Dexmedetomidine improves the acute stress reactivity of male rat through interventions of serum- and glucocorticoid-inducible kinase 1 and nNOS in the bed nucleus of the stria terminalis.

Moderate acute stress responses are beneficial for adaptation and maintenance of homeostasis. Exposure of male rat to stress induces effects in the bed nucleus of the stria terminalis (BNST), for it can be activated by the same stimuli that induce activation of the hypothalamic-pituitary-adrenal axis. However, the underlying mechanism of the BNST on male stress reactivity remains unclear. In this study, we explored whether systematic administration of dexmedetomidine (DEXM) altered the acute stress reactivity through its effect on the BNST. Male Sprague-Dawley rats in the stress (STRE) group, DEXM group, and the DEXM + GSK-650394 (GSK, an antagonist of serum- and glucocorticoid-inducible kinase 1 (SGK1)) group, except those in the vehicle (VEH) group, underwent 1-h restraint plus water-immersion (RPWI) exposure. All the rats proceeded the open field test (OFT) 24 h before RPWI and 1 h after RPWI. After the second OFT, the rats received VEH, DEXM (75 µg/kg i.p.), or were pretreated with GSK (2 µM i.p.) 0.5 h ahead of DEXM respectively. The third OFT was conducted 6 h after drug administration and then the rats were sacrificed. The rats that experienced RPWI showed dramatically elevated serum corticosterone (CORT), multiplied neuronal nitric oxide synthase (nNOS) and SGK1 in the BNST, and terrible OFT behavior. We discovered when the nNOS and SGK1 were decreased in the rat BNST through DEXM treatment, the serum CORT was reduced and the OFT manifestation was ameliorated, whereas these were restrained by GSK application. Our results reveal that modest interventions to SGK1 and nNOS in the BNST improve the male rat reactivity to acute stress, and DEXM was one modulator of these effects.

Pharmacokinetics of remimazolam after intravenous infusion in anaesthetised children.

BACKGROUND: The pharmacokinetic properties of the new benzodiazepine remimazolam have been studied only in adults. We investigated the pharmacokinetics of remimazolam after i.v. infusion in anaesthetised paediatric patients. METHODS: Twenty-four children (2-6 yr, ASA physical status 1-2, BMI 15-18 kg m-2) undergoing general anaesthesia with sevoflurane were enrolled. During surgery, remimazolam was administered as an i.v. infusion over 1 h at 5 mg kg-1 h-1 for 5 min, followed by 1.5 mg kg-1 h-1 for 55 min. Plasma concentrations of remimazolam and its metabolite CNS7054 were determined from arterial blood samples using ultra-high performance liquid chromatography-mass spectrometry. Pharmacokinetic modelling was performed by population analysis. RESULTS: Pharmacokinetics were best described by a three-compartment model for remimazolam and a two-compartment model for CNS7054 linked by a transit compartment. Remimazolam showed a high clearance of 15.9 (12.9, 18.2) ml kg-1 min-1 (median, Q25, Q75), a small central volume of distribution of 0.11 (0.08, 0.14) L kg-1 and a short terminal half-life of 67 (49, 85) min. The context-sensitive half-time after an infusion of 4 h was 17 (12, 21) min. The metabolite CNS7054 showed a low clearance of 0.89 (0.33, 1.40) ml kg-1 min-1, a small central volume of distribution of 0.011 (0.005, 0.016) L kg-1, and a long terminal half-life of 321 (230, 770) min. CONCLUSIONS: Remimazolam in children was characterised by a high clearance and short context-sensitive half-time. When normalised to weight, pharmacokinetic properties were similar to those reported for adults. CLINICAL TRIAL REGISTRATION: ChiCTR2200057629.

RPL21 interacts with LAMP3 to promote colorectal cancer invasion and metastasis by regulating focal adhesion formation.

BACKGROUND: Metastasis is the leading cause of death among patients with colorectal cancer (CRC). Therefore, it is important to explore the molecular mechanisms of metastasis to develop effective therapeutic targets for CRC. In the present study, ribosomal protein L21 (RPL21) was considered as being involved in promoting CRC metastasis, yet the underlying mechanism requires further investigation. METHODS: Immunohistochemistry, western blotting, and quantitative reverse transcription polymerase chain reaction were performed to measure the expression of RPL21 and lysosome-associated membrane protein 3 (LAMP3) in CRC tissues and cells. Wound healing, transwell migration, and invasion assays were performed to study the migration and invasion of cultured CRC cells. An orthotopic CRC mouse model was developed to investigate the metastatic ability of CRC. Transcriptome sequencing was conducted to identify the genes related to RPL21. The dual-luciferase reporter gene assay was performed to determine the transcriptional activity of transcription factor EB (TFEB). The GST/His pull-down assay was performed to investigate the specific binding sites of RPL21 and LAMP3. The cell adhesion assay was performed to determine the adhesion ability of CRC cells. Immunofluorescence staining was performed to observe focal adhesions (FAs). RESULTS: RPL21 was highly expressed in CRC, contributing to tumor invasiveness and poor patient prognosis. Functionally, RPL21 promoted the migration and invasion of CRC cells in vitro and tumor metastasis in vivo. Moreover, LAMP3 was identified as being highly related to RPL21 and was essential in promoting the migration and invasion of CRC cells. Mechanistically, RPL21 activated the transcriptional function of TFEB to upregulate LAMP3 expression. RPL21 directly bound to the aa 341-416 domain of LAMP3 via its aa 1-40 and aa 111-160 segments. The combination of RPL21 and LAMP3 enhanced the stability of the RPL21 protein by suppressing the degradation of the ubiquitin-proteasome system. Furthermore, RPL21 and LAMP3 promoted the formation of immature FAs by activating the FAK/paxillin/ERK signaling pathway. CONCLUSIONS: RPL21 promoted invasion and metastasis by regulating FA formation in a LAMP3-dependent manner during CRC progression. The interaction between RPL21 and LAMP3 may function as a potential therapeutic target against CRC.

Direct inhibition of bisphenols on human and rat 11ß-hydroxysteroid dehydrogenase 2: Structure-activity relationship and docking analysis.

Bisphenols (BPs) as endocrine-disrupting compounds have drawn attention to their health hazards. Whether a BP interferes with glucocorticoid metabolism remains unclear. 11ß-Hydroxysteroid dehydrogenase 2 (11ß-HSD2) is a key glucocorticoid-metabolizing enzyme that controls fetal glucocorticoid levels across the placental barrier and mineralocorticoid receptor specificity in the kidney. In this study, 11 BPs were tested to inhibit human placental and rat renal 11ß-HSD2 and were analyzed for inhibitory potency, mode action, and docking parameters. BPs had inhibitory potency against human 11ß-HSD2: BPFL>BPAP>BPZ>BPB>BPC>BPAF>BPA>TDP and the IC10 values were 0.21, 0.55, 1.04, 2.04, 2.43, 2.57, 14.43, and 22.18 µM, respectively. All BPs are mixed inhibitors except BPAP, which is a competitive inhibitor for human 11ß-HSD2. Some BPs also inhibited rat renal 11ß-HSD2, with BPB (IC50, 27.74 ± 0.95) > BPZ (42.14 ± 0.59) > BPAF (54.87 ± 1.73) > BPA (77.32 ± 1.20) > other BPs (about 100 µM). Docking analysis showed that all BPs bound to the steroid-binding site, interacting with the catalytic residue Tyr232 of both enzymes and the most potent human 11ß-HSD2 inhibitor BPFL acts possibly due to its large fluorene ring that has hydrophobic interaction with residues Glu172 and Val270 and π-stacking interaction with catalytic residue Tyr232. The increase in the size of substituted alkanes and halogenated groups in the methane moiety of the bridge of BPs increases its inhibitory potency. Regressions of the lowest binding energy with inhibition constant indicated that there was an inverse regression. These results indicated that BPs significantly inhibited human and rat 11ß-HSD2 activity and that there were species-dependent differences.

Metabolomic characterization of semen from asthenozoospermic patients using ultra-high-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry.

Asthenozoospermia (AS) is a common factor of male infertility, and its pathogenesis remains unclear. The purpose of this study was to investigate the differential seminal plasma metabolic pattern in asthenozoospermic men and to identify potential biomarkers in relation to spermatogenic dysfunction using sensitive ultra-high-performance liquid chromatography-tandem quadruple time-of-flight MS (UHPLC-Q-TOF/MS). The samples of seminal plasma from patients with AS (n = 20) and healthy controls (n = 20) were checked and differentiated by UHPLC-Q-TOF/MS. Compared with the control group, the AS group showed a total of nine significantly different metabolites, including increases in creatinine, uric acid, N6 -methyladenosine (m6 A), uridine, and taurine and decreases in carnitine, nicotinamide, N-acetylputrescine and l-palmitoylcarnitine. By analyzing the correlation among these metabolites and clinical computer-assisted semen analysis reports, we found that m6 A is significantly correlated with not only the four decreased metabolites but also with sperm count, motility, and curvilinear velocity. Furthermore, nicotinamide was shown to correlate with other identified metabolites, indicating its important role in the metabolic pathway of AS. Current results implied that sensitive untargeted seminal plasma metabolomics could identify distinct metabolic patterns of AS and would help clinicians by offering novel cues for discovering the pathogenesis of male infertility.

AKAP-9 promotes colorectal cancer development by regulating Cdc42 interacting protein 4.

Our previous studies have shown that PRKA kinase anchor protein 9 (AKAP-9) is involved in colorectal cancer (CRC) cell proliferation and migration in vitro. However, whether or not AKAP-9 is important for CRC development or metastasis in vivo remains unknown. In the present study, we found that AKAP-9 expression was significantly higher in human colorectal cancer tissues than the paired normal tissues. In fact, AKAP-9 level correlated with the CRC infiltrating depth and metastasis. Moreover, the higher AKAP-9 expression was associated with the lower survival rate in patients. In cultured CRC cells, knockdown of AKAP-9 inhibited cell proliferation, invasion, and migration. AKAP-9 deficiency also attenuated CRC tumor growth and metastasis in vivo. Mechanistically, AKAP-9 interacted with cdc42 interacting protein 4 (CIP4) and regulated its expression. CIP4 levels were interrelated to the AKAP-9 level in CRC cells. Functionally, AKAP-9 was essential for TGF-ß1-induced epithelial-mesenchymal transition of CRC cells, and CIP4 played a critical role in mediating the function of AKAP-9. Importantly, CIP4 expression was significantly up-regulated in human CRC tissues. Taken together, our results demonstrated that AKAP-9 facilitates CRC development and metastasis via regulating CIP4-mediated epithelial-mesenchymal transition of CRC cells.

MALAT1 promotes colorectal cancer cell proliferation/migration/invasion via PRKA kinase anchor protein 9.

Our previous studies have shown that the 3' end of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is involved in colorectal cancer (CRC) cell proliferation and migration/invasion in vitro. The role and mechanism of MALAT1 in CRC metastasis in vivo, however, remain largely unknown. In the present study, we found that MALAT1 was up-regulated in human primary CRC tissues with lymph node metastasis. Overexpression of MALAT1 via RNA activation promoted CRC cell proliferation, invasion and migration in vitro, and stimulated tumor growth and metastasis in mice in vivo. Conversely, knockdown of MALAT1 inhibited CRC tumor growth and metastasis. MALAT1 regulated at least 243 genes in CRC cells in a genome-wide expression profiling. Among these genes, PRKA kinase anchor protein 9 (AKAP-9) was significantly up-regulated at both mRNA and protein levels. AKAP-9 was highly expressed in CRC cells with metastatic potential and human primary CRC tissues with lymph node metastasis, but not in normal cells or tissues. Importantly, knockdown of AKAP-9 blocked MALAT1-mediated CRC cell proliferation, migration and invasion. These data indicate that MALAT1 may promote CRC tumor development via its target protein AKAP-9.

NDRG1 attenuates epithelial-mesenchymal transition of nasopharyngeal cancer cells via blocking Smad2 signaling.

N-myc downstream-regulated gene 1 (NDRG1) has been implicated in tumorigenesis and metastasis in different cancers. However, its role in nasopharyngeal carcinoma remains unknown. We found that NDRG1 expression level was high in nasopharyngeal cancer 5-8F cells but low in 5-8F-LN cells with lymphatic metastasis potential. Knockdown of NDRG1 by shRNA promoted 5-8F cell proliferation, migration, and invasion in vitro and its tumorigenesis in vivo. Moreover, NDRG1 deficiency induced an epithelial-mesenchymal transition (EMT) of 5-8F cells as shown by an attenuation of E-cadherin and an induction of N-cadherin and vimentin expression. NDRG1 knockdown also enhanced Smad2 expression and phosphorylation. Smad2 signaling was attenuated in 5-8F cells but was significantly activated in 5-8F-LN cells. Knockdown of Smad2 restored E-cadherin but attenuated N-cadherin expression in NDRG1-deficient 5-8F cells, suggesting a reduction of EMT. Consistently, blockade of Smad2 in 5-8F-LN cells increased E-cadherin while diminishing N-cadherin and vimentin expression. These data indicate that Smad2 mediates the NDRG1 deficiency-induced EMT of 5-8F cells. In tumors derived from NDRG1-deficient 5-8F cells, E-cadherin expression was inhibited while vimentin and Smad2 were increased in a large number of cancer cells. Most importantly, NDRG1 expression was attenuated in human nasopharyngeal carcinoma tissues, resulted in a lower survival rate in patients. The NDRG1 was further decreased in the detached nasopharyngeal cancer cells, which was associated with a further reduced survival rate in patients with lymphatic metastasis. Taken together, these results demonstrated that NDRG1 prevents nasopharyngeal tumorigenesis and metastasis via inhibiting Smad2-mediated EMT of nasopharyngeal cells.

Optimization of an accelerated solvent extraction dispersive liquid-liquid microextraction method for the separation and determination of essential oil from Ligusticum chuanxiong Hort by gas chromatography with mass spectrometry.

In this study, an accelerated solvent extraction dispersive liquid-liquid microextraction coupled with gas chromatography and mass spectrometry was established and employed for the extraction, concentration and analysis of essential oil constituents from Ligusticum chuanxiong Hort. Response surface methodology was performed to optimize the key parameters in accelerated solvent extraction on the extraction efficiency, and key parameters in dispersive liquid-liquid microextraction were discussed as well. Two representative constituents in Ligusticum chuanxiong Hort, (Z)-ligustilide and n-butylphthalide, were quantitatively analyzed. It was shown that the qualitative result of the accelerated solvent extraction dispersive liquid-liquid microextraction approach was in good agreement with that of hydro-distillation, whereas the proposed approach took far less extraction time (30 min), consumed less plant material (usually <1 g, 0.01 g for this study) and solvent (<20 mL) than the conventional system. To sum up, the proposed method could be recommended as a new approach in the extraction and analysis of essential oil.

Neonatal di-(2-ethylhexyl)phthalate exposure induces permanent alterations in secretory CRH neuron characteristics in the hypothalamus paraventricular region of adult male rats.

Corticotrophin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) play a critical role in the modulation of the hypothalamic-pituitary-adrenal (HPA) axis. Early-life exposure to di-(2-ethylhexyl) phthalate (DEHP) has been associated with an increased risk of developing psychiatric disorders in adulthood. The present work was designed to explore the impact of neonatal exposure to DEHP on adult PVN CRH neuronal activity. DEHP or vehicle was given to male rat pups from PND16 to PND22. Then, anxiety-like behaviors, serum corticosterone and testosterone, immunohistochemistry, western blotting, fluorescence in situ hybridization and acute ex vivo slice electrophysiological recordings were used to evaluate the influence of DEHP on adult PVN secretory CRH neurons. Neonatal DEHP-exposed rats exhibited enhanced anxiety-like behaviors in adults, with an increase in CORT. Secretory CRH neurons showed higher spontaneous firing activity but could be inhibited by GABAAR blockers. CRH neurons displayed fewer firing spikes, prolonged first-spike latency, depolarizing shifts in GABA reversal potential and strengthened GABAergic inputs, as indicated by increases in the frequency and amplitude of sIPSCs. Enhancement of GABAergic transmission was accompanied by upregulated expression of GAD67 and downregulated expression of GABABR1, KCC2 and GAT1. These findings suggest that neonatal exposure to DEHP permanently altered the characteristics of secretory CRH neurons in the PVN, which may contribute to the development of psychiatric disorders later in life.

Visible light-induced metal-free chemoselective oxidative cleavage of benzyl C-heteroatom (N, S, Se) bonds utilizing organoboron photocatalysts.

The oxidation process is widely explored and used to synthesize diverse organic chemicals. Herein, a unified metal-free photooxidative platform for the cleavage of C-heteroatom bonds has been developed. In these reactions, the aminoquinolate diarylboron (AQDAB) complex is utilized as the photocatalyst, instigating the oxidation process induced by visible light. The cleavage of C-heteroatom bonds can be achieved chemoselectively, affording the formal carbonylation product of C-N, C-S, and C-Se bonds. This method provides a channel for connecting amines, thiols, or selenides with the carbonyl compounds directly, broadening the potential applications of oxidation as a synthetic tool.

One-step cladding metal oxide nanowires with a carbon-dots-embedded ZnO amorphous layer toward boosted photoelectrochemical water oxidation.

An effective method was proposed for constructing carbon dots (CDs)-sensitized multijunction composite photoelectrodes via one-step cladding a CDs-embedded ZnO amorphous overlayer on vertically aligned metal oxide nanowires. This strategy involved the double role of hexamethylenetetramine (HMTA) in the ethylene glycol (EG) solvent mixed with a controllable trace amount of water. In the water-deficient synthetic system, a limited portion of HMTA served as the pH buffer and hydroxyl source to force the hydrolytic process of zinc ions for the production of ZnO. The precipitated ZnO clusters were instantly capped by EG molecules through the activated alkoxidation reaction, and further crosslinked into an amorphous network surrounding the individual nanowires. Meanwhile, the excess HMTA was simultaneously depleted as the precursor for producing CDs in the EG solution through thermal condensation, which were packed in the gradually formed aggregates. We revealed that a CDs-embedded amorphous ZnO overlayer with an appropriate proportion of ingredient could be tailored through an optimal tradeoff between hydrolysis and condensation of HMTA. Benefiting from the synergy of the amorphous ZnO layer and the embedded CDs, the multijunction composite photoanodes exhibited significantly improved PEC performance and stability for water oxidation.

Effects of phenolic compounds on 3ß-hydroxysteroid dehydrogenase activity in human and rat placenta: Screening, mode of action, and docking analysis.

Human 3ß-hydroxysteroid dehydrogenase type I (HSD3B1) and rat type IV (HSD3B4) in placentas catalyze the conversion of pregnenolone to progesterone, which plays a key role in maintaining pregnancy. Many phenolic compounds potentially inhibit HSD3B in placentas as endocrine disruptors. In this study, the effects of 16 phenolic compounds on the activity of human HSD3B1 and rat HSD3B4 were determined and the structure-activity relationship was compared. HSD3B1 in human placental microsomes and HSD3B4 in rat placental microsomes were used to measure their activities and pregnenolone and NAD+ were used as substrates. Of the 16 phenolic compounds, 4-nonylphenol, pentabromophenol, and 2-bromophenol resulted in residual human HSD3B1 activity lower than 50 % and 4-nonylphenol and pentabromophenol resulted in residual rat HSD3B4 activity lower than 50 %. 4-Nonylphenol, pentabromophenol, and 2-bromophenol were mixed inhibitors of human HSD3B1, with Ki values of 2.31, 3.58 and 4.86 µM, respectively, while 4-nonylphenol and pentabromophenol were noncompetitive inhibitors of rat HSD3B4 with Ki values of 20.86 and 141.8 µM. Molecular docking showed that 4-nonylphenol, pentabromophenol, and 2-bromophenol docked to the active sites of human HSD3B1 and rat HSD3B4, and the shift of residue S125 in human HSD3B1 to T125 in rat HSD3B4 could explain the species-dependent difference in their inhibitory potency and mode of action. This study demonstrates that 4-nonylphenol, pentabromophenol, and 2-bromophenol are mixed inhibitors of human placental HSD3B1, while 4-nonylphenol and pentabromophenol are noncompetitive inhibitors of rat HSD3B4, possibly blocking the placental steroidogenesis.

Sea urchin-like amorphous MgNiCo mixed metal hydroxide nanoarrays for efficient overall water splitting under industrial electrolytic conditions.

Exploring amorphous mixed transition metal hydroxide electrocatalysts with high performance and stability for overall water splitting is a difficult challenge under industrial electrolytic conditions. Herein, a sea urchin-like amorphous MgNiCo hydroxide (MgxNi1-xCo-OH, 0 < x < 1), self-assembled from nanowire arrays, is synthesized by the hydrothermal process. The synergistic effect between Mg and Ni/Co adjusts their crystal structure and morphology, which can improve the inherent activity and provide more active sites. Benefiting from the favorable structural features, Mg0.5Ni0.5Co-OH exhibits superior electrocatalytic oxygen and hydrogen evolution reaction (OER and HER) activity with a low overpotential of 277 and 110 mV (10 mA cm-2) in 1 M KOH at 25 °C. Furthermore, overpotentials of 239 and 197 mV are required to achieve a current density of 50 mA cm-2 for the OER and HER under simulated industrial electrolysis conditions (5 M KOH at 65 °C). Notably, Mg0.5Ni0.5Co-OH remarkably accelerates water splitting with a low voltage of 1.938 and 1.699 V for 50 mA cm-2 in 1 M KOH at 25 °C and 5 M KOH at 65 °C, respectively. This work presents a novel amorphous strategy to design and construct sea urchin-like mixed metal hydroxide bifunctional efficient electrocatalysts for industrial applications.

Benzophenone-1 and -2 UV-filters potently inhibit human, rat, and mouse gonadal 3ß-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis.

Benzophenone (BP) ultraviolet (UV) -filters have been widely used to prevent adverse effects of UV. Whether they can disrupt gonadal steroidogenesis remains unclear. Gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSD) catalyse the conversion of pregnenolone to progesterone. This study explored the effect of 12 BPs on human, rat, and mouse 3ß-HSD isoforms, and analysed the structure-activity relationship (SAR) and underlying mechanisms. The inhibitory potency was BP-1 (IC50, 5.66 ± 0.95 µM) > BP-2 (5.84 ± 2.22 µM) > BP-6 (185.8 ± 115.2 µM) > BP3-BP12 on human KGN 3ß-HSD2, BP-2 (5.90 ± 1.02 µM) > BP-1 (7.55 ± 1.26 µM) > BP3-B12 on rat testicular 3ß-HSD1, and BP-1 (15.04 ± 5.20 µM) > BP-2 (22.64 ± 11.81 µM) > BP-6（125.1 ± 34.65 µM）> BP-7 (161.1 ± 102.4 µM) > other BPs on mouse testicular 3ß-HSD6. BP-1 is a mixed inhibitor of human, rat, and mouse 3ß-HSDs, and BP-2 is a mixed inhibitor of human and rat 3ß-HSDs and a noncompetitive inhibitor of mouse 3ß-HSD6. 4-Hydroxyl substitution in the benzene ring plays a key role in enhancing potency of inhibiting human, rat, and mouse gonadal 3ß-HSDs. BP-1 and BP-2 can penetrate human KGN cells to inhibit progesterone secretion at ≥ 10 µM. Docking analysis revealed that the 4-hydroxyl group of BP-1 and BP-2 forms hydrogen bonds with residue Ser123 of human 3ß-HSD2 and residue Asp127 of rat 3ß-HSD1. In conclusion, this study demonstrates that BP-1 and BP-2 are the most potent inhibitors of human, rat, and mouse gonadal 3ß-HSDs and that there is a significant SAR difference.

Inhibition of human and rat placental 3ß-hydroxysteroid dehydrogenase/Δ5,4-isomerase activities by insecticides and fungicides: Mode action by docking analysis.

Many insecticides and fungicides are endocrine-disrupting compounds, which possibly interfere with the placental endocrine system. In the placenta, 3ß-hydroxysteroid dehydrogenase/Δ5,4-isomerase type 1 (HSD3B1) is the major steroidogenic enzyme, which makes progesterone from pregnenolone to support the placental stability. In this study, we screened 12 classes of insecticides and fungicides to inhibit placental HSD3B1 activity and compared them to the rat homolog type 4 (HSD3B4) isoform. Human HSD3B1 activity and rat HSD3B4 activity were measured in the presence of 200 nM pregnenolone and 0.2 mM NAD+ and 100 µM of test chemical. Triclosan, triflumizole, dichlone, and oxine at 100 µM significantly inhibited human HSD3B1 activity with the residual activity being less than 50% of the control. Further study showed that the half-maximal inhibitory concentration (IC50) values of triclosan, triflumizole, dichlone, and oxine were 85.53 ± 9.14, 73.75 ± 3.42, 2.54 ± 0.40, and 102.93 ± 6.10 µM, respectively. In the presence of pregnenolone, triclosan, triflumizole, and dichlone were mixed inhibitors of HSD3B1, while oxine was a noncompetitive inhibitor. In the presence of NAD+, triclosan exhibited competitive inhibition while triflumizole possessed uncompetitive inhibition. Docking analysis showed that triclosan bound NAD+-binding site, while triflumizole, dichlone, and oxine mostly bound steroid-binding site. When the effect of these insecticides on rat placental HSD3B4 activity was screened in the presence of 200 nM pregnenolone, atrazine, triclosan, triflumizole, oxine, cyprodinil, and diphenyltin at 100 µM significantly inhibited rat HSD3B4 activity, with IC50 values of triclosan, triflumizole, oxine, and cyprodinil were 82.99 ± 6.48, 35.45 ± 2.73, 105.59 ± 12.04, and 43.37 ± 3.00 µM, respectively. The mode action analysis showed that triflumizole and cyprodinil were almost competitive inhibitors, while triclosan and oxine were almost noncompetitive inhibitors of rat HSD3B4. Docking analysis showed that triclosan and oxine bound cofactor NAD+ binding residues more than steroid-binding residues of rat HSD3B4 while triflumizole and cyprodinil bound most pregnenolone-interactive residues. In conclusion, some insecticides such as triclosan, triflumizole, and oxine can effectively inhibit both human and rat placental HSD3B activity and they have unique mode action due to the structure difference.

Curcuminoids inhibit human and rat placental 3ß-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine, curcuma longa L has been applied to treat pain and tumour-related symptoms for over thousands of years. Curcuminoids, polyphenolic compounds, are the main pharmacological component from the rhizome of Curcuma longa L. Pharmacological investigations have found that curcuminoids have many pharmacological activities of anti-inflammatory, anti-tumour, and anti-metastasis. AIM OF THE STUDY: 3ß-Hydroxysteroid dehydrogenase (3ß-HSD1) catalyses the production of steroid precursors for androgens and estrogens, which play an essential role in cancer metastasis. We explored the potency and mode of action of curcuminoids and their metabolites of inhibiting 3ß-HSD1 activity and compared the species difference between human and rat. MATERIALS AND METHODS: In this study, we investigated the direct inhibition of 6 curcuminoids on human placental 3ß-HSD1 activity and compared the species-dependent difference in human 3ß-HSD1 and rat placental homolog 3ß-HSD4. RESULTS: The inhibitory potency of curcuminoids on human 3ß-HSD1 was demethoxycurcumin (IC50, 0.18 µM) > bisdemethoxycurcumin (0.21 µM)>curcumin (2.41 µM)> dihydrocurcumin (4.13 µM)>tetrahydrocurcumin (15.78 µM)>octahydrocurcumin (ineffective at 100 µM). The inhibitory potency of curcuminoids on rat 3ß-HSD4 was bisdemethoxycurcumin (3.34 µM)>dihydrocurcumin (5.12 µM)>tetrahydrocurcumin (41.82 µM)>demethoxycurcumin (88.10 µM)>curcumin (137.06 µM)> octahydrocurcumin (ineffective at 100 µM). Human choriocarcinoma JAr cells with curcuminoid treatment showed that these chemicals had similar potency to inhibit progesterone secretion under basal and 8bromo-cAMP stimulated conditions. Docking analysis showed that all chemicals bind pregnenolone-binding site with mixed/competitive mode for 3ß-HSD. CONCLUSION: Some curcuminoids are potent human placental 3ß-HSD1 inhibitors, possibly being potential drugs to treat prostate cancer and breast cancer.

Inhibition of Resveratrol Analogs on Human and Rat 3ß-Hydroxysteroid Dehydrogenases: Structure-Activity Relationship and Docking Analysis.

Resveratrol and its analogs are phytochemicals. Human 3ß-hydroxysteroid dehydrogenase 1 (3ß-HSD1) synthesizes steroid hormones for normal pregnancy or promoting cancer metastasis. Whether they inhibit 3ß-HSD1 remains unclear. In this study, the inhibitory potency, mode of action, structure-activity relationship, and docking parameters of resveratrol and its analogs on 3ß-HSD1 and rat homolog 3ß-HSD4 were analyzed. The inhibitory potency of these chemicals on human 3ß-HSD1 was 4,4'-dihydroxystilbene (IC50, 3.68 µM) > pinostilbene (8.07 µM) > pinosylvin (10.60 µM) > lunularin (26.84 µM) > resveratrol (30.20 µM) > dihydroresveratrol (>100 µM) = oxyresveratrol (>100 µM) > dihydropinosylvin (ineffective at 100 µM). Resveratrol analogs and metabolites are mixed or competitive inhibitors of human 3ß-HSD1. Resveratrol and 4,4'-dihydroxystilbene inhibited progesterone secretion by human JAr cells at ≥1 µM. Resveratrol (IC50, 32.09 µM) and pinosylvin (34.71 µM) significantly inhibited rat placental 3ß-HSD4 activity. Docking analysis shows that resveratrol analogs and metabolites bind the steroid-binding sites of human 3ß-HSD1 and rat 3ß-HSD4 and interact with the catalytic residues Ser125/Thr125 and Tyr155. The negative correlation of LogP and IC50 values for human 3ß-HSD1 indicates that lipophilicity of chemicals plays a critical role in the inhibitory effect of chemicals. In conclusion, 4,4'-dihydroxystilbene, pinostilbene, and pinosylvin effectively inhibit human 3ß-HSD1 depending on their lipophilicity, thereby acting as potential therapeutic agents.

The analysis of pesticides and fungicides in the inhibition of human and rat placental 3ß-hydroxysteroid dehydrogenase activity: Mode of inhibition and mechanism.

3ß-Hydroxysteroid dehydrogenase/steroid Δ5,4-isomerase 1 (3ß-HSD1) plays a critical role in the biosynthesis of progesterone from pregnenolone in the human placenta to maintain normal pregnancy. Whether they inhibit placental 3ß-HSD1 and mode of inhibition remains unclear. In this study, we screened 21 pesticides and fungicides in five classes to inhibit human 3ß-HSD1 and compared them to rat homolog 3ß-HSD4. 3ß-HSD activity was measured by catalyzing pregnenolone to progesterone in the presence of NAD+. Of the 21 chemicals, azoles (difenoconazole), thiocarbamates (thiram and ferbam) and organochlorine (hexachlorophene) significantly inhibited human 3ß-HSD1 with half maximal inhibitory concentration (IC50) values of 2.77, 0.24, 0.68, and 17.96 µM, respectively. We also found that difenoconazole, ferbam and hexachlorophene are mixed/competitive inhibitors of 3ß-HSD1 while thiram is a mixed/noncompetitive inhibitor. Docking analysis showed that difenoconazole and hexachlorophene bound steroid-binding site. Difenoconazole and hexachlorophene except thiram and ferbam also significantly inhibited rat 3ß-HSD4 activity with IC50 of 1.12 and 2.28 µM, respectively. Thiram and ferbam significantly inhibited human 3ß-HSD1 possibly by interfering with cysteine residues, while they had no effects on rat 3ß-HSD4. In conclusion, some pesticides potently inhibit placental 3ß-HSD, leading to the reduction of progesterone formation.

Benzophenone-type ultraviolet filters inhibit human and rat placental 3ß-hydroxysteroid dehydrogenases: Structure-activity relationship and in silico docking analysis.

Benzophenones (BPs) are a class of chemicals found in various personal care and cosmetic products, such as sunscreens and lotions. Their usage is known to cause reproductive and hormonal health risks, but the exact mechanism of action remains unknown. In this study, we investigated the effects of BPs on human and rat placental 3ß-hydroxysteroid dehydrogenases (3ß-HSDs), which play a crucial role in the biosynthesis of steroid hormones, particularly progesterone. We tested inhibitory effects of 12 BPs, and performed structure-activity relationship (SAR) and in silico docking analysis. The potency of BPs to inhibit human 3ß-HSD1 (h3ß-HSD1) is BP-1 (IC50, 8.37 µM)>BP-2 (9.06 µM)>BP-12 (94.24 µM)>BP-7 (1160 µM) >BP-8 (1257 µM) >BP-6 (1410 µM) > other BPs (ineffective at 100 µM). The potency of BPs on rat r3ß-HSD4 is BP-1 (IC50, 4.31 µM)>BP-2 (117.3 µM)>BP-6 (669 µM) >BP-3 (820 µM)>other BPs (ineffective at 100 µM). BP-1, BP-2, and BP-12 are mixed h3ß-HSD1 inhibitors and BP-1 is a mixed r3ß-HSD4 inhibitor. LogP, lowest binding energy, and molecular weight were positively associated with IC50 for h3ß-HSD1, while LogS was negatively associated with IC50. The 4-OH substitution in the benzene ring plays a key role in enhancing the effectiveness of inhibiting h3ß-HSD1 and r3ß-HSD4, possibly through increasing water solubility and decreasing lipophilicity by forming hydrogen bonds. BP-1 and BP-2 inhibited progesterone production in human JAr cells. Docking analysis shows that 2-OH of BP-1 forms hydrogen bonds with catalytic residue Ser125 of h3ß-HSD1 and Thr125 of r3ß-HSD4. In conclusion, this study demonstrates that BP-1 and BP-2 are moderate inhibitors of h3ß-HSD1 and BP-1 is a moderate inhibitor of r3ß-HSD4. There is a significant SAR differences for 3ß-HSD homologues between BPs and distinct species-dependent inhibition of placental 3ß-HSDs.