Alpha-hydroxysteroid Dehydrogenase Research Articles

Polystyrene microplastic exposure modulates gut microbiota and gut-liver axis in gilthead seabream (Sparus aurata)

Microplastics (MPs) are a threat of growing concern for living organisms as they exist in all ecosystems. The bidirectional communication between the gut, its microbiota, and the liver, has been conceptualized as gut-liver axis and may be influenced by environmental factors. MPs can cause intestinal and hepatic injuries, but there is still limited research exploring their impact on gut-liver axis. The aim of this study was to assess the effects of MP ingestion on gut-liver axis balance in gilthead seabream (Sparus aurata) fed with a diet enriched with polystyrene (PS)-MPs (0, 25, or 250 mg/kg b.w./day) for 21 days. PS-MPs affected the composition of gut microbiota, enhancing the evenness of gut microbial species. We also observed the impoverishment of core microbiota, suggesting reduced stability and permanence of microbiota members. Furthermore, PS-MPs reduced predominant bacteria in the gut of gilthead seabreams, increasing low-abundance species, including potential harmful taxa. On the other hand, PS-MPs increased the gene expression of immune and inflammatory mediators (i.e., TLR2, TLR5, and COX-2) in the liver. PS-MP exposure also increased serum triglycerides and bile acids (BAs) without modifying cholesterol. Moreover, the hepatic BA metabolism was impacted by PS-MPs which increased the expression of genes involved in primary BA kinetic (i.e., CYP27A1 and LXRa), which in turn can modulate intestinal microbial community. Indeed, PICRUSt2 mapping of BA-related functions predicted the increase of factors involved in BA metabolism. Specifically, K01442 (choloylglycine hydrolase) and K00076 (7α-hydroxysteroid dehydrogenase) were augmented by PS-MPs, suggesting a possible adaptation or co-evolution of gut microbiota to the modified hepatic BA metabolism. Thus, the obtained results showed that ingested PS-MPs impact the gut microbiota architecture and functions, the hepatic innate immunity, and the BA metabolism, suggesting the involvement of the gut-liver axis in MP-induced toxicity.

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

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

Hybrid Molecules Containing Methotrexate, Vitamin D, and Platinum Derivatives: Synthesis, Characterization, In Vitro Cytotoxicity, In Silico ADME Docking, Molecular Docking and Dynamics.

Designing hybrid-based drugs is one promising strategy for developing effective anticancer drugs that explore combination therapy to enhance treatment efficacy, overcome the development of drug resistance, and lower treatment duration. Bisphosphonates and Vitamin D are commonly administered drugs for the treatment of bone diseases and the prevention of bone metastases. Platinum-based and methotrexate are widely used anticancer drugs in clinics. However, their use is hampered by adverse side effects. Hybrid-based compounds containing either bisphosphonate, vitamin D, platinum-based, or methotrexate were synthesized and characterized using FTIR, 1H-,31P, 13C-NMR, and UHPLC-HRMS which confirmed their successful synthesis. The hydroxyapatite bone binding assay revealed a promising percentage binding affinity of the bisphosphonate hybrid compounds. In vitro cytotoxicity assays on MCF-7 and HT-29 cell lines revealed a promising cytotoxic effect of hybrid 19 at 50 and 100 μg/mL on HT-29 and hybrid 15 on MCF-7 at 100 μg/mL. Molecular docking and dynamics simulation analysis revealed a binding affinity of -9.70 kcal/mol for hybrid 15 against Human 3 alpha-hydroxysteroid dehydrogenase type 3, showing its capability to inhibit Human 3 alpha-hydroxysteroid dehydrogenase type 3. The Swiss ADME, ProTox-II, GUSAR (General Unrestricted Structure-Activity Relationships), and molecular docking and dynamics studies revealed that these compounds are promising anticancer compounds.

Cofactor-dependence alteration of 7β-hydroxysteroid dehydrogenase: Enhancing one-pot synthesis efficiency of chenodeoxycholic acid to ursodeoxycholic acid through cofactor self-recycling

NAD+-dependent 7α-hydroxysteroid dehydrogenase (7α-HSDH) and NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH) are involved in the biosynthesis of chenodeoxycholic acid (CDCA) to ursodeoxycholic acid (UDCA). To realize the one-pot synthesis of CDCA to UDCA through NAD+-NADH cycling, we aimed to improve the binding ability of Hyphomicrobium sp. 7β-HSDH to NADH. The 7β-HSDH structure was modeled and some potential residues to improve NADH affinity near conserved cofactor binding regions were screened, including Ala22, Gln23, Asn24, Asp44, Leu45, and Asn46. The dominant mutant A22T/Q23E/L45A/N46E significantly enhanced the binding affinity for NADH, resulting in a 44.9-fold increase in its kcat/Km value. It increased enzymatic activity by 65.2-fold and catalyzed the synthesis of UDCA at a yield of 77.6 % with 5 g/L 7K-LCA and 12.5 mM NADH. Molecular dynamics simulations indicated increased interactions of mutated 7β-HSDH and the ligand NADH by their spatially reduced binding distance and reaction energy. The modified cofactor-dependence of 7β-HSDH realized efficient one-pot synthesis of CDCA to UDCA through strengthening cofactor-recycling and reducing the use of cofactor, achieving 90.1 % UDCA yield and 54.1 g/L/d spatiotemporal yield when coupled with 7α-HSDH with only 0.5 mM NAD+ as coenzyme. This work also supplies a universal cofactor-dependence engineering technique for homologous HSDH enzymes.

B-034 Performance Evaluation of the Total Bile Acids (TBA) Assay on Mindray BS-2800M Analyzer

Abstract Background Bile acids (BA) are synthesized in the liver and secreted into bile. The function of BA is to promote absorption of lipids including fat-soluble vitamins. During liver diseases such as viral hepatitis and liver injury by hepatotoxic drugs, and cholestasis condition, BA secretion to bile is impaired, causing serum BA level to rise. Therefore, the determination of serum BA can be used as a sensitive indicator of liver function. TBA reagent contains NADH that requires pH ≥ 9 to be stable that is usually difficult to be maintained during reagent opened and stored on the analyzer. Meanwhile, 3α-Hydroxysteroid Dehydrogenase (3α-HSD) is required for the reaction. 3α-HSD is sensitive to pH condition. Minor changes in pH results in significant changes of enzyme activity. We formulated our TBA reagent with prolonged stability when reagent is onboard on the analyzer. This study evaluated the performance of the new Mindray TBA assay on Mindray BS-2800M. Methods TBA assay was based on the method with coupled Thio-NAD and NADH reaction for BA. The R1 contains Thio-NAD. The R2 contains NADH and 3α-HSD. R1 is incubated with serum sample containing BA for 5 min at 37°C and R2 is then added. During further incubation in the presence of Thio-NAD, 3α-HSD converts BA to 3-ketosteroids and Thio-NADH. The reaction is reversible, and 3α-HSD can convert 3-keto steroids and Thio-NADH to BA and Thio-NAD. In the presence of excess NADH, enzyme cycling occurs efficiently and the rate of formation of Thio-NADH is determined by measuring specific change of absorbance at 412 nm. Two-point-line calibration is used by this assay. The TBA concentrations for the test samples are obtained by the absorbances of test samples from the calibration line. The performances of precision, linearity, onboard stability, interference (hemolysis, lipid and bilirubin), correlation are evaluated following CLSI protocols. Results The precision study was run according to EP05A3 with two commercial controls (about 21 and 36μmol/L) and two serum sample pools(about 9.9 and 24.2 μmol/L)on the Mindray BS-2800M system. The repeatability and within-lab CVs ranged from 0.74% to 1.97%, respectively. The linearity of the assay was determined as from 2.0 μmol/L to 180 μmol/L. The reagent onboard stability is for 28 days without recalibration that is superior to TBA reagents from other manufacturers. The interference of hemolysis, lipid and bilirubin are with less than ±10% impact with hemolysate Hemoglobin up to 500 mg/dL, Intralipids up to 500 mg/dL, and Conjugated bilirubin up to 30 mg/dL, Unconjugated bilirubin up to 30mg/dL. This assay (Y) also correlated well with SEKISUI TBA assay (x), (Y = 0.9965X + 0.2694, r= 0.9993). Conclusions From this evaluation, we concluded that Mindray TBA assay can measure serum TBA precisely and accurately with good performances, especially for onboard stability. It is suitable for use in routine clinical laboratories.

Enhancing the specific activity of 3α-hydroxysteroid dehydrogenase through cross-regional combinatorial mutagenesis

3α-Hydroxysteroid dehydrogenase (3α-HSD) from Comamonas testosteroni is widely used in clinical settings to measure serum total bile acid levels. However, its low enzymatic activity leads to high operational costs. In this study, we employed a combinatorial mutagenesis approach to systematically identify potential key mutation sites within the enzyme. The enzyme molecule was segmented into distinct regions, and a comprehensive strategy integrating substrate pocket engineering, binding energy calculations, and deep learning techniques was used. Through experimental verification, single-point mutants from the mutation library with enhanced enzymatic activity by at least 1.5-fold were identified. Through iterative combinatorial mutations of them, the optimal mutant H119A/R201G/R216L was obtained. This mutant exhibited a specific activity of 34.18 U/mg towards deoxycholic acid, representing a 6.85-fold increase over the wild-type (WT) enzyme. Additionally, the optimal temperature of the mutant increased from 35 °C to 40 °C, and its turnover number and catalytic efficiency increased by 6.4-fold and 9.4-fold, respectively. Quantum mechanics/molecular mechanics (QM/MM) calculations indicated that the energy barrier of the dehydrogenase reaction was reduced in the H119A/R201G/R216L mutant compared to that of the WT enzyme. Specifically, the R201G mutation significantly reduced the electric field strength along the 3α-hydroxyl group, facilitating its deprotonation. This study provides insights into enhancing enzymatic efficiency through strategic mutagenesis and elucidates mechanistic changes that optimize enzyme performance for clinical and biotechnological applications.

Ginsenoside Rh4 inhibits colorectal cancer via the modulation of gut microbiota-mediated bile acid metabolism

IntroductionDysbiosis of the gut microbiota is emerging as a pivotal factor in the pathogenesis of colorectal cancer (CRC). Ginsenoside Rh4 (Rh4) is an active compound isolated from ginseng with beneficial effects in modulating intestinal inflammation and gut microbiota dysbiosis, but how Rh4 regulates the gut microbiota to alleviate CRC remains underexplored. ObjectivesWe investigated the impact of Rh4 on CRC and the mechanism of its action in inhibiting CRC via modulation of gut microbiota. MethodsWe used the AOM/DSS model and employed transcriptomics, genomics and metabolomics techniques to explore the inhibitory impact of Rh4 on CRC. Furthermore, we employed experiments involving antibiotic treatment and fecal microbiota transplantation (FMT) to investigate the role of the gut microbiota. Finally, we elucidated the pivotal role of key functional bacteria and metabolites regulated by Rh4 in CRC. ResultsOur research findings indicated that Rh4 repaired intestinal barrier damage caused by CRC, alleviated intestinal inflammation, and inhibited the development of CRC. Additionally, Rh4 inhibited CRC in a gut microbiota-dependent manner. Rh4 increased the diversity of gut microbiota, enriched the probiotic Akkermansia muciniphila (A. muciniphila), and alleviated gut microbiota dysbiosis caused by CRC. Subsequently, Rh4 regulated A. muciniphila-mediated bile acid metabolism. A. muciniphila promoted the production of UDCA by enhancing the activity of 7α-hydroxysteroid dehydrogenase (7α-HSDH). UDCA further activated FXR, modulated the TLR4-NF-κB signaling pathway, thus inhibiting the development of CRC. ConclusionOur results confirm that Rh4 inhibits CRC in a gut microbiota-dependent manner by modulating gut microbiota-mediated bile acid metabolism and promoting the production of UDCA, which further activates the FXR receptor and regulates the TLR4-NF-κB signaling pathway. Our results confirm that Rh4 has the potential to be used as a modulator of gut microbiota for preventing and treatment of CRC.

Enzyme-Driven LC-HRMS Approach for Specific Recognition of 12α-Hydroxy Bile Acids.

The synthesis of 12α-hydroxylated bile acids (12HBAs) and non-12α-hydroxylated bile acids (non-12HBAs) occurs via classical and alternative pathways, respectively. The composition of these BAs is a crucial index for pathophysiologic assessment. However, accurately differentiating 12HBAs and non-12HBAs is highly challenging due to the limited standard substances. Here, we innovatively introduce 12α-hydroxysteroid dehydrogenase (12α-HSDH) as an enzymatic probe synthesized by heterologous expression in Escherichia coli, which can specifically and efficiently convert 12HBAs in vitro under mild conditions. Coupled to the conversion rate determined by liquid chromatography-high resolution mass spectrometry (LC-HRMS), this enzymatic probe allows for the straightforward distinguishing of 210 12HBAs and 312 non-12HBAs from complex biological matrices, resulting in a BAs profile with a well-defined hydroxyl feature at the C12 site. Notably, this enzyme-driven LC-HRMS approach can be extended to any molecule with explicit knowledge of enzymatic transformation. We demonstrate the practicality of this BAs profile in terms of both revealing cross-species BAs heterogeneity and monitoring the alterations of 12HBAs and non-12HBAs under asthma disease. We envisage that this work will provide a novel pattern to recognize the shift of BA metabolism from classical to alternative synthesis pathways in different pathophysiological states, thereby offering valuable insights into the management of related diseases.

Inhibitory influence of agmatine on catamenial-like seizure susceptibility in progesterone withdrawn female rats

Catamenial epilepsy is a condition marked by an increased occurrence of seizures that coincide with the menstrual cycle in women. Our study explored the role of neurotransmitter/neuromodulator agmatine in progesterone withdrawal induced increased seizure susceptibility that is one of the causes of catamenial epilepsy. Additionally, it investigates potential interactions between agmatine and the central neurosteroid system. The experiments involved the use of female Sprague-Dawley rats. To mime the seizure susceptibility as seen in catamenial epilepsy, rats were treated with PMSG and b-HCG which cause a higher release of progesterone and later injected with Finasteride on the 11th day of post-human chorionic gonadotropin administration to cause a sudden drop of progesterone. On the 12th day, seizure susceptibility was assessed through a low-dose (35 mg/kg) pentylenetetrazol (PTZ) injection. The results revealed that agmatine treatment showed a protective effect against seizures. Furthermore, this study explored the impact of neurosteroids such as allopregnanolone or progesterone (a precursor of allopregnanolone) and neurosteroidogenic drugs like FGIN 1–27 or metyrapone (an 11β-hydroxylase inhibitor) on the anticonvulsant effect of agmatine. The findings indicated that prior administration of these substances significantly enhanced the anticonvulsant effect of agmatine. In contrast, inhibiting neurosteroid production with drugs like trilostane (a 3β-hydroxysteroid dehydrogenase inhibitor) or indomethacin (a 3α-hydroxysteroid dehydrogenase inhibitor) completely nullified agmatine's effect. Seizures were associated with elevated progesterone levels and reduced allopregnanolone levels, which were normalized by agmatine treatment. Notably, agmatine exhibited seizure protection even in ovariectomized rats, affirming the involvement of neurosteroids in its anti-seizure effects in progesterone withdrawal increased seizure susceptibility. In conclusion, these findings emphasize central neurosteroid involvement in agmatine's pharmacological effects against seizure susceptibility in progesterone withdrawal in female rats.

Ultrasensitive electrochemical detection of bile acids via ZIF-67-MOF-derived CoNi(OH)x/CeO2/COOH-MWCNTs composite electrodes

Fast and ultra-sensitive assays are essential for the assessment of the efficacy of traditional Chinese medicines. Here, an ultrasensitive and rapid electrochemical detection of bile acid in traditional Chinese medicine using composite electrodes is presented. Cobalt-nickel hydroxide and cerium dioxide (CoNi(OH)x/CeO2) nanomaterials were synthesized in situ by a two-step reflux method with cobalt-containing metal–organic framework (ZIF-67), as precursor. It was ultrasonically complexed with carboxylated carbon nanotubes (COOH-MWCNTs) to form CoNi(OH)x/CeO2/COOH-MWCNTs composites, for facile direct detection of bile acids in traditional Chinese medicine. Characterization methods were used to verify the successful creation of the composite CoNi(OH)x/CeO2/COOH-MWCNTs, a material which demonstrated excellent electrochemical properties. The experimental results showed that the electrode responded well when bile acids, nicotinamide adenine dinucleotide, and 3α hydroxysteroid dehydrogenase were simultaneously present in the solution, and that the peak current had a good linear relationship (R2 = 0.988) with the concentration of bile acids (0.05–10.0 µM). The sensor has an outstanding detection limit of 1.0 nM, 2–3 orders of magnitude more sensitive than the concentration. Therefore, it can be used for rapid on-site detection of bile acids in real traditional Chinese medicines.

Characterization of a Novel Thermostable 7α-Hydroxysteroid Dehydrogenase.

7α-Hydroxysteroid dehydrogenase (7α-HSDH) plays a pivotal role in vivo in the biotransformation of secondary bile acids and has great potential in industrial biosynthesis due to its broad substrate specificity. In this study, we expressed and characterized a novel thermostable 7α-HSDH (named Sa 7α-HSDH). The DNA sequence was derived from the black bear gut microbiome metagenomic sequencing data, and the coding sequence of Sa 7α-HSDH was chemically synthesized. The heterologous expression of the enzyme was carried out using the pGEX-6p-1 vector. Subsequently, the activity of the purified enzyme was studied by measuring the absorbance change at 340 nm. Finally, the three-dimensional structure was predicted with AlphaFold2. Coenzyme screening results confirmed it to be NAD(H) dependent. Substrate specificity test revealed that Sa 7α-HSDH could catalyze taurochenodeoxycholic acid (TCDCA) with catalytic efficiency (kcat/Km) 3.81 S-1 mM-1. The optimum temperature of Sa 7α-HSDH was measured to be 75°C, confirming that it belongs to thermophilic enzymes. Additionally, its thermostability was assessed using an accelerated stability test over 32 hours. The catalytic activity of Sa 7α-HSDH remained largely unchanged for the first 24 hours and retained over 90% of its functionality after 32 hours at 50°C. Sa 7α-HSDH exhibited maximal activity at pH 10. The effect of metal ions-K+, Na+, Mg2+ and Cu2+-on the enzymatic activity of Sa 7α-HSDH was investigated. Only Mg2+ was observed to enhance the enzyme's activity by 27% at a concentration of 300 mM. Neither K+ nor Na+ had a significant influence on activity. Only Cu2+ was found to reduce enzyme activity. We characterized the thermostable 7α-HSDH, which provides a promising biocatalyst for bioconversion of steroids at high reaction temperatures.

Butorphanol inhibits androgen biosynthesis and metabolism in rat immature Leydig cells in vitro.

Butorphanol is a synthetic opioid analgesic medication that is primarily used for the management of pain. Butorphanol may have an inhibitory effect on androgen biosynthesis and metabolism in rat immature Leydig cells. The objective of this study was to investigate the influence of butorphanol on androgen secretion by rat Leydig cells isolated from the 35-day-old male rats. Rat Leydig cells were cultured with 0.5-50 μM butorphanol for 3h in vitro. Butorphanol at 5 and 50μM significantly inhibited androgen secretion in immature Leydig cells. At 50μM, butorphanol also blocked the effects of luteinizing hormone (LH) and 8bromo-cAMP-stimulated androgen secretion and 22R-hydroxycholesterol- and pregnenolone-mediated androgen production. Further analysis of the results showed that butorphanol downregulated the expression of genes involved in androgen production, including Lhcgr (LH receptor), Cyp11a1 (cholesterol side-chain cleavage enzyme), Srd5a1 (5α-reductase 1), and Akr1c14 (3α-hydroxysteroid dehydrogenase). Additionally, butorphanol directly inhibited HSD3B1 (3β-hydroxysteroid dehydrogenase 1) and SRD5A1 activity. In conclusion, butorphanol may have side effects of inhibiting androgen biosynthesis and metabolism in Leydig cells.

Engineering the cofactor binding site of 7α-hydroxysteroid dehydrogenase for improvement of catalytic activity, thermostability, and alteration of substrate preference

Hydroxysteroid dehydrogenases (HSDHs) are crucial for bile acid metabolism and influence the size of the bile acid pool and gut microbiota composition. HSDHs with high activity, thermostability, and substrate selectivity are the basis for constructing engineered bacteria for disease treatment. In this study, we designed mutations at the cofactor binding site involving Thr15 and Arg16 residues of HSDH St-2-2. The T15A, R16A, and R16Q mutants exhibited 7.85-, 2.50-, and 4.35-fold higher catalytic activity than the wild type, respectively, while also displaying an altered substrate preference (from taurocholic acid (TCA) to taurochenodeoxycholic acid (TCDCA)). These mutants showed lower Km and higher kcat values, indicating stronger binding to the substrate and resulting in 3190-, 3123-, and 3093-fold higher kcat/Km values for TCDCA oxidation. Furthermore, the Tm values of the T15A, R16A, and R16Q mutants were found to increase by 4.3 °C, 6.0 °C, and 7.0 °C, respectively. Molecular structure analysis indicated that reshaped internal hydrogens and surface mutations could improve catalytic activity and thermostability, and altered interactions among the catalytic triad, cofactor binding sites, and substrates could change substrate preference. This work provides valuable insights into modifying substrate preference as well as enhancing the catalytic activity and thermostability of HSDHs by targeting the cofactor binding site.

Efficient synthesis of 7-oxo-lithocholic acid using a newly identified 7α-hydroxysteroid dehydrogenase

7-Oxo-lithocholic acid (7-oxo-LCA) is a key precursor for the enzymatic synthesis of the therapeutic bile acid ursodeoxycholic acid (UDCA). Chenodeoxycholic acid (CDCA) can be readily converted to 7-oxo-LCA by the 7α-hydroxysteroid dehydrogenase (7α-HSDH). However, the poor substrate tolerance of the current 7α-HSDH limits their practical applications. In this study, a new and natively NAD(+)-dependent Sm7α-HSDH from Shewanella morhuae was identified, which exhibited a better substrate tolerance compared with other 7α-HSDHs. The purified Sm7α-HSDH showed a high oxidative activity of 281 U mg−1protein toward CDCA, with a catalytic efficiency (kcat/Km) of 7.47 × 103 mM−1 s−1. In a preparative biotransformation (100 mL scale), 200 mM (∼80 g L−1) CDCA was almost completely oxidized to 7-oxo-LCA in 1 h, with a 78% isolated yield and a space-time yield (STY) of up to 1418 g L−1 d−1, which is so far the highest productivity for biosynthesis of 7-oxo-LCA from CDCA. These results demonstrate the great potential of Sm7α-HSDH as a promising biocatalyst for UDCA synthesis.

Identification of novel metabolites of abiraterone in human serum and their metabolic pathways.

Two novel abiraterone (Abi, 3β-OH-Abi) metabolites in human serum were identified as 3α-OH-Abi and Δ5-Abi (D5A). Both metabolites were confirmed by their retention times on LC/MS and their product-ion mass spectra on LC-MS/MS compared to those of authentic compounds, which were chemically synthesized. The plausible metabolic pathways of these two metabolites are as follows: Abi is first oxidized to D5A by 3β-hydroxysteroid dehydrogenase (3β-HSD) and then irreversibly converted to Δ4-Abi (D4A) by ∆5-∆4 isomerase. Presumably, D5A detection is difficult because of its rapid conversion to D4A and its low concentration in serum samples. In contrast, the low concentration 3α-OH-Abi was generated by reducing the remaining D5A using 3α-hydroxysteroid dehydrogenase (3α-HSD).

Regulation of 20α-Hydroxysteroid Dehydrogenase Expression in Term Pregnant Human Myometrium Ex Vivo

Metabolic inactivation of progesterone within uterine myocytes by 20α-hydroxysteroid dehydrogenase (20α-HSD) has been postulated as a mechanism contributing to functional progesterone withdrawal at term. In humans, 20α-HSD is encoded by the gene AKR1C1. Myometrial AKR1C1 mRNA abundance has been reported to increase significantly during labor at term. In spontaneous preterm labor, however, we previously found no increase in AKR1C1 mRNA level in the myometrium except for preterm labor associated with clinical chorioamnionitis. This suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. In this study, we have determined the effects of various treatments of therapeutic relevance on AKR1C1 expression in pregnant human myometrium in an ex vivo culture system. AKR1C1 expression increased spontaneously during 48 h culture (p < 0.0001), consistent with the myometrium transitioning to a labor-like phenotype ex vivo, as reported previously. Serum supplementation, prostaglandin F2α, phorbol myristate acetate, and mechanical stretch had no effect on the culture-induced increase, whereas progesterone (p = 0.0058) and cAMP (p = 0.0202) further upregulated AKR1C1 expression. In contrast, culture-induced upregulation of AKR1C1 expression was dose-dependently repressed by three histone/protein deacetylase inhibitors: trichostatin A at 5 (p = 0.0172) and 25 µM (p = 0.0115); suberoylanilide hydroxamic acid at 0.5 (p = 0.0070), 1 (p = 0.0045), 2.5 (p = 0.0181), 5 (p = 0.0066) and 25 µM (p = 0.0014); and suberoyl bis-hydroxamic acid at 5 (p = 0.0480) and 25 µM (p = 0.0238). We propose the inhibition of histone/protein deacetylation helps to maintain the anti-inflammatory, pro-quiescence signaling of progesterone in pregnant human myometrium by blocking its metabolic inactivation. Histone deacetylase inhibitors may represent a class of agents that preserve or restore the progesterone sensitivity of the pregnant uterus.

PGF2alpha Inhibits 20alpha-HSD Expression by Suppressing CK1alpha-induced ERK and SP1 Activation in the Corpus Luteum of Pregnant Mice.

Prostaglandin F2α (PGF2α) is a luteolytic hormone that promotes parturition in mammals at the end of pregnancy by reducing progesterone secretion from the corpus luteum (CL). In rodents and primates, PGF2α rapidly converts progesterone to 20α-hydroxyprogesterone (20α-OHP) by promoting 20α-hydroxysteroid dehydrogenase (20α-HSD) expression. However, the specific mechanism of 20α-HSD regulation by PGF2α remains unclear. Casein Kinase 1α (CK1α) is a CK1 family member that regulates a variety of physiological functions, including reproductive development. Here, we investigated the effects of CK1α on pregnancy in female mice. Our experiments showed that CK1α is expressed in mouse CL, and its inhibition enhanced progesterone metabolism, decreased progesterone levels, and affected mouse embryo implantation. Further, CK1α mediated the effect of PGF2α on 20α-HSD in mouse luteal cells in vitro. Our results are the first to show that CK1α affects the 20α-HSD mRNA level by affecting the ERK signalling pathway to regulate the expression of the transcription factor SP1. These findings improve our understanding of PGF2α regulation of 20α-HSD.

Cell-free fat extract restores hair loss: a novel therapeutic strategy for androgenetic alopecia

BackgroundAndrogenetic alopecia (AGA) is one of the most common hair loss diseases worldwide. However, current treatments including medicine, surgery, and stem cells are limited for various reasons. Cell-free fat extract (CEFFE), contains various cell factors, may have potential abilities in treating AGA. This study aims to evaluate the safety, effectiveness and the underlying mechanism of CEFFE in treating AGA.MethodsSex hormone evaluation, immunogenicity assay and genotoxicity assay were conducted for CEFFE. In vivo study, male C57BL/6 mice were injected subcutaneously with dihydrotestosterone (DHT) and were treated with different concentration of CEFFE for 18 days (five groups and n = 12 in each group: Control, Model, CEFFELow, CEFFEMiddle, CEFFEHigh). Anagen entry rate and hair coverage percentage were analyzed through continuously taken gross photographs. The angiogenesis and proliferation of hair follicle cells were evaluated by hematoxylin–eosin, anti-CD31, and anti-Ki67 staining. In vitro study, dermal papilla cells (DPCs) were incubated with different concentrations of CEFFE, DHT, or CEFFE + DHT, followed by CCK-8 assay and flow cytometry to evaluate cell proliferation cycle and apoptosis. The intracellular DHT level were assessed by enzyme-linked immunosorbent assay. The expression of 5α-reductase type II, 3α-hydroxysteroid dehydrogenase and androgen receptor were assessed through reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or/and western blot.ResultsIn CEFFE-treated mice, an increase in the anagen entry rate and hair coverage percentage was observed. The number of CD31-positive capillaries and Ki67-positive cells were increased, suggesting that CEFFE promoted the proliferation of DPCs, modulated the cell cycle arrest, inhibited apoptosis caused by DHT, reduced the intracellular concentration of DHT in DPCs, and downregulated the expression of AR.ConclusionsCEFFE is a novel and effective treatment option for AGA through producing an increased hair follicle density and hair growth rate. The proposed mechanisms are through the DHT/AR pathway regulation and regional angiogenesis ability.

Design of St-2-2 7α-HSDH mutants for altering substrate preference and thermostability

Tauroursodeoxycholic acid (TUDCA) is the main active ingredient in bear bile powder. 7α-hydroxysteroid dehydrogenase (7α-HSDH), which selectively catalyzes the oxidation of taurochenodeoxycholic acid (TCDCA) to produce TUDCA is important when using chicken bile powder as a substrate. St-2-2 is an NADP(H)-dependent 7α-HSDH with higher activity on taurocholic acid (TCA) than on TCDCA. Here, we designed mutants of St-2-2 to alter the substrate preference (from TCA to TCDCA). Interestingly, mutant I255G had 3.38-fold increased catalytic activity toward TCDCA compared with St-2-2 and decreased catalytic activity to 4.6% that of St-2-2 toward TCA. In addition, I255T had higher thermostability. For TCDCA, I255G and I255T had lower KM values (0.07 mM, 0.13 mM), higher kcat values (201.2 s−1, 184.8 s−1), and lower ∆∆G values (-3.80 kJ mol−1, -2.20 kJ mol−1) than those of St-2-2. Increased α-helix content, more interactions between surrounding residues, and lower RMSD values indicated increased thermostability of I255T. Both I255G and I255T had newly formed hydrogen bond interactions between the catalytic center and substrates, suggesting stronger binding to TCDCA thus improving catalytic activity. This is the first report about altering substrate preference, catalytic activity, and thermostability through single-site mutation of 7α-HSDH.

Functional and morphological maturation of the full-sized and mini-pig corpus luteum by programmed cell death mechanism.

The formation and function of the corpus luteum (CL) increase the likelihood of pregnancy and efficiently manage implantation. Apoptosis must occur at an appropriate time in the formation of the CL. This also affects its function. However, it is still unclear if the type of apoptosis affects the function. We conducted morphological analysis of the CL collected on day 15 between the middle and late oestrous phases of Yorkshire pigs and mini-pigs, and measured the difference in hormone expression and apoptosis using an immunoassay method and messenger RNA level. The CL cells were more uniform in the Yorkshire pigs than in the mini-pigs, and the composition of the CL was also fuller. The expression of luteinising hormone was higher in the Yorkshire pigs. Apoptosis and the rate of action of matrix metalloproteinases (MMPs) were different between the two pig types. Expression of MMPs was higher in the Yorkshire pigs than in the mini-pigs. However, the expression of caspase 3 and 20alpha-hydroxysteroid dehydrogenase, a progesterone inhibitor, was potentiated in the mini-pigs. Autophagy throughout the CL was more extensive in the Yorkshire pigs than in the mini-pigs, suggesting that autophagy and cell reorganisation by MMPs were highly correlated. The occurrence of autophagy in the formation and function of the CL may affect the action of hormones and expression of cell reconstitution factors.