Not exclusively the activity, but the sweet spot: a dehydrogenase point mutation synergistically boosts activity, substrate tolerance, thermal stability and yield.

Catalytic activity is undoubtedly a key focus in enzyme engineering. The complicated reaction conditions hinder some enzymes from industrialization even though they have relatively promising activity. This has occurred to some dehydrogenases. Hydroxysteroid dehydrogenases (HSDHs) specifically catalyze the conversion between hydroxyl and keto groups, and hold immense potential in the synthesis of steroid medicines. We underscored the importance of 7α-HSDH activity, and analyzed the overall robustness and underlying mechanisms. Employing a high-throughput screening approach, we comprehensively assessed a mutation library, and obtained a mutant with enhanced enzymatic activity and overall stability/tolerance. The superior mutant (I201M) was identified to harbor improved thermal stability, substrate susceptibility, cofactor affinity, as well as the yield. This mutant displayed a 1.88-fold increase in enzymatic activity, a 1.37-fold improvement in substrate tolerance, and a 1.45-fold increase in thermal stability when compared with the wild type (WT) enzyme. The I201M mutant showed a 2.25-fold increase in the kcat/KM ratio (indicative of a stronger binding affinity for the cofactor). This mutant did not exhibit the highest enzyme activity compared with all the tested mutants, but these improved characteristics contributed synergistically to the highest yield. When a substrate at 100 mM was present, the 24 h yield by I201M reached 89.7%, significantly higher than the 61.2% yield elicited by the WT enzyme. This is the first report revealing enhancement of the catalytic efficiency, cofactor affinity, substrate tolerance, and thermal stability of NAD(H)-dependent 7α-HSDH through a single-point mutation. The mutated enzyme reached the highest enzymatic activity of 7α-HSDH ever reported. High enzymatic activity is undoubtedly crucial for enabling the industrialization of an enzyme. Our findings demonstrated that, when compared with other mutants boasting even higher enzymatic activity, mutants with excellent overall robustness were superior for industrial applications. This principle was exemplified by highly active enzymes such as 7α-HSDH.

The metabolic activation of pentachlorophenol to chloranil as a potent inhibitor of human and rat placental 3ß-hydroxysteroid dehydrogenases.

Pentachlorophenol (PCP) is a widely used pesticide. However, whether PCP and its metabolite chloranil have endocrine-disrupting effects by inhibiting placental 3ß-hydroxysteroid dehydrogenase 1 (3ß-HSD1) remains unclear. The study used in vitro assays with human and rat placental microsomes to measure 3ß-HSD activity as well as human JAr cells to evaluate progesterone production. The results showed that PCP exhibited moderate inhibition of human 3ß-HSD1, with an IC50 value of 29.83 µM and displayed mixed inhibition in terms of mode of action. Conversely, chloranil proved to be a potent inhibitor, demonstrating an IC50 value of 147 nM, and displaying a mixed mode of action. PCP significantly decreased progesterone production by JAr cells at 50 µM, while chloranil markedly reduced progesterone production at ≥1 µM. Interestingly, PCP and chloranil moderately inhibited rat placental homolog 3ß-HSD4, with IC50 values of 27.94 and 23.42 µM, respectively. Dithiothreitol (DTT) alone significantly increased human 3ß-HSD1 activity. Chloranil not PCP mediated inhibition of human 3ß-HSD1 activity was completely reversed by DTT and that of rat 3ß-HSD4 was partially reversed by DTT. Docking analysis revealed that both PCP and chloranil can bind to the catalytic domain of 3ß-HSDs. The difference in the amino acid residue Cys83 in human 3ß-HSD1 may explain why chloranil is a potent inhibitor through its interaction with the cysteine residue of human 3ß-HSD1. In conclusion, PCP is metabolically activated to chloranil as a potent inhibitor of human 3ß-HSD1.

Ginkgo biloba extract protects against tartrazine-induced testicular toxicity in rats: involvement of antioxidant, anti-inflammatory, and anti-apoptotic mechanisms.

The use of additives, especially colorants, in food and pharmaceutical industry is increasing dramatically. Currently, additives are classified as contaminants of emerging concern (CECs). Concerns have been raised about the potential hazards of food additives to reproductive organs and fertility. The present study investigates the reproductive toxicity of tartrazine (TRZ), a synthetic colorant, in male rats and aims to explore the curative effect of Ginkgo biloba extract (EGb) against TRZ-induced testicular toxicity. Twenty-four rats were divided into four groups: the control (0.5 ml distilled water), the EGb group (100 mg/kg EGb alone), the TRZ group (7.5 mg/kg TRZ alone), and the TRZ-EGb group (7.5 mg/kg TRZ plus 100 mg/kg EGb). The doses were administered orally in distilled water once daily for 28 days. Toxicity studies of TRZ investigated testicular redox state, serum gonadotropins, and testosterone levels, testicular 17 ß-hydroxysteroid dehydrogenase activity, sperm count and quality, levels of inflammatory cytokines, and caspase-3 expression as an apoptotic marker. Also, histopathological alterations of the testes were examined. TRZ significantly affected the testicular redox status as indicated by the increase in malondialdehyde and the decrease in reduced glutathione, superoxide dismutase, and catalase. It also disrupted serum gonadotropins (follicle stimulating hormone and luteinizing hormone) and testosterone levels and the activity of testicular 17ß-hydroxysteroid dehydrogenase. Additionally, TRZ adversely affected sperm count, motility, viability, and abnormality. Levels of tumor necrosis factor-α, interleukin-1ß, interleukin-6, and expression of caspase-3 were increased in the testes. Histopathological examination of the testes supported the alterations mentioned above. Administration of EGb significantly ameliorated TRZ-induced testicular toxicity in rats. In conclusion, EGb protected against TRZ-induced testicular toxicity through antioxidant, anti-inflammatory, and anti-apoptotic mechanisms.

Comparison of structure-activity relationship for bisphenol analogs in the inhibition of gonadal 3ß-hydroxysteroid dehydrogenases among human, rat, and mouse.

Bisphenol A (BPA) is a widely used plastic material and its potential endocrine disrupting effect has restricted its use and increasing use of BPA alternatives has raised health concerns. However, the effect of bisphenol alternatives on steroidogenesis remains unclear. The objective of this study was to compare inhibitory potencies of 10 BPA alternatives in the inhibition of gonadal 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in three species (human, rat and mouse). The inhibitory potency for human 3ß-HSD2, rat 3ß-HSD1, and mouse 3ß-HSD6 ranged from bisphenol FL (IC50, 3.32 µM for human, 5.19 µM for rat, and 3.26 µM for mouse) to bisphenol E, F, and thiodiphenol (ineffective at 100 µM). Most BPA alternatives were mixed inhibitors of gonadal 3ß-HSD and they dose-dependently inhibited progesterone formation in KGN cells. Molecular docking analysis showed that all BPA analogs bind to steroid and NAD+ active sites. Lipophilicity of BPA alternatives was inversely correlated with IC50 values. In conclusion, BPA alternatives mostly can inhibit gonadal 3ß-HSDs and lipophilicity determines their inhibitory strength.

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.

Hydroxysteroid Dehydrogenase-Catalyzed Highly Regio-, Chemo-, and Enantioselective Hydrogenation of 3-Keto in Steroids.

A highly selective hydrogenation of 3-keto in steroids to 3-hydroxyl steroids catalyzed by hydroxysteroid dehydrogenases (HSDHs) was demonstrated. The Ct3α-HSDH-catalyzed hydrogenation generated 3α-hydroxyl steroids as the main enantiopure isomers in high yields, while the Ss3ß-HSDH catalytic system afforded 3ß-hydroxyl steroids in excellent yields. In both catalytic systems, the hydrogenation proceeded regioselectively at 3-keto with 7-, 11-, 17-, and 20-keto almost unreacted, and chemoselectively with the C═C bond and ester group unattacked. Our HSDH-promoted hydrogenation showed advantages like high regio-, chemo-, and enantioselectivity, good yields, mild conditions, a wide substrate scope, and being suitable for gram-scale synthesis. Notably, bioactive molecules like dehydroepiandrosterone, brienolone, and alfaxalone were obtained facilely in high yields via our hydrogenation approach.

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.

Carbon Chain Length Determines Inhibitory Potency of Perfluoroalkyl Sulfonic Acids on Human Placental 3ß-Hydroxysteroid Dehydrogenase 1: Screening, Structure-Activity Relationship, and In Silico Analysis.

Objective: This study aimed to compare 9 perfluoroalkyl sulfonic acids (PFSA) with carbon chain lengths (C4-C12) to inhibit human placental 3ß-hydroxysteroid dehydrogenase 1 (3ß-HSD1), aromatase, and rat 3ß-HSD4 activities. Methods: Human and rat placental 3ß-HSDs activities were determined by converting pregnenolone to progesterone and progesterone secretion in JEG-3 cells was determined using HPLC/MS-MS, and human aromatase activity was determined by radioimmunoassay. Results: PFSA inhibited human 3ß-HSD1 structure-dependently in the order: perfluorooctanesulfonic acid (PFOS, half-maximum inhibitory concentration, IC 50: 9.03 ± 4.83 µmol/L) > perfluorodecanesulfonic acid (PFDS, 42.52 ± 8.99 µmol/L) > perfluoroheptanesulfonic acid (PFHpS, 112.6 ± 29.39 µmol/L) > perfluorobutanesulfonic acid (PFBS) = perfluoropentanesulfonic acid (PFPS) = perfluorohexanesulfonic acid (PFHxS) = perfluorododecanesulfonic acid (PFDoS) (ineffective at 100 µmol/L). 6:2FTS (1H, 1H, 2H, 2H-perfluorooctanesulfonic acid) and 8:2FTS (1H, 1H, 2H, 2H-perfluorodecanesulfonic acid) did not inhibit human 3ß-HSD1. PFOS and PFHpS are mixed inhibitors, whereas PFDS is a competitive inhibitor. Moreover, 1-10 µmol/L PFOS and PFDS significantly reduced progesterone biosynthesis in JEG-3 cells. Docking analysis revealed that PFSA binds to the steroid-binding site of human 3ß-HSD1 in a carbon chain length-dependent manner. All 100 µmol/L PFSA solutions did not affect rat 3ß-HSD4 and human placental aromatase activity. Conclusion: Carbon chain length determines inhibitory potency of PFSA on human placental 3ß-HSD1 in a V-shaped transition at PFOS (C8), with inhibitory potency of PFOS > PFDS > PFHpS > PFBS = PFPS = PFHxS = PFDoS = 6:2FTS = 8:2FTS.

Machine-Learning-Guided Engineering of an NADH-Dependent 7ß-Hydroxysteroid Dehydrogenase for Economic Synthesis of Ursodeoxycholic Acid.

Enzymatic synthesis of ursodeoxycholic acid (UDCA) catalyzed by an NADH-dependent 7ß-hydroxysteroid dehydrogenase (7ß-HSDH) is more economic compared with an NADPH-dependent 7ß-HSDH when considering the much higher cost of NADP+/NADPH than that of NAD+/NADH. However, the poor catalytic performance of NADH-dependent 7ß-HSDH significantly limits its practical applications. Herein, machine-learning-guided protein engineering was performed on an NADH-dependent Rt7ß-HSDHM0 from Ruminococcus torques. We combined random forest, Gaussian Naïve Bayes classifier, and Gaussian process regression with limited experimental data, resulting in the best variant Rt7ß-HSDHM3 (R40I/R41K/F94Y/S196A/Y253F) with improvements in specific activity and half-life (40 °C) by 4.1-fold and 8.3-fold, respectively. The preparative biotransformation using a "two stage in one pot" sequential process coupled with Rt7ß-HSDHM3 exhibited a space-time yield (STY) of 192 g L-1 d-1, which is so far the highest productivity for the biosynthesis of UDCA from chenodeoxycholic acid (CDCA) with NAD+ as a cofactor. More importantly, the cost of raw materials for the enzymatic production of UDCA employing Rt7ß-HSDHM3 decreased by 22% in contrast to that of Rt7ß-HSDHM0, indicating the tremendous potential of the variant Rt7ß-HSDHM3 for more efficient and economic production of UDCA.

Rhamnazin ameliorates 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin-evoked testicular toxicity by restoring biochemical, spermatogenic and histological profile in male albino rats.

2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is a potential environmental toxin that has the ability to affect male reproductive tract. Rhamnazin is a naturally present flavone that displays multiple medicinal properties. Therefore, the current study was designed to determine the mitigative role of rhamnazin against TCDD induced reproductive damage. 48 adult male albino rats were randomly separated into four groups: control, TCDD (10 µgkg-1), TCDD + rhamnazin (10 µgkg-1 + 5 mgkg-1 respectively) and rhamnazin (5 mgkg-1). The trial was conducted for 56 days. TCDD intoxication notably affected superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GSR) and catalase (CAT) activities, besides reactive oxygen species (ROS) and malondialdehyde (MDA) concentrations were augmented. TCDD administration also lowered sperm motility, viability, sperm number, while it augmented the sperm morphological (tail, neck/midpiece and head) anomalies. Moreover, it decreased the levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and plasma testosterone. Moreover, TCDD reduced steroidogenic enzymes i.e., 17-beta hydroxysteroid dehydrogenase (17ß-HSD), steroidogenic acute regulatory protein (StAR) and 3-beta hydroxysteroid dehydrogenase (3ß-HSD) as well as B-cell lymphoma 2 (Bcl-2) expressions, but increased the expressions of Bcl-2-associated X protein (Bax) and cysteine-aspartic acid protease (Caspase-3). Furthermore, TCDD exposure also induced histopathological anomalies in testicular tissues. However, the supplementation of rhamnazin recovered all the mentioned damages in the testicles. The outcomes revealed that rhamnazin can ameliorate TCDD induced reproductive toxicity due to its anti-oxidant, anti-apoptotic and androgenic nature.

Resistance to Resveratrol Treatment in Experimental PTSD Is Associated with Abnormalities in Hepatic Metabolism of Glucocorticoids.

Glucocorticoids are metabolized by the CYP3A isoform of cytochrome P450 and by 11-ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD-1). Experimental data suggest that post-traumatic stress disorder (PTSD) is associated with an increase in hepatic 11ß-HSD-1 activity and a concomitant decrease in hepatic CYP3A activity. Trans-resveratrol, a natural polyphenol, has been extensively studied for its antipsychiatric properties. Recently, protective effects of trans-resveratrol were found in relation to PTSD. Treatment of PTSD rats with trans-resveratrol allowed the rats to be divided into two phenotypes. The first phenotype is treatment-sensitive rats (TSR), and the second phenotype is treatment-resistant rats (TRRs). In TSR rats, trans-resveratrol ameliorated anxiety-like behavior and reversed plasma corticosterone concentration abnormalities. In contrast, in TRR rats, trans-resveratrol aggravated anxiety-like behavior and decreased plasma corticosterone concentration. In TSR rats, hepatic 11ß-HSD-1 activity was suppressed, with a concomitant increase in CYP3A activity. In TRR rats, the activities of both enzymes were suppressed. Thus, the resistance of PTSD rats to trans-resveratrol treatment is associated with abnormalities in hepatic metabolism of glucocorticoids. The free energy of binding of resveratrol, cortisol, and corticosterone to the human CYP3A protein was determined using the molecular mechanics Poisson-Boltzmann surface area approach, indicating that resveratrol could affect CYP3A activity.

Characterization of the pH-dependent protein stability of 3α-hydroxysteroid dehydrogenase/carbonyl reductase by differential scanning fluorimetry.

The characterization of protein stability is essential for understanding the functions of proteins. Hydroxysteroid dehydrogenase is involved in the biosynthesis of steroid hormones and the detoxification of xenobiotic carbonyl compounds. However, the stability of hydroxysteroid dehydrogenases has not yet been characterized in detail. Here, we determined the changes in Gibbs free energy, enthalpy, entropy, and heat capacity of unfolding for 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) by varying the pH and urea concentration through differential scanning fluorimetry and presented pH-dependent protein stability as a function of temperature. 3α-HSD/CR shows the maximum stability of 30.79 kJ mol-1 at 26.4°C, pH 7.6 and decreases to 7.74 kJ mol-1 at 25.7°C, pH 4.5. The change of heat capacity of 30.25 ± 1.38 kJ mol-1 K-1 is obtained from the enthalpy of denaturation as a function of melting temperature at varied pH. Two proton uptakes are linked to protein unfolding from residues with differential pKa of 4.0 and 6.5 in the native and denatured states, respectively. The large positive heat capacity change indicated that hydrophobic interactions played an important role in the folding of 3α-HSD/CR. These studies reveal the mechanism of protein unfolding in HSD and provide a convenient method to extract thermodynamic parameters for characterizing protein stability using differential scanning fluorimetry.

Characterization of a novel porcine carbonyl reductase activated by glutathione: Relationship to carbonyl reductase 1, 3α/ß-hydroxysteroid dehydrogenase and prostaglandin 9-ketoreductase.

A porcine gene, LOC100622246, encodes carbonyl reductase [NADPH] 1 (pCBR-N1), whose function remains unknown. Previously, three porcine carbonyl reductases, carbonyl reductase 1 (pCBR1), 3α/ß-hydroxysteroid dehydrogenase (p3α/ß-HSD) and prostaglandine-9-keto reductase (pPG-9-KR), were purified from neonatal testis, adult testis and adult kidney, respectively. However, the relationship of pCBR-N1 with the three enzymes is still unknown. Here, we compare the properties of the recombinant pCBR-N1 and pCBR1. The two enzymes reduced various carbonyl compounds including 5α-dihydrotestosterone, which was converted to its 3α- and 3ß-hydroxy-metabolites. Compared to pCBR1, pCBR-N1 exhibited higher Km and kcat values for most substrates, but more efficiently reduced prostaglandin E2. pCBR-N1 was inhibited by known inhibitors of p3α/ß-HSD (hexestrol and indomethacin), but not by pCBR1 inhibitors. pCBR-N1 was highly expressed than pCBR1 in the several tissues of adult domestic and microminiature pigs. The results, together with partial amino acid sequence match between pCBR-N1 and pPG-9-KR, reveal that pCBR-N1 is identical to p3α/ß-HSD and pPG-9-KR. Notably, pCBR-N1, but not pCBR1, reduced S-nitrosoglutathione and glutathione-adducts of alkenals including 4-oxo-2-nonenal with Km of 8.3-32 µM, and its activity toward non-glutathionylated substrates was activated 2- to 9-fold by 1 mM glutathione. Similar activation by glutathione was also observed for human CBR1. Site-directed mutagenesis revealed that the differences in kinetic constants and glutathione-mediated activation between pCBR-N1 and pCBR1 are due to differences in residue 236 and two glutathione-binding residues (at positions 97 and 193), respectively. Thus, pCBR-N1 is a glutathione-activated carbonyl reductase that functions in the metabolism of endogenous and xenobiotic carbonyl compounds.

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.

[HSDL2 overexpression promotes rectal cancer progression by regulating cancer cell cycle and promoting cell proliferation].

OBJECTIVE: To analyze the expression of hydroxysteroid dehydrogenase like 2 (HSDL2) in rectal cancer tissues and the effect of changes in HSDL2 expression level on proliferation of rectal cancer cells. METHODS: Clinical data and tissue samples of 90 patients with rectal cancer admitted to our hospital from January 2020 to June 2022 were collected from the prospective clinical database and biological specimen database. The expression level of HSDL2 in rectal cancer and adjacent tissues was detected by immunohistochemistry, and based on the median level of HSDL2 expression, the patients were divided into high expression group (n=45) and low expression group (n=45) for analysis the correlation between HSDL2 expression level and the clinicopathological parameters. GO and KEGG enrichment analyses were performed to explore the role of HSDL2 in rectal cancer progression. The effects of changes in HSDL2 expression levels on rectal cancer cell proliferation, cell cycle and protein expressions were investigated in SW480 cells with lentivirus-mediated HSDL2 silencing or HSDL2 overexpression using CCK-8 assay, flow cytometry and Western blotting. RESULTS: The expressions of HSDL2 and Ki67 were significantly higher in rectal cancer tissues than in the adjacent tissues (P < 0.05). Spearman correlation analysis showed that the expression of HSDL2 protein was positively correlated with Ki67, CEA and CA19-9 expressions (P < 0.01). The rectal cancer patients with high HSDL2 expressions had significantly higher likelihood of having CEA ≥5 µg/L, CA19-9 ≥37 kU/L, T3-4 stage, and N2-3 stage than those with a low HSDL2 expression (P < 0.05). GO and KEGG analysis showed that HSDL2 was mainly enriched in DNA replication and cell cycle. In SW480 cells, HSDL2 overexpression significantly promoted cell proliferation, increased cell percentage in S phase, and enhanced the expression levels of CDK6 and cyclinD1 (P < 0.05), and HSDL2 silencing produced the opposite effects (P < 0.05). CONCLUSION: The high expression of HSDL2 in rectal cancer participates in malignant progression of the tumor by promoting the proliferation and cell cycle progress of the cancer cells.

Impaired alveolar macrophage 11ß-hydroxysteroid dehydrogenase type 1 reductase activity contributes to increased pulmonary inflammation and mortality in sepsis-related ARDS.

Background: Acute Respiratory Distress Syndrome (ARDS) is a devastating pulmonary inflammatory disorder, commonly precipitated by sepsis. Glucocorticoids are immunomodulatory steroids that can suppress inflammation. Their anti-inflammatory properties within tissues are influenced by their pre-receptor metabolism and amplification from inactive precursors by 11ß-hydroxysteroid dehydrogenase type-1 (HSD-1). We hypothesised that in sepsis-related ARDS, alveolar macrophage (AM) HSD-1 activity and glucocorticoid activation are impaired, and associated with greater inflammatory injury and worse outcomes. Methods: We analysed broncho-alveolar lavage (BAL) and circulating glucocorticoid levels, AM HSD-1 reductase activity and Receptor for Advanced Glycation End-products (RAGE) levels in two cohorts of critically ill sepsis patients, with and without ARDS. AM HSD-1 reductase activity was also measured in lobectomy patients. We assessed inflammatory injury parameters in models of lung injury and sepsis in HSD-1 knockout (KO) and wild type (WT) mice. Results: No difference in serum and BAL cortisol: cortisone ratios are shown between sepsis patients with and without ARDS. Across all sepsis patients, there is no association between BAL cortisol: cortisone ratio and 30-day mortality. However, AM HSD-1 reductase activity is impaired in patients with sepsis-related ARDS, compared to sepsis patients without ARDS and lobectomy patients (0.075 v 0.882 v 0.967 pM/hr/106 AMs, p=0.004). Across all sepsis patients (with and without ARDS), impaired AM HSD-1 reductase activity is associated with defective efferocytosis (r=0.804, p=0.008) and increased 30-day mortality. AM HSD-1 reductase activity negatively correlates with BAL RAGE in sepsis patients with ARDS (r=-0.427, p=0.017). Following intra-tracheal lipopolysaccharide (IT-LPS) injury, HSD-1 KO mice demonstrate increased alveolar neutrophil infiltration, apoptotic neutrophil accumulation, alveolar protein permeability and BAL RAGE concentrations compared to WT mice. Caecal Ligation and Puncture (CLP) injury in HSD-1 KO mice results in greater peritoneal apoptotic neutrophil accumulation compared to WT mice. Conclusions: AM HSD-1 reductase activity does not shape total BAL and serum cortisol: cortisone ratios, however impaired HSD-1 autocrine signalling renders AMs insensitive to the anti-inflammatory effects of local glucocorticoids. This contributes to the decreased efferocytosis, increased BAL RAGE concentrations and mortality seen in sepsis-related ARDS. Upregulation of alveolar HSD-1 activity could restore AM function and improve clinical outcomes in these patients.

Effect of peripheral neural stimulation with allopregnanolone on ovarian physiology.

Neuroactive steroids can rapidly regulate multiple physiological functions in the central and peripheral nervous systems. The aims of the present study were to determine whether allopregnanolone (ALLO), administered in low nanomolar and high micromolar concentrations, can: (i) induce changes in the ovarian progesterone (P4) and estradiol (E2) release; (ii) modify the ovarian mRNA expression of Hsd3b1 (3ß-hydroxysteroid dehydrogenase, 3ß-HSD)3ß-, Akr1c3 (20α-hydroxysteroid dehydrogenase, 20α-HSD), and Akr1c14 (3α-hydroxy steroid oxidoreductase, 3α-HSOR)); and (iii) modulate the ovarian expression of progesterone receptors A and B, α and ß estrogenic receptors, luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR). To further characterize ALLO peripheral actions, the effects were evaluated using a superior mesenteric ganglion-ovarian nervous plexus-ovary (SMG-ONP-O) and a denervated ovary (DO) systems. ALLO SMG administration increased P4 concentration in the incubation liquid by decreasing ovarian 20α-HSD mRNA, and it also increased ovarian 3α-HSOR mRNA expression. In addition, ALLO neural peripheral modulation induced an increase in the expression of ovarian LHR, PRA, PRB, and ERα. Direct ALLO administration to the DO decreased E2 and increased P4 concentration in the incubation liquid. The mRNA expression of 3ß-HSD decreased and 20α-HSD increased. Further, ALLO in the OD significantly changed ovarian FSHR and PRA expression. This is the first evidence of ALLO's direct effect on ovarian steroidogenesis. Our results provide important insights about how this neuroactive steroid interacts both with the PNS and the ovary, and these findings might help devise some of the pleiotropic effects of neuroactive steroids on female reproduction. Moreover, ALLO modulation of ovarian physiology might help uncover novel treatment approaches for reproductive diseases.

20α-Hydroxysteroid Dehydrogenase Expression in the Human Myometrium at Term and Preterm Birth: Relationships to Fetal Sex and Maternal Body Mass Index.

The mechanism by which human labor is initiated in the presence of elevated circulating progesterone levels remains unknown. Recent evidence indicates that the progesterone-metabolizing enzyme, 20α-hydroxysteroid dehydrogenase (20α-HSD), encoded by the gene AKR1C1, may contribute to functional progesterone withdrawal. We found that AKR1C1 expression significantly increased with labor onset in term myometrium, but not in preterm myometrium. Among preterm laboring deliveries, clinically diagnosed chorioamnionitis was associated with significantly elevated AKR1C1 expression. AKR1C1 expression positively correlated with BMI before labor and negatively correlated with BMI during labor. Analysis by fetal sex showed that AKR1C1 expression was significantly higher in women who delivered male babies compared to women who delivered female babies at term, but not preterm. Further, in pregnancies where the fetus was female, AKR1C1 expression positively correlated with the mother's age and BMI at the time of delivery. In conclusion, the increase in myometrial AKR1C1 expression with term labor is consistent with 20α-HSD playing a role in local progesterone metabolism to promote birth. Interestingly, this role appears to be specific to term pregnancies where the fetus is male. Upregulated AKR1C1 expression in the myometrium at preterm in-labor with clinical chorioamnionitis suggests that increased 20α-HSD activity is a mechanism through which inflammation drives progesterone withdrawal in preterm labor. The link between AKR1C1 expression and maternal BMI may provide insight into why maternal obesity is often associated with dysfunctional labor. Higher myometrial AKR1C1 expression in male pregnancies may indicate fetal sex-related differences in the mechanisms that precipitate labor onset at term.

Comparative study on the gene expression of corticosterone metabolic enzymes in embryonic tissues between Tibetan and broiler chickens.

Glucocorticoids (GCs) are an essential mediator hormone that can regulate animal growth, behavior, the phenotype of offspring, and so on, while GCs in poultry are predominantly corticosterones. The biological activity of GCs is mainly regulated by the intracellular metabolic enzymes, including 11ß-hydroxysteroid dehydrogenases 1 (11ß-HSD1), 11ß-hydroxysteroid dehydrogenases 2 (11ß-HSD2), and 20-hydroxysteroid dehydrogenase (20-HSD). To investigate the embryonic mechanisms of phenotypic differences between breeds, we compared the expression of corticosterone metabolic enzyme genes in the yolk-sac membrane and chorioallantoic membrane (CAM). We described the tissue distribution and ontogenic patterns of corticosterone metabolic enzymes during embryonic incubation between Tibetan and broiler chickens. Forty fertilized eggs from Tibetan and broiler chickens were incubated under hypoxic and normoxic conditions, respectively. Real-time fluorescence quantitative PCR was used to examine the expression of 11ß-HSD1/2, and 20-HSD mRNA in embryonic tissues. The results showed that the expression levels of yolk-sac membrane mRNA of 11ß-HSD2 and 20-HSD in Tibetan chickens on E14 (embryonic day of 14) were significantly lower than those of broiler chickens (P < 0.05), and these genes expression of CAM in Tibetan chickens were higher than those of broiler chickens (P < 0.05). In addition, the three genes in the yolk-sac membrane and CAM were followed by a down-regulation on E18 (embryonic day of 18). The 11ß-HSD1 and 11ß-HSD2 genes followed a similar tissue-specific pattern: the expression level was more abundantly in the liver, kidney, and intestine, with relatively lower abundance in the hypothalamus and muscle, and the expression level of 20-HSD genes in all tissues tested was higher. In the liver, 20-HSD of both Tibetan and broiler chickens showed different ontogeny development patterns, and hepatic mRNA expression of 20-HSD in broiler chickens was significantly higher than that of Tibetan chickens of the same age from E14 to E18 (P < 0.05). This study preliminarily revealed the expression levels of cortisol metabolic genes in different tissues during the development process of Tibetan and broiler chicken embryos. It provided essential information for in-depth research of the internal mechanism of maternal GCs programming on offspring.

SRSF3-Mediated Ki67 Exon 7-Inclusion Promotes Head and Neck Squamous Cell Carcinoma Progression via Repressing AKR1C2.

Ki67 is a well-known proliferation marker with a large size of around 350 kDa, but its biological function remains largely unknown. The roles of Ki67 in tumor prognosis are still controversial. Ki67 has two isoforms generated by alternative splicing of exon 7. The roles and regulatory mechanisms of Ki67 isoforms in tumor progression are not clear. In the present study, we surprisingly find that the increased inclusion of Ki67 exon 7, not total Ki67 expression level, was significantly associated with poor prognosis in multiple cancer types, including head and neck squamous cell carcinoma (HNSCC). Importantly, the Ki67 exon 7-included isoform is required for HNSCC cell proliferation, cell cycle progression, cell migration, and tumorigenesis. Unexpectedly, Ki67 exon 7-included isoform is positively associated with intracellular reactive oxygen species (ROS) level. Mechanically, splicing factor SRSF3 could promote exon 7 inclusion via its two exonic splicing enhancers. RNA-seq revealed that aldo-keto reductase AKR1C2 is a novel tumor-suppressive gene targeted by Ki67 exon 7-included isoform in HNSCC cells. Our study illuminates that the inclusion of Ki67 exon 7 has important prognostic value in cancers and is essential for tumorigenesis. Our study also suggested a new SRSF3/Ki67/AKR1C2 regulatory axis during HNSCC tumor progression.