Active efflux transport of glucuronides out of cells is a critical process in elimination of drugs and food-derived compounds. Wushanicaritin, a natural polyphenol from Epimedium species, has shown many biological activities. However, the transporters responsible for excretion of wushanicaritin glucuronides still remain undefined. Herein, chemical inhibitors (Ko143, MK571, dipyridamole and leukotriene C4) and single stable knocked-down efflux transporters (BCRP, MRP1, MRP3 and MRP4) were used to determine the contributions of efflux transporters to glucuronide efflux and cellular glucuronidation in UGT1A1-overexpressing HeLa cells (HeLa1A1). Knock-down of transporters was performed by stable transfection of short hairpin RNA (shRNA) using lentiviral vectors. The HeLa1A1 cell lysate catalyzed wushanicaritin glucuronidation, generating wushanicaritin-3-O-glucuronide and wushanicaritin-7-O-glucuronide. Ko143 (a dual inhibitor of BCRP, 5-20 μM) caused a marked decrease in excretion rate (maximal 53.4%) and increase of intracellular glucuronides (maximal 86.0%), while MK-571 (an inhibitor of MRPs, 5-20 μM) resulted in a significant reduction in excretion rate (maximal 64.6%) and rise of intracellular glucuronides (maximal 98.0%). By contrast, dipyridamole and leukotriene C4 showed no inhibitory effects on glucuronide excretion. Furthermore, shRNA-mediated silencing of a target transporter led to a marked reduction in the excretion rate of wushanicaritin glucuronides (maximal 33.8% for BCRP; 25.9% for MRP1; 26.7% for MRP3; 39.3% for MRP4). Transporter silencing also led to substantial decreases in efflux clearance (maximal 61.5% for BCRP; 48.7% for MRP1; 35.1% for MRP3; 63.1% for MRP4). In conclusion, chemical inhibition and gene silencing results suggested that BCRP, MRP1, MRP3 and MRP4 were significant contributors to excretion of wushanicaritin glucuronides.

SummaryHypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death in young individuals. A potential role of mtDNA mutations in HCM is known. However, the underlying molecular mechanisms linking mtDNA mutations to HCM remain poorly understood due to lack of cell and animal models. Here, we generated induced pluripotent stem cell-derived cardiomyocytes (HCM-iPSC-CMs) from human patients in a maternally inherited HCM family who carry the m.2336T>C mutation in the mitochondrial 16S rRNA gene (MT-RNR2). The results showed that the m.2336T>C mutation resulted in mitochondrial dysfunctions and ultrastructure defects by decreasing the stability of 16S rRNA, which led to reduced levels of mitochondrial proteins. The ATP/ADP ratio and mitochondrial membrane potential were also reduced, thereby elevating the intracellular Ca2+ concentration, which was associated with numerous HCM-specific electrophysiological abnormalities. Our findings therefore provide an innovative insight into the pathogenesis of maternally inherited HCM.

Endometriosis is one of the most common disorders that causes infertility in women. Iron is overloaded in endometriosis peritoneal fluid (PF), with harmful effects on early embryo development. However, the mechanism by which endometriosis peritoneal fluid affects embryonic development remains unclear. Hence, this study investigated the effect of iron overload on mouse embryos and elucidated the molecular mechanism. Iron overload in endometriosis PF disrupted blastocyst formation, decreased GPX4 expression and induced lipid peroxidation, suggesting that iron overload causes embryotoxicity and induces ferroptosis. Moreover, mitochondrial damage occurs in iron overload-treated embryos, presenting as decreased ATP levels, increased ROS levels and MMP hyperpolarization. The cytotoxicity of iron overload is attenuated by the ferroptosis inhibitor Fer-1. Furthermore, Smart-seq analysis revealed that HMOX1 is upregulated in embryo ferroptosis and that HMOX1 suppresses ferroptosis by maintaining mitochondrial function. This study provides new insight into the mechanism of endometriosis infertility and a potential target for future endometriosis infertility treatment efforts.

BackgroundHypertrophic cardiomyopathy (HCM) is a primary disorder characterised by asymmetric thickening of septum and left ventricular wall, with a prevalence of 0.2% in the general population.ObjectiveTo describe a novel mitochondrial DNA mutation and its association with the pathogenesis of HCM.Methods and resultsAll maternal members of a Chinese family with maternally transmitted HCM exhibited variable severity and age at onset, and were implanted permanent pacemakers due to complete atrioventricular block (AVB). Nuclear gene screening (MYH7, MYBPC3, TNNT2 and TNNI3) was performed, and no potential pathogenic mutation was identified. Mitochondrial DNA sequencing analysis identified a novel homoplasmic 16S rRNA 2336T>C mutation. This mutation was exclusively present in maternal members and absent in non-maternal members. Conservation index by comparison to 16 other vertebrates was 94.1%. This mutation disturbs the 2336U-A2438 base pair in the stem–loop structure of 16S rRNA domain III, which is involved in the assembly of mitochondrial ribosome. Oxygen consumption rate of the lymphoblastoid cells carrying 2336T>C mutation had decreased by 37% compared with controls. A reduction in mitochondrial ATP synthesis and an increase in reactive oxidative species production were also observed. Electron microscopic analysis indicated elongated mitochondria and abnormal mitochondrial cristae shape in mutant cells.ConclusionsIt is suggested that the 2336T>C mutation is one of pathogenic mutations of HCM. This is the first report of mitochondrial 16S rRNA 2336T>C mutation and an association with maternally inherited HCM combined with AVB. Our findings provide a new insight into the pathogenesis of HCM.

Corylin, an phenolic compound from Psoralea corylifolia, has been reported with various pharmacological properties but has poor bioavailability due to massive metabolism. In this study, twelve metabolites of corylin mainly involving in oxidation, hydration, glucuronidation and sulfation were detected in mice. Furthermore, the oxidation and hydration of corylin (M4) in human liver microsomes (HLM) and human intestine microsomes (HIM) were both efficient with high CL (intrinsic clearance) values of 24.29 and 42.85 μL/min/mg, respectively. CYP1A1, 1B1 and 2C19 contributed most for M4 with the CL values of 26.63, 33.09 and 132.41 μL/min/mg, respectively. Besides, M4 was strongly correlated with phenacetin-N-deacetylation (r = 0.885, p = 0.0001) and tolbutamide-4-oxidation (r = 0.727, p = 0.001) in twelve individual HLMs, respectively. In addition, corylin was efficiently glucuronidated (M7) in HLM (125.33 μL/min/mg) and in HIM (108.74 μL/min/mg). UGT1A1 contributed the most for M7 with the CL value of 122.32 μL/min/mg. Meanwhile, M7 was significantly correlated with β-estradiol-3-O-glucuronidation (r = 0.742, p = 0.006) in twelve individual HLMs. Moreover, the metabolism of corylin showed marked species differences. Taken together, corylin was subjected to massive first-pass metabolism in liver and intestine, while CYP1A1, 1B1, 2C19 and UGT1A1 were the main contributors. Finally, the proposed metabolic pathway of corylin involed CYP and UGT isoforms were summarized, which could help to understand the metabolic fate of corylin in vivo.

Norbakuchinic acid (NBKA) is the most abundant metabolite of bakuchiol (a hepatotoxicity and nephrotoxicity component inPsoralea corylifoliaL.) in plasma and urine.

ObjectiveThe aim of this meta-analysis is to explore the beneficial role of granulocyte colony-stimulating factor (G-CSF) on infertile women under artificial reproduction technology treatment.MethodMedline, Embase and ISI Web of Science databases were searched to identify relevant randomized control trials. Studies before July, 2017 were included for primary screening. Meta-analysis of the total and subgroup patients was conducted, and relative risks (RRs) and their 95% confidence intervals (95% CI) were calculated by a fixed-effect model if no heterogeneity (evaluated as I2 statistic) existed. Otherwise, a random-effects model was adopted. Subgroup analysis was performed by administrating route or clinical indication. Egger test and influence analysis were conducted to evaluate the publication bias and study power, respectively.ResultsThe final selection enrolled 10 RCTs, involving 1016 IVF-ET cycles (521 distributed to the G-CSF group and 495 to the control). Compared with control group, G-CSF administration could significantly improve clinical pregnancy rate (CPR, RR 1.89, 95% CI 1.53–2.33), while it had no beneficial effect on embryo implantation rate (IR, RR 1.84, 95% CI 0.84–4.03). The subgroup analysis by administration route showed that both uterine infusion and subcutaneous injection can produce a substantial increase in CPR, with the pooled RRs (95% CI) 1.46 (1.04–2.05) and 2.23 (1.68–2.95), respectively. Nevertheless, most of included RCTs dealt with the RIF subjects, and the pooled analysis of this data showed a higher PR and IR in G-CSF group as compared to that in the control, with the RRs (95% CI) 2.07 (1.64–2.61) and 1.52 (1.08–2.14), respectively. Egger regression test did not demonstrate any significance for the publication bias.ConclusionG-CSF administration has a beneficial role on the clinical outcome after embryo transfer by both routes of local infusion and systematic administration, especially for the cases with RIF. Further RCTs are needed to investigate the role of G-CSF in thin endometrium patients.Electronic supplementary materialThe online version of this article (10.1007/s00404-018-4892-4) contains supplementary material, which is available to authorized users.

Neobavaisoflavone (NBIF), a phenolic compound isolated from Psoralea corylifolia L., possesses several significant biological properties. However, the pharmacokinetic behaviors of NBIF have been characterized as rapid oral absorption, high clearance, and poor oral bioavailability. We found that NBIF underwent massive glucuronidation and oxidation by human liver microsomes (HLM) in this study with the intrinsic clearance (CL) values of 12.43, 10.04, 2.01, and 6.99 μL/min/mg for M2, M3, M4, and M5, respectively. Additionally, the CL values of G1 and G2 by HLM were 271.90 and 651.38 μL/min/mg, respectively, whereas their respective parameters were 59.96 and 949.01 μL/min/mg by human intestine microsomes (HIM). Reaction phenotyping results indicated that CYP1A1, 1A2, 2C8, and 2C19 were the main contributors to M4 (34.96 μL/min/mg), M3 (29.45 μL/min/mg), M3 (13.16 μL/min/mg), and M2 (63.42 μL/min/mg), respectively. UGT1A1, 1A7, 1A8, and 1A9 mainly catalyzed the formation of G1 (250.87 μL/min/mg), G2 (438.15 μL/min/mg), G1 (92.68 μL/min/mg), and G2 (1073.25 μL/min/mg), respectively. Activity correlation analysis assays showed that phenacetin-N-deacetylation was strongly correlated to M3 (r = 0.860, p = 0.003) and M4 (r = 0.775, p = 0.014) in nine individual HLMs, while significant activity correlations were detected between paclitaxel-6-hydroxylation and M2 (r = 0.675, p = 0.046) and M3 (r = 0.829, p = 0.006). There was a strong correlation between β-estradiol-3-O-glucuronide and G1 (r = 0.822, p = 0.007) and G2 (r = 0.689, p = 0.040), as well as between propofol-O-glucuronidation and G1 (r = 0.768, p = 0.016) and G2 (r = 0.860, p = 0.003). Moreover, the phase I metabolism and glucuronidation of NBIF revealed marked species differences, and mice are the best animal model for investigating the metabolism of NBIF in humans. Taken together, characterization of NBIF-related metabolic pathways involving in CYP1A1, 1A2, 2C8, 2C19, and UGT1A1, 1A7, 1A8, 1A9 are helpful for understanding the pharmacokinetic behaviors and conducting in-depth pharmacological studies.