kcal/mol) shared a binding pattern with BPA. ADMET analysis of the top 10 compounds, followed by molecular dynamics simulations, revealed toxicity and dynamic behavior. Experimental studies demonstrated that only BPA disrupts DHT-induced AR dimerization, thereby affecting AR's function due to its binding nature. This similarity to DHT was observed during computational analysis. These findings emphasize the importance of targeted strategies to mitigate BPA toxicity, offering crucial insights for interventions in human health and environmental well-being.

[Application effect of a dual release system of androgen and its antagonist in the repair of full-thickness burn wounds in mice].

Objective: To explore the optimal ratio of dihydrotestosterone and hydroxyflutamide (hereinafter referred to as DH), construct a dual release system of androgen and its antagonist, and analyze the application effect of this system in the repair of full-thickness burn wounds in mice. Methods: This study was an experimental study. The HaCaT cells were divided into blank group (without drug culture), low baseline group, medium baseline group, and high baseline group according to the random number table (the same grouping method below), and the last three groups of cells were cultured by adding three different ratios of DH. Under a medium ratio, the mass of dihydrotestosterone in the three baseline groups from low to high was 1.4, 2.8, and 4.0 µg, respectively, and the mass of hydroxyflutamide was 1.2, 1.6, and 2.0 µg, respectively. On this basis, under a small ratio, the mass of dihydrotestosterone was reduced by half and the mass of hydroxyflutamide was increased by half; under a large ratio, the mass of dihydrotestosterone was increased by half and the mass of hydroxyflutamide was reduced by half. After culture of 2 days, the cell proliferation level was detected by cell counting kit 8 (n=4). Sixteen 6-8-week-old male BALB/c mice were used to establish a full-thickness burn wound on the back and divided into blank group, small ratio group, medium ratio group, and large ratio group, with 4 mice in each group. On post injury day (PID) 7, normal saline containing different ratios of DH was locally dropped to the wounds of mice in the last three groups of mice (the total mass of DH in the three ratio groups from small to large was 127.5, 165.0, and 202.5 µg, respectively, and the mass ratios of dihydrotestosterone to hydroxyflutamide (hereinafter referred to as drug mass ratio) were 8∶9, 8∶3, and 8∶1, respectively), afterwards, the administration was repeated every 48 hours until PID 27; normal saline was dropped to the wound of mice in blank group at the aforementioned time points. The wound healing status on PID 0 (immediately), 7, 14, 21, and 28 was observed, and the wound healing rates on PID 7, 14, 21, and 28 were calculated (n=4). On PID 28, the wound tissue was taken, which was stained with hematoxylin and eosin for observing re-epithelialization and with Masson for observing collagen fibers, and the proportion of collagen fibers was analyzed (n=3). Twenty 6-8-week-old male BALB/c mice were used to establish a full-thickness burn wound on the back and divided into ordinary scaffold group, small proportion scaffold group, medium proportion scaffold group, and large proportion scaffold group (with 5 mice in each group). On PID 7, the wound was continuously dressed with a polycaprolactone scaffold without drug and a polycaprolactone scaffold containing DH with a drug mass ratio of 1∶3, 1∶1, or 3∶1 (i.e. the dual release system of androgen and its antagonist, with total mass of DH being about 1.7 mg) prepared by using electrospinning technology until the end of the experiment. Histopathological analyses of tissue (n=3) at the same time points as those in the previous animal experiment were performed. On PID 7 and 14, the wound exudates were collected and the relative abundance of bacterial communities was analyzed using 16S ribosomal RNA high-throughput sequencing (n=3). Results: After culture of 2 days, under a small ratio, the proliferation levels of HaCaT cells in low baseline group and high baseline group were significantly higher than the level in blank group (P<0.05). As the time after injury prolonged, the wounds of all four groups of mice continued to shrink. On PID 14, the wound healing rate of mice in large ratio group was 72.5% (61.7%, 75.1%), which was close to 53.3% (49.5%, 64.4%) in blank group (P>0.05); the wound healing rates of mice in small and medium ratio groups were 74.2% (71.0%, 84.2%) and 70.4% (65.1%, 74.4%), respectively, which were significantly higher than the rate in blank group (with both Z values being -2.31, P<0.05). On PID 21, the wound healing rate of mice in small ratio group was significantly higher than that in blank group (Z=-2.31, P<0.05). On PID 28, the wounds of mice in the three ratio groups were completely re-epithelialized and the epidermis was thicker than that in blank group; compared with that in blank group, the collagen fiber content in the wound tissue of mice in the three ratio groups was higher and arranged more orderly, and the proportions of collagen fibers in the wound tissue of mice in small and large ratio groups were significantly increased (P<0.05). On PID 28, the wounds of mice in ordinary scaffold group were partially epithelialized, while the wounds of mice in the three proportion scaffold groups were almost completely epithelialized. Among them, the wounds of mice in small proportion scaffold group had the thickest epidermis. The proportion of collagen fibers in the wound tissue of mice in small proportion scaffold group was significantly increased compared with that in ordinary scaffold group (P<0.05). On PID 7, the bacterial communities with high relative abundance in the wound exudation of mice in the four groups included bacteria of Corynebacterium, Staphylococcus, and Rhodococcus. On PID 14, the bacterial communities with high relative abundance in the wound exudation of mice in the four groups included bacteria of Stenotrophomonas, Rhodococcus, and Staphylococcus, and the number of bacterial species in the wound exudation of mice in the three proportion scaffold groups was more than that in ordinary scaffold group. Conclusions: When the drug mass ratio is relatively small, DH has the effect of promoting the proliferation of HaCaT cells. The ratio of 8∶9 is the optimal mass ratio of dihydrotestosterone to hydroxyflutamide, and DH with this mass ratio can promote re-epithelialization and collagen deposition of full-thickness burn wounds in mice, and promote wound healing. The constructed dual release system of androgen and its antagonist with DH in a 1∶3 drug mass ratio contributes to the re-epithelialization and collagen deposition of the full-thickness burn wounds in mice, and can improve the diversity of wound microbiota.

Dihydrotestosterone reduces neuroinflammation in spinal cord injury through NF-κB and MAPK pathway.

Neuroinflammation induced by microglia following spinal cord injury (SCI) leads to secondary neurologic injury. Androgens including testosterone and dihydrotestosterone (DHT) show as endogenous neuroprotective factors against multiple neurologic diseases, while their therapeutic role in SCI-induced neuroinflammation and underlying mechanism remains elusive. In the study, we aimed to investigate the role of DHT against microglia-induced neuroinflammation in SCI and evaluate its protective treatment. BV2 cells were activated by neuroinflammation via LPS in vitro. Adult male C57BL/6 mice were used to establish the SCI model. BV2 cells and SCI mice were administrated DHT. Microglia activation, pro-inflammatory factors, p38 and p65 phosphorylation, glial scar, fibrotic scar, histology, and locomotor function recovery were measured, respectively. We demonstrated that DHT administration attenuates neuroinflammation in microglia through inhibition of p38 and p65 pathways. Moreover, DHT reduces microglia and astrocyte accumulation, cord fibrosis and histologic damage. Besides, DHT ameliorates locomotor functional recovery after SCI. DHT is verified to play a neuroprotective role in SCI, which fights against neuroinflammation by inhibition of p38 and p65 pathways. Therefore, Mel is defined as a promising factor in protecting neural tissue after SCI.

Androgen receptor actions on AgRP neurons are not a major cause of reproductive and metabolic impairments in peripubertally androgenized mice.

Excess levels of circulating androgens during prenatal or peripubertal development are an important cause of polycystic ovary syndrome (PCOS), with the brain being a key target. Approximately half of the women diagnosed with PCOS also experience metabolic syndrome; common features including obesity, insulin resistance and hyperinsulinemia. Although a large amount of clinical and preclinical evidence has confirmed this relationship between androgens and the reproductive and metabolic features of PCOS, the mechanisms by which androgens cause this dysregulation are unknown. Neuron-specific androgen receptor knockout alleviates some PCOS-like features in a peripubertal dihydrotestosterone (DHT) mouse model, but the specific neuronal populations mediating these effects are undefined. A candidate population is the agouti-related peptide (AgRP)-expressing neurons, which are important for both reproductive and metabolic function. We used a well-characterised peripubertal androgenized mouse model and Cre-loxP transgenics to investigate whether deleting androgen receptors specifically from AgRP neurons can alleviate the induced reproductive and metabolic dysregulation. Androgen receptors were co-expressed in 66% of AgRP neurons in control mice, but only in <2% of AgRP neurons in knockout mice. The number of AgRP neurons was not altered by the treatments. Only 20% of androgen receptor knockout mice showed rescue of DHT-induced androgen-induced anovulation and acyclicity. Furthermore, androgen receptor knockout did not rescue metabolic dysfunction (body weight, adiposity or glucose and insulin tolerance). While we cannot rule out developmental compensation in our model, these results suggest peripubertal androgen excess does not markedly influence Agrp expression and does not dysregulate reproductive and metabolic function through direct actions of androgens onto AgRP neurons.

ß-Nicotinamide Mononucleotide Promotes Cell Proliferation and Hair Growth by Reducing Oxidative Stress.

ß-Nicotinamide mononucleotide (NMN) has shown promising effects on intestinal health, and it is extensively applied as an anti-aging and Alzheimer's disease therapeutic, due to its medicinal properties. The effects of NMN on the growth of mouse hair were observed after hair removal. The results indicated that NMN can reverse the state of hair follicle atrophy, hair thinning, and hair sparsity induced by dihydrotestosterone (DHT), compared to that of minoxidil. In addition, the action mechanisms of NMN promoting hair growth in cultured human dermal papilla cells (HDPCs) treated with DHT were investigated in detail. The incubation of HDPCs with DHT led to a decrease in cell viability and the release of inflammatory mediators, including interleukin-6 (IL-6), interleukin-1Beta (IL-1ß) and tumor necrosis factor Alpha (TNF-α). It was found that NMN can significantly lower the release of inflammatory factors induced by DHT in HDPCs. HDPCs cells are protected from oxidative stress damage by NMN, which inhibits the NF-κB p65 inflammatory signaling pathway. Moreover, the levels of androgen receptor (AR), dickkopf-1 (DKK-1), and ß-catenin in the HDPCs were assessed using PCR, indicating that NMN can significantly enhance the expression of VEGF, reduced IL-6 levels and suppress the expression of AR and DKK-1, and notably increase ß-catenin expression in DHT-induced HDPCs.

Determination of urinary androgens in women with polycystic ovary syndrome using LC-QqQ/MS and the application of thin film solid-phase microextraction (TF-SPME).

Hyperandrogenism is one of the most pronounced symptoms of Polycystic Ovary Syndrome (PCOS) and seems to play a key role in the pathogenesis of this complex disorder. Nevertheless, there is still a lack of consistent results regarding common steroid predictors of PCOS. Therefore, a liquid chromatography tandem mass spectrometry (HPLC-QqQ/MS) method was developed and validated to determine the concentrations of four classic androgens: androstenedione (An-dione), testosterone (T), 5α-dihydrotestosterone (DHT) and androsterone (An) in urine samples obtained from women with PCOS and healthy controls. The limits of detection were between 0.04 and 0.09 ng/mL, while the limits of quantification ranged from 0.1 to 0.3 ng/mL respectively. As a pre-treatment procedure prior to analysis, hydrolysis using ß-glucuronidase and thin film solid-phase microextraction (TF-SPME) was applied. The methodology was employed to perform targeted metabolomics of urinary steroids in women with PCOS and healthy controls. All measured androgens: An-dione (p < 0.0001), T (p = 0.0001), DHT (p < 0.0001) and An (p = 0.0002) showed significantly higher concentrations in the urine of women with PCOS. The largest difference in the mean concentration was found for DHT, which was 2.8 times higher in the PCOS group (13.9 ± 14.1 ng/mg creatinine) in comparison to healthy controls (4.9 ± 3.4 ng/mg creatinine). The results of receiver operating characteristic curve indicated that determination of the panel of three urinary androgens: T+DHT+An-dione with, under the study assumptions, was the best predictor of PCOS diagnosis (AUC of ROC curve = 0.91 (95 % CI: 0.8212-0.9905). The application of an LC-MS/MS-based analysis, together with highly sensitive extraction techniques like TF-SPME, is a suitable approach to perform fast assays and obtain reliable results - crucial in the search for valuable and significant steroids predictors of PCOS.

Therapeutic maintenance effect of finasteride 1 mg every other month regimen in androgenetic alopecia and study on the difference in response to finasteride treatment: A prospective cohort study.

Finasteride is commonly used for androgenetic alopecia (AGA) treatment. The aim of this study was to assess the therapeutic maintenance effect of a finasteride every other month (EOM) regimen and analyze clinical and laboratory differences in patients with AGA according to their treatment response. One hundred males with AGA who received finasteride 1 mg daily treatment for a year were enrolled in the study. At 1 year follow-up, treatment responses of patients who completed the visit schedule were assessed using five scales. The patients were assigned to good or bad response groups according to their assessment. Further, they were randomly divided into two groups (daily vs. EOM) and treated with finasteride (1 mg) for 1 more year. At 2 years follow-up, treatment efficacy was assessed. At 1-year follow-up, 36 patients completed the schedule, including eight and three patients in the good and bad response groups, respectively. At the 2-year follow-up, 23 patients completed the schedule, with nine in the daily group and 14 in the EOM group. Changes in global photographic assessment in the second year were 1.33 and 1.29 for the daily and EOM groups, respectively. The daily group showed an elevated hair density and lower concentration of dihydrotestosterone (DHT) and the DHT to testosterone ratio (DHT/T). However, the EOM group showed decreased hair density and elevated DHT and DHT/T. Following treatment response assessment after 1 year of treatment, the good response group showed early onset which was associated with maternal AGA. Analysis of serum androgen hormone magnitude of DHT reduction was much greater (54.4% vs. 44.4%). DHT/T was higher in the bad response group (1.98 vs. 2.33). We concluded that the finasteride EOM regimen showed similar maintenance effects to the daily regimen.

A plasma membrane-associated form of the androgen receptor enhances nuclear androgen signaling in osteoblasts and prostate cancer cells.

Androgen binding to the androgen receptor (AR) in the cytoplasm induces the AR to translocate to the nucleus, where it regulates the expression of target genes. Here, we found that androgens rapidly activated a plasma membrane-associated signaling node that enhanced nuclear AR functions. In murine primary osteoblasts, dihydrotestosterone (DHT) binding to a membrane-associated form of AR stimulated plasma membrane-associated protein kinase G type 2 (PKG2), leading to the activation of multiple kinases, including ERK. Phosphorylation of AR at Ser515 by ERK increased the nuclear accumulation and binding of AR to the promoter of Ctnnb1, which encodes the transcription factor ß-catenin. In male mouse osteoblasts and human prostate cancer cells, DHT induced the expression of Ctnnb1 and CTNN1B, respectively, as well as ß-catenin target genes, stimulating the proliferation, survival, and differentiation of osteoblasts and the proliferation of prostate cancer cells in a PKG2-dependent fashion. Because ß-catenin is a master regulator of skeletal homeostasis, these results explain the reported male-specific osteoporotic phenotype of mice lacking PKG2 in osteoblasts and imply that PKG2-dependent AR signaling is essential for maintaining bone mass in vivo. Our results suggest that widely used pharmacological PKG activators, such as sildenafil, could be beneficial for male and estrogen-deficient female patients with osteoporosis but detrimental in patients with prostate cancer.

Troxerutin dampened hypothalamic neuroinflammation via microglial IL-22/IL-22R1/IRF3 activation in dihydrotestosterone-induced polycystic ovary syndrome rats.

BACKGROUND: Polycystic ovary syndrome (PCOS) is the most common reproductive-endocrine condition in premenopausal women. Troxerutin, a common clinical anti-coagulant agent, was shown to work as a strong IL-22 boosting agent counteracting the hyperactivated gonadotrophin releasing hormone (GnRH) neurons and heightened GnRH release, the neuroendocrine origin of PCOS with unknown mechanism in rats. Exploring the off-label use of troxerutin medication for PCOS is thus sorely needed. METHODS: Serum IL-22 content and hypothalamic IL-22 protein were detected. Inflammatory factor levels in hypothalamo-pituitary were evaluated. Immunofluorescence staining was employed to determine the activation and M1/M2-prone polarization of microglia in arcuate hypothalamus and median eminence. RNA-sequencing and transcriptome analysis were applied to explore the potential driver of microglia M2-polarization in response to IL-22 bolstering effect. The function of microglial IL-22/IL-22R1/IRF3 system was further verified using in vivo knockdown of IL-22R1 and a potent IRF3 inhibitor in BV2 microglial cell lines in vitro. RESULTS: Troxerutin augmented serum IL-22 content, and its consequent spillover into the hypothalamus led to the direct activation of IL-22R1/IRF3 system on microglia, thereby promoted microglia M2 polarization in arcuate hypothalamus and median eminence, dampened hypothalamic neuroinflammation, inhibited hyperactive GnRH and rescued a breadth of PCOS-like traits in dihydrotestosterone (DHT) rats. The salutary effects of troxerutin treatment on hypothalamic neuroinflammation, microglial M1/2 polarization, GnRH secretion and numerous PCOS-like features were blocked by in vivo knockdown of IL-22R1. Moreover, evidence in vitro illustrated that IL-22 supplement to BV-2 microglia cell lines promoted M2 polarization, overproduction of anti-inflammatory marker and limitation of pro-inflammatory factors, whereas these IL-22 effects were blunted by geldanamycin, a potent IRF3 inhibitor. CONCLUSION: Here, the present study reported the potential off-label use of troxerutin medication, a common clinical anti-coagulant agent and an endogenous IL-22 enhancer, for multiple purposes in PCOS. The rational underlying the application of troxerutin as a therapeutic choice in PCOS derived from its activity as an IL-22 memetic agent targeting the neuro-endocrine origin of PCOS, and its promotive impact on microglia M2 polarization via activating microglial IL-22R1/IRF3 system in the arcuate hypothalamus and median eminence of DHT female rats.

Dihydrotestosterone induces arterial stiffening in female mice.

BACKGROUND: Androgens are important sex hormones in both men and women and are supplemented when endogenous levels are low, for gender transitioning, or to increase libido. Androgens also circulate at higher levels in women with polycystic ovarian syndrome, a condition that increases the risk for cardiovascular diseases including hypertension and arterial stiffness. Since our previous work shows an important role for the G protein-coupled estrogen receptor (GPER) in arterial stiffness, we hypothesized that other hormones including androgens may impact arterial stiffness in female mice via downregulation of GPER. METHODS: The impact of the non-aromatizable androgen dihydrotestosterone (DHT), the glucocorticoid dexamethasone, and the progestin medroxyprogesterone acetate (all 100 nM for 24 h) on GPER and ERα expression was assessed in cultured vascular smooth muscle cells using droplet digital PCR (ddPCR). To assess the in vivo impact of the DHT-induced downregulation of GPER, female ovary-intact C57Bl/6 mice at 15-16 weeks of age were treated with silastic capsules containing DHT for 4 weeks, one with a dosage expected to mimic human male DHT levels and another to double the expected human concentration (n = 8-9/group). RESULTS: In cultured vascular smooth muscle cells, GPER mRNA was decreased by DHT (P = 0.001) but was not impacted by dexamethasone or medroxyprogesterone. In contrast, ERα expression in cultured cells was significantly suppressed by all three hormones (P < 0.0001). In control mice or mice treated with a single or double dose of DHT, a dose-dependent increase in body weight was observed (control 22 ± 2 g, single dose 24 ± 2 g, double dose 26 ± 2 g; P = 0.0002). Intracarotid stiffness measured via pulse wave velocity showed a more than two-fold increase in both DHT-treated groups (control 1.9 ± 0.3 m/s, single dose 4.3 ± 0.8 m/s, double dose 4.8 ± 1.0 m/s). This increase in arterial stiffness occurred independent of changes in blood pressure (P = 0.59). Histological analysis of aortic sections using Masson's trichrome showed a significant decrease in collagen between the control group (24 ± 5%) and the double dose group (17 ± 3%, P = 0.007), despite no changes in aortic wall thickness or smooth muscle content. Lastly, ddPCR showed that in vivo DHT treatment decreased aortic expression of both GPER (control 20 ± 5, single dose 10.5 ± 5.6, double dose 10 ± 4 copies/ng; P = 0.001) and ERα (control 54 ± 2, single dose 24 ± 13, and double dose 23 ± 12 copies/ng; P = 0.003). CONCLUSIONS: These findings indicate that androgen promotes arterial stiffening and cardiovascular damage in female mice and is associated with decreased estrogen receptor expression. These data are important for transgender men, women using testosterone for fitness or reduced libido, as well as patients with polycystic ovarian syndrome.

The current study investigated the impact of other hormones on estrogen receptor expression and its impact on vascular health. In both cultured vascular cells and in vivo vascular tissue, dihydrotestosterone decreased the expression of estrogen receptors. Female mice treated with dihydrotestosterone also displayed increased body weight and arterial stiffness despite no change in blood pressure. These findings indicate that increases in testosterone may impact vascular health, which may be important clinically for transgender men, women using testosterone for fitness or reduced libido, as well as patients with polycystic ovarian syndrome.

Changes in pituitary gonadotropin subunits and hypothalamic Kiss-1 gene expression by administration of sex steroids in ovary-intact female rats.

OBJECTIVE: We examined how the sex steroids influence the synthesis of gonadotropins. MATERIALS AND METHODS: The effects of sex steroids estradiol (E2), progesterone (P4), and dihydrotestosterone (DHT) in pituitary gonadotroph cell model (LßT2 cells) in vitro and ovary-intact rats in vivo were examined. The effects of sex steroids on Kiss1 gene expression in the hypothalamus were also examined in ovary-intact rats. RESULTS: In LßT2 cells, E2 increased common glycoprotein alpha (Cga) and luteinizing hormone beta (Lhb) subunit promoter activity as well as their mRNA expression. Although gonadotropin subunit promoter activity was not modulated by P4, Cga and Lhb mRNA expression was increased by P4. DHT inhibited Cga and Lhb mRNA expression with a concomitant decrease in their promoter activity. During the 2-week administration of exogenous E2 to ovary-intact rats, the estrous cycle determined by vaginal smears was disrupted. P4 or DHT administration completely eliminated the estrous cycle. Protein expression of all three gonadotropin subunits within the pituitary gland was inhibited by E2 or P4 treatment in vivo; however, DHT reduced Cga expression but did not modulate Lhb or follicle-stimulating hormone beta subunit expression. E2 administration significantly repressed Kiss1 mRNA expression in a posterior hypothalamic region that included the arcuate nucleus. P4 and DHT did not modulate Kiss1 mRNA expression in this region. In contrast, P4 administration significantly inhibited Kiss1 mRNA expression in the anterior region of the hypothalamus that included the anteroventral periventricular nucleus. The expression of gonadotropin-releasing hormone (Gnrh) mRNA in the anterior hypothalamic region, where the preoptic area is located, appeared to be decreased by treatment with E2 and P4. CONCLUSION: Our findings suggest that sex steroids have different effects in the hypothalamus and pituitary gland.

Exploring rat corpus cavernosum alterations induced by finasteride treatment and withdrawal.

Despite its efficacy for treating androgenetic alopecia, finasteride, an inhibitor of 5α-reductase (i.e., the enzyme converting testosterone, T, into dihydrotestosterone, DHT), is associated with several side effects including sexual dysfunction (e.g., erectile dysfunction). These side effects may persist after drug suspension, inducing the so-called post-finasteride syndrome (PFS). The effects of subchronic treatment with finasteride (i.e., 20 days) and its withdrawal (i.e., 1 month) in rat corpus cavernosum have been explored here. Data obtained show that the treatment was able to decrease the levels of the enzyme 5α-reductase type II in the rat corpus cavernosum with increased T and decreased DHT levels. This local change in T metabolism was linked to mechanisms associated with erectile dysfunction. Indeed, by targeted metabolomics, we reported a decrease in the nitric oxide synthase (NOS) activity, measured by the citrulline/arginine ratio and confirmed by the decrease in NO2 levels, and a decrease in ornithine transcarbamylase (OTC) activity, measured by citrulline/ornithine ratio. Interestingly, the T levels are negatively correlated with NOS activity, while those of DHT are positively correlated with OTC activity. Finasteride treatment also induced alterations in the levels of other molecules involved in the control of penile erection, such as norepinephrine and its metabolite, epinephrine. Indeed, plasma levels of norepinephrine and epinephrine were significantly increased and decreased, respectively, suggesting an impairment of these mediators. Interestingly, these modifications were restored by suspension of the drug. Altogether, the results reported here indicate that finasteride treatment, but not its withdrawal, affects T metabolism in the rat corpus cavernosum, and this alteration was linked to mechanisms associated with erectile dysfunction. Data here reported could also suggest that the PFS sexual side effects are more related to dysfunction in a sexual central control rather than peripheral compromised condition.

Alcohol does not influence trust in others or oxytocin, but increases positive affect and risk-taking: a randomized, controlled, within-subject trial.

BACKGROUND: Alcohol consumption to facilitate social interaction is an important drinking motive. Here, we tested whether alcohol influences trust in others via modulation of oxytocin and/or androgens. We also aimed at confirming previously shown alcohol effects on positive affect and risk-taking, because of their role in facilitating social interaction. METHODS: This randomized, controlled, within-subject, parallel group, alcohol-challenge experiment investigated the effects of alcohol (versus water, both mixed with orange juice) on perceived trustworthiness via salivary oxytocin (primary and secondary endpoint) as well as testosterone, dihydrotestosterone, positive affect, and risk-taking (additional endpoints). We compared 56 male participants in the alcohol condition (1.07 ± 0.18 per mille blood alcohol concentration) with 20 in the control condition. RESULTS: The group (alcohol versus control condition) × time (before [versus during] versus after drinking) interactions were not significantly associated with perceived trustworthiness (η2 < 0.001) or oxytocin (η2 = 0.003). Bayes factors provided also substantial evidence for the absence of these effects (BF01 = 3.65; BF01 = 7.53). The group × time interactions were related to dihydrotestosterone (η2 = 0.018 with an increase in the control condition) as well as positive affect and risk-taking (η2 = 0.027 and 0.007 with increases in the alcohol condition), but not significantly to testosterone. DISCUSSION: The results do not verify alcohol effects on perceived trustworthiness or oxytocin in male individuals. However, they indicate that alcohol (versus control) might inhibit an increase in dihydrotestosterone and confirm that alcohol amplifies positive affect and risk-taking. This provides novel mechanistic insight into social facilitation as an alcohol-drinking motive.