Structure-activity relationship and mechanistic study of organotins as inhibitors of human, pig, and rat gonadal 3ß-hydroxysteroid dehydrogenases.

Organotins have been widely used in various industrial applications. This study investigated the structure-activity relationship as inhibitors of human, pig, and rat gonadal 3ß-hydroxysteroid dehydrogenases (3ß-HSD). Human KGN cell, pig, and rat testis microsomes were utilized to assess the inhibitory effects of 18 organotins on the conversion of pregnenolone to progesterone. Among them, diphenyltin, triethyltin, and triphenyltin exhibited significant inhibitory activity against human 3ß-HSD2 with IC50 values of 114.79, 106.98, and 5.40 µM, respectively. For pig 3ß-HSD, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin demonstrated inhibitory effects with IC50 values of 172.00, 100.19, 87.00, 5.75, and 1.65 µM, respectively. Similarly, for rat 3ß-HSD1, dipropyltin, diphenyltin, triethyltin, tributyltin, and triphenyltin displayed inhibitory activity with IC50 values of 81.35, 43.56, 55.55, 4.09, and 0.035 µM, respectively. They were mixed inhibitors of pig and rat 3ß-HSD, while triphenyltin was identified as a competitive inhibitor of human 3ß-HSD2. The mechanism underlying the inhibition of organotins on 3ß-HSD was explored, revealing that they may disrupt the enzyme activity by binding to cysteine residues in the catalytic sites. This proposition was supported by the observation that the addition of dithiothreitol reversed the inhibition caused by all organotins except for triethyltin, which was partially reversed. In conclusion, this study provides valuable insights into the structure-activity relationship of organotins as inhibitors of human, pig, and rat gonadal 3ß-HSD. The mechanistic investigation suggests that these compounds likely exert their inhibitory effects through binding to cysteine residues in the catalytic sites.

Schwann cell-derived amphiregulin enhances nerve regeneration via supporting the proliferation and migration of Schwann cells and the elongation of axons.

Peripheral nerves have limited regeneration ability following nerve injury. Applying growth factors with neurotrophic roles is beneficial for accelerating peripheral nerve regeneration. Here we show that after rat sciatic nerve injury, growth factor amphiregulin (AREG) is upregulated in Schwann cells of sciatic nerves. Elevated AREG stimulates the proliferation and migration of Schwann cells by activating ERK1/2 cascade. Schwann cell-secreted AREG further facilitates the outgrowth of neurites and the elongation of injured axons. Administration of AREG to injured sciatic nerves stimulates the proliferation of Schwann cells to replace lost cell population, encourages the migration of Schwann cells to form cell cords, and facilitates the regrowth of axons. Overall, our results identify AREG as an important neurotrophic factor and thus provide a promising therapeutic avenue towards peripheral nerve injury.

Cell populations in neonatal rat peripheral nerves identified by single-cell transcriptomics.

Peripheral nerves connect central nerves with target tissues and organs and execute vital signal transduction functions. Although sub-types of neurons have been defined, the heterogeneity of cell populations in peripheral nerves, especially Schwann cells, has not been well demonstrated. Here, we collected sciatic nerves (SN) and dorsal root ganglia (DRG) from neonatal (1-day old) rats and classified cell populations by high-coverage single-cell sequencing. A total of 10 types of cells, including endothelial cells, erythrocytes, fibroblasts, monocytic cells, neurons, neutrophils, pericytes, satellite cells, Schwann cells, and vascular smooth muscle cells, were identified by transcriptome-based cell typing. The comparisons of cells in neonatal rat SN and DRG revealed distinct atlas in different tissue localizations. Investigations of ligand-receptor interactions showed that there existed direct cell-cell communications between endothelial cells and fibroblasts in SN and among endothelial cells, fibroblasts, and vascular smooth muscle cells in DRG. Schwann cells in neonatal rats were further sub-grouped to four sub-types, including LOC100134871 and Hbb expressing Schwann cell sub-type 1, Cldn19 and Emid1 expressing Schwann cell sub-type 2, Timp3 and Col5a3 expressing Schwann cell sub-type 3, and Cenpf and Mki67 expressing Schwann cell sub-type 4. These Schwann cell sub-types exhibited distinct genetic features and functional enrichments. Collectively, our results illustrated the diversity and cellular complexity of peripheral nerves at the neonatal stage and revealed the heterogeneity of Schwann cells in the peripheral nervous system.

Establishment of quantitative methodology for sophoridine analysis and determination of its pharmacokinetics and bioavailability in rat.

OBJECTIVE: The aim of this study is to develop a rapid and sensitive UPLC-MS/MS approach to determine the sophoridine (SOP) level in rat plasma and the pharmacokinetics of the substance. SIGNIFICANCE: Sophoridine is used as an anti-inflammatory, anti-virus, anti-microbial, and anti-tumor alkaloid. It is essential to explore specific detection methods for the quantitative analysis of SOP in the blood circulation. METHODS: The rat plasma samples were prepared by one-step protein precipitation with acetonitrile. Subsequently, the samples were separated by chromatography using a UPLC BEH C18 reversed-phase with an initial mobile phase of methanol and 0.1% formic acid aqueous solution. The gradient elution was performed at a fixed flow rate of 0.4 mL/min, and multiple reaction monitoring (MRM) mode with an electrospray positive ionization source was employed to detect the transitions of m/z 249.1 → 84.2 for SOP and m/z 264.3 → 69.8 for dendrobine (IS). The entire process required 3.5 min for each sample. RESULTS: A linear correlation was established over the range of 2-2000 ng/mL (r2≥0.9954) for SOP in rat plasma with a lower limit of quantification (LLOQ) at 2 ng/mL. The range of accuracy was tested between 94.90% and 100.80%, and the relative standard deviations (RSDs) toward both intra- and inter-day precision were <10%. Thus, this method was successfully applied to a pharmacokinetic study, and the subsequent results demonstrated a low absolute bioavailability of 2.32%. CONCLUSION: The present study established a reliable method that quantified the SOP concentration in rat plasma after administering a dose of 2 mg/kg intravenously or 20 mg/kg orally.

Association of polymorphisms in MALAT1 with the risk of endometriosis in Southern Chinese women.

Endometriosis is a common estrogen-dependent inflammatory disease characterized by the presence of endometrial-like tissue outside the uterine cavity, which causes infertility and pelvic pain. Polymorphisms in MALAT1 have been demonstrated to play crucial roles in many diseases. However, the roles of MALAT1 polymorphisms in the etiology of endometriosis have not been well documented. We genotyped three MALAT1 polymorphisms in 555 endometriosis patients and 535 female control participants using quantitative polymerase chain reaction with TaqMan probes. To estimate the associations between MALAT1 polymorphisms and endometriosis risk, an unconditional logistic regression model was conducted to calculate an odds ratio (OR) and the 95% confidence interval (CI), adjusting for age, abortion history, number of deliveries, Body Mass Index (BMI), and The International Federation of Gynecology and Obstetrics (FIGO) stage. We found that the MALAT1 rs591291 C > T polymorphism significantly enhanced endometriosis risk (heterogeneous: adjusted OR = 1.36, 95% CI = 1.00-1.85, P = 0.050; homogenous: adjusted OR = 1.55, 95% CI = 1.03-2.33, P = 0.037; dominant: adjusted OR = 1.41, 95% CI = 1.05-1.88, P = 0.021). In stratification analyses, these associations were more predominant in the patients younger than 35 years old, with a relatively high number of deliveries and with a BMI between 25 and 29.9. Compared with wild-type CCG haplotype carriers, individuals with TCC haplotypes had a higher risk of developing endometriosis. The MALAT1 rs591291 C > T polymorphism was associated with a significant increase in endometriosis risk.

Association of polymorphisms in MALAT1 with the risk of endometrial cancer in Southern Chinese women.

BACKGROUND: Endometrial cancer is the most common gynecologic malignancy worldwide. Polymorphisms in MALAT1 have been demonstrated to play critical roles in cancer. However, the roles of MALAT1 polymorphisms in the etiology of endometrial cancer have not been well documented. METHODS: We genotyped three MALAT1 polymorphisms in 249 endometrial cancer cases and 446 cancer-free female controls using quantitative polymerase chain reaction with TaqMan probes. To estimate the association between MALAT1 polymorphisms (rs591291 C>T, rs664589 C>G, and rs4102217 G>C) and the risk of endometrial cancer, an unconditional logistic regression model was conducted to calculate the odds ratio (OR) and the 95% confidence interval (CI), adjusting for surgery history, menopause, number of deliveries, BMI, and FIGO stage. RESULTS: We found that the MALAT1 rs664589 C>G polymorphism was significantly associated with endometrial cancer risk (heterogeneous: adjusted OR = 0.57, 95% CI = 0.34-0.93, P = .026; homogenous: adjusted OR = 3.74, 95% CI = 1.12-12.45, P = .032; and recessive: adjusted OR = 4.06, 95% CI = 1.22-13.48, P = .022). Stratified analysis further demonstrated that the MALAT1 rs664589 C>G polymorphism significantly increased the risk of endometrial cancer susceptibility in patients with no history of surgery, more deliveries, BMI between 25 and 29.9, and FIGO stages II-III. Compared with the wild-type GCG haplotype carriers, individuals with CGG haplotypes had a higher risk of developing endometrial cancer. CONCLUSION: The MALAT1 rs664589 C>G polymorphism was associated with a significant increase in endometrial cancer risk.

Correlation Analysis of Phenolic Contents and Antioxidation in Yellow- and Black-Seeded Brassica napus.

Brassica napus L. is rich in phenolic components and it has natural antioxidant characteristics which are important to human health. In the present study, the total phenolic and flavonoid contents of developing seeds of yellow- and black-seeded B. napus were compared. Both phenolic and flavonoid contents were significantly higher at 5 weeks after flowering (WAF) in black seeds (6.44 ± 0.97 mg EE/g phenolics and 3.78 ± 0.05 mg EE/g flavonoids) than yellow seeds (2.80 ± 0.13 mg/g phenolics and 0.83 ± 0.01 mg/g flavonoids). HPLC⁻DAD⁻ESI/MS analysis revealed different content of 56 phenolic components between yellow and black-seeded B. napus, including kaempferol-3-O-glucoside, isorhamnetin-3-O-glucoside, quercetin-3-O-sophoroside, procyanidin B2 ([DP 2]), which were significantly reduced in yellow seeds compared with black seeds. Applying the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical assay, we found maximum clearance of DPPH and ABTS in the late developmental stages of yellow and black seeds. Additionally, the ferric reducing antioxidant power (FRAP) value maximized at 5 WAF in black seeds (432.52 ± 69.98 µmol Fe (II)/g DW) and 6 WAF in yellow seeds (274.08 ± 2.40 µmol Fe (II)/g DW). Generally, antioxidant ability was significantly reduced in yellow-seeded B. napus compared to black rapeseed, and positive correlations between antioxidation and flavonoid content were found in both yellow- and black-seeded B. napus.

Enhanced inhibition of human and rat aromatase activity by benzene ring substitutions in bisphenol A: QSAR structure-activity relationship and in silico docking analysis.

Bisphenol A (BPA) is a widely used plastic material, but its potential endocrine disrupting effect has restricted its use. The BPA alternatives have raised concerns. This study aimed to compare inhibitory potencies of 11 BPA analogues on human and rat placental aromatase (CYP19A1). The inhibitory potency on human CYP19A1 ranged from bisphenol H (IC50, 0.93 µM) to tetramethyl BPA and tetrabromobisphenol S (ineffective at 100 µM) when compared to BPA (IC50, 73.48 µM). Most of them were mixed/competitive inhibitors and inhibited estradiol production in human BeWo cells. Molecular docking analysis showed all BPA analogues bind to steroid active site or in between steroid and heme of CYP19A1 and form a hydrogen bond with catalytic residue Met374. Pharmacophore analysis showed that there were 4 hydrophobic regions for BPA analogues, with bisphenol H occupying 4 regions. Bivariate correlation analysis showed that LogP (lipophilicity) and LogS (water solubility) of BPA analogues were correlated with their IC50 values. Computerized drug metabolism and pharmacokinetics analysis showed that bisphenol H, tetrabromobisphenol A, and tetrachlorobisphenol A had low solubility, which might explain their weaker inhibition on estradiol production on BeWo cells. In conclusion, BPA analogues mostly can inhibit CYP19A1 and the lipophilicity determines their inhibitory strength.

Effects of organochlorine pesticides on human and rat 17ß-hydroxysteroid dehydrogenase 1 activity: Structure-activity relationship and in silico docking analysis.

The objective of this study was to examine the effect of 11 organochlorine pesticides on human and rat 17ß-Hydroxysteroid dehydrogenase 1 (17ß-HSD1) in human placental and rat ovarian microsome and on estradiol production in BeWo cells. The results showed that the IC50 values for endosulfan, fenhexamid, chlordecone, and rhothane on human 17ß-HSD1 were 21.37, 73.25, 92.80, and 117.69 µM. Kinetic analysis revealed that endosulfan acts as a competitive inhibitor, fenhexamid as a mixed/competitive inhibitor, chlordecone and rhothane as a mixed/uncompetitive inhibitor. In BeWo cells, all insecticides except endosulfan significantly decreased estradiol production at 100 µM. For rats, the IC50 values for dimethomorph, fenhexamid, and chlordecone were 11.98, 36.92, and 109.14 µM. Dimethomorph acts as a mixed inhibitor, while fenhexamid acts as a mixed/competitive inhibitor. Docking analysis revealed that endosulfan and fenhexamid bind to the steroid-binding site of human 17ß-HSD1. On the other hand, chlordecone and rhothane binds to a different site other than the steroid and NADPH-binding site. Dimethomorph binds to the steroid/NADPH binding site, and fenhexamid binds to the steroid binding site of rat 17ß-HSD1. Bivariate correlation analysis showed a positive correlation between IC50 values and LogP for human 17ß-HSD1, while a slight negative correlation was observed between IC50 values and the number of HBA. ADMET analysis provided insights into the toxicokinetics and toxicity of organochlorine pesticides. In conclusion, this study identified the inhibitory effects of 3-4 organochlorine pesticides and binding mechanisms on human and rat 17ß-HSD1, as well as their impact on hormone production.

Bisphenol analogues inhibit human and rat 17ß-hydroxysteroid dehydrogenase 1: 3D-quantitative structure-activity relationship (3D-QSAR) and in silico docking analysis.

Bisphenols, estrogenic endocrine-disrupting chemicals, disrupt at least one of three endocrine pathways (estrogen, androgen, and thyroid). 17ß-Hydroxysteroid dehydrogenase 1 (17ß-HSD1) is a steroidogenic enzyme that catalyzes the activation of estradiol from estrone in human placenta and rat ovary. However, whether bisphenols inhibit 17ß-HSD1 and the mode of action remains unclear. This study we screened 17 bisphenols for inhibiting human 17ß-HSD1 in placental microsomes and rat 17ß-HSD1 in ovarian microsomes and determined 3D-quantitative structure-activity relationship (3D-QSAR) and mode of action. We observed some bisphenols with substituents were found to significantly inhibit both human and rat 17ß-HSD1 with the most potent inhibition on human enzyme by bisphenol H (IC50 = 0.90 µM) when compared to bisphenol A (IC50 = 113.38 µM). Rat enzyme was less sensitive to the inhibition of bisphenols than human enzyme with bisphenol H (IC50 = 32.94 µM) for rat enzyme. We observed an inverse correlation between IC50 and hydrophobicity (expressed as Log P). Docking analysis showed that they bound steroid-binding site of 17ß-HSD1. The 3D-QSAR models demonstrated that hydrophobic region, hydrophobic aromatic, ring aromatic, and hydrogen bond acceptor are key factors for the inhibition of steroid synthesis activity of 17ß-HSD1.

Dysregulated miR-29a-3p/PMP22 Modulates Schwann Cell Proliferation and Migration During Peripheral Nerve Regeneration.

Schwann cells switch to a repair phenotype following peripheral nerve injury and create a favorable microenvironment to drive nerve repair. Many microRNAs (miRNAs) are differentially expressed in the injured peripheral nerves and play essential roles in regulating Schwann cell behaviors. Here, we examine the temporal expression patterns of miR-29a-3p after peripheral nerve injury and demonstrate significant up-regulation of miR-29a-3p in injured sciatic nerves. Elevated miR-29a-3p inhibits Schwann cell proliferation and migration, while suppressed miR-29a-3p executes reverse effects. In vivo injection of miR-29a-3p agomir to rat sciatic nerves hinders the proliferation and migration of Schwann cells, delays the elongation and myelination of axons, and retards the functional recovery of injured nerves. Mechanistically, miR-29a-3p modulates Schwann cell activities via negatively regulating peripheral myelin protein 22 (PMP22), and PMP22 extensively affects Schwann cell metabolism. Our results disclose the vital role of miR-29a-3p/PMP22 in regulating Schwann cell phenotype following sciatic nerve injury and shed light on the mechanistic basis of peripheral nerve regeneration.

Biocompatible Assessment of Erythrocyte Membrane-Camouflaged Polymeric PLGA Nanoparticles in Pregnant Mice: Both on Maternal and Fetal/Juvenile Mice.

Purpose: Poly(lactic-co-glycolic) acid (PLGA) nanoparticles coated with the membrane of red blood cells (RBC-NP) have been applied in various biomedical fields. Despite the well-documented great biocompatibility, the potential toxicity of RBC-NP on maternal mice or their developing fetuses during pregnancy, or juvenile mice post-birth, remains unclear, which warrants a systematic evaluation. Methods: We fabricate an RBC-NP with approximately 50 nm in diameter (RBC-NP-50). Upon RBC-NP-50, pregnant mice are intravenously injected with this nanoparticle either at a single high dose of 400 mg/kg (1HD) or a low dose of 200 mg/kg for 3 times (3LD). Afterwards, the biocompatible assessments are performed at 48 h after the final injection or 21 d post-birth/partum both on maternal and fetal/juvenile mice. Results: RBC-NP-50 is capable of accumulating in the placenta and then passing through the blood-fetal barrier (BFB) into the fetus. On 48 h after RBC-NP-50 exposure, no significant dose-dependent toxicity is observed in maternal mice including blood biochemistry, inflammatory factors, progesterone level, histological analysis, etc, whereas fetal brains reveal remarkable differentially expressed genes analyzed by transcriptome sequencing. On 21 d post-birth, those genes' expression in juvenile mice is alleviated, along with negligible differences in behavioral evaluations including surface righting test, negative geotaxis test, cliff avoidance test, and olfactory orientation test. Conclusion: These results indicate that RBC-NP is considered to be generally safe and biocompatible both for maternal mice and fetus during pregnancy, and for the subsequent juvenile mice post-birth, although future studies will need to examine higher dosage or longer-term measurements.

miR-328a-3p stimulates endothelial cell migration and tubulogenesis.

Endothelial cells have important biological roles after peripheral nerve injury by forming blood vessels within the nerve gap and guiding Schwann cell migration. MicroRNAs (miRNAs/miRs) affect cellular behavior and regulate a wide variety of physiological and pathological activities, including peripheral nerve regeneration. Emerging studies have identified the essential roles of miRNAs in the phenotype modulation of Schwann cells, while the effects of miRNAs on endothelial cells have remained to be thoroughly investigated. miR-328a-3p was differentially expressed in peripheral nerve stumps after nerve injury. In the present study, the effects of miR-328a-3p on biological functions of endothelial cells were determined by transfecting cultured human umbilical vein endothelial cells (HUVECs) with miR-328a-3p mimics or inhibitor. Transfection with miR-328a-3p mimics led to slightly decreased HUVEC proliferation and robustly increased HUVEC migration and tubulogenesis, while transfection with miR-328a-3p inhibitor led to opposite results. Using bioinformatics analysis, potential regulators and effectors of miR-328a-3p were further discovered and a miR-328a-3p-centered competing endogenous RNA network was constructed. Collectively, the present study demonstrated that dysregulated miR-328a-3p after peripheral nerve injury may affect the migration and angiogenesis of endothelial cells and contribute to peripheral nerve regeneration.

The Association of Polymorphisms in Base Excision Repair Genes with Ovarian Cancer Susceptibility in Chinese Women: A Two-Center Case-Control Study.

Base excision repair (BER) acts upon the most important mechanism of the DNA repair system, protecting DNA stability and integrity from the mutagenic and cytotoxic effects. Multiple researches have indicated that single-nucleotide polymorphisms (SNPs) in the BER-related gene may be associated with the susceptibility of ovarian cancer. However, the results are controversial. In this two-center case-control study, 19 potentially functional SNPs in six BER-related genes (hOGG1, APE1, PARP1, FEN1, LIG3 and XRCC1) was genotyped in 196 ovarian cancer cases and 272 cancer-free controls. And, their associations with ovarian cancer risk were assessed by unconditional logistic regression analyses. We found that PARP1 rs8679 and hOGG1 rs293795 polymorphisms were associated with a decreased risk of ovarian cancer under dominant model (adjusted OR=0.39, 95% CI=0.17-0.90, P=0.026; and adjusted OR=0.36, 95% CI=0.13-0.99, P=0.049, respectively). Stratification analysis demonstrated that this association was more pronounced in the subgroups of lower BMI and patients with early menarche and serous carcinoma. Moreover, LIG3 rs4796030 AA/AC variant genotypes performed an increased risk of ovarian cancer under recessive model (adjusted OR=1.54, 95% CI=1.01-2.35, P=0.046), especially in the subgroups of higher BMI, early clinic stage and the carcinoma at the left. These results suggested that PARP1, hOGG1 and LIG3 polymorphisms might impact on the risk of ovarian cancer. However, more researches with larger and different ethnic populations are warranted to support our findings.

Characteristics of cytokines in the sciatic nerve stumps and DRGs after rat sciatic nerve crush injury.

BACKGROUND: Cytokines are essential cellular modulators of various physiological and pathological activities, including peripheral nerve repair and regeneration. However, the molecular changes of these cellular mediators after peripheral nerve injury are still unclear. This study aimed to identify cytokines critical for the regenerative process of injured peripheral nerves. METHODS: The sequencing data of the injured nerve stumps and the dorsal root ganglia (DRGs) of Sprague-Dawley (SD) rats subjected to sciatic nerve (SN) crush injury were analyzed to determine the expression patterns of genes coding for cytokines. PCR was used to validate the accuracy of the sequencing data. RESULTS: A total of 46, 52, and 54 upstream cytokines were differentially expressed in the SNs at 1 day, 4 days, and 7 days after nerve injury. A total of 25, 28, and 34 upstream cytokines were differentially expressed in the DRGs at these time points. The expression patterns of some essential upstream cytokines are displayed in a heatmap and were validated by PCR. Bioinformatic analysis of these differentially expressed upstream cytokines after nerve injury demonstrated that inflammatory and immune responses were significantly involved. CONCLUSIONS: In summary, these findings provide an overview of the dynamic changes in cytokines in the SNs and DRGs at different time points after nerve crush injury in rats, elucidate the biological processes of differentially expressed cytokines, especially the important roles in inflammatory and immune responses after peripheral nerve injury, and thus might contribute to the identification of potential treatments for peripheral nerve repair and regeneration.