ß-hydroxybutyrate administered at reperfusion reduces infarct size and preserves cardiac function by improving mitochondrial function through autophagy in male mice.

Ischemia/reperfusion (I/R) injury after revascularization contributes ∼50% of infarct size and causes heart failure, for which no established clinical treatment exists. ß-hydroxybutyrate (ß-OHB), which serves as both an energy source and a signaling molecule, has recently been reported to be cardioprotective when administered immediately before I/R and continuously after reperfusion. This study aims to determine whether administering ß-OHB at the time of reperfusion with a single dose can alleviate I/R injury and, if so, to define the mechanisms involved. We found plasma ß-OHB levels were elevated during ischemia in STEMI patients, albeit not to myocardial protection level, and decreased after revascularization. In mice, compared with normal saline, ß-OHB administrated at reperfusion reduced infarct size (by 50%) and preserved cardiac function, as well as activated autophagy and preserved mtDNA levels in the border zone. Our treatment with one dose ß-OHB reached a level achievable with fasting and strenuous physical activity. In neonatal rat ventricular myocytes (NRVMs) subjected to I/R, ß-OHB at physiologic level reduced cell death, increased autophagy, preserved mitochondrial mass, function, and membrane potential, in addition to attenuating reactive oxygen species (ROS) levels. ATG7 knockdown/knockout abolished the protective effects of ß-OHB observed both in vitro and in vivo. Mechanistically, ß-OHB's cardioprotective effects were associated with inhibition of mTOR signaling. In conclusion, ß-OHB, when administered at reperfusion, reduces infarct size and maintains mitochondrial homeostasis by increasing autophagic flux (potentially through mTOR inhibition). Since ß-OHB has been safely tested in heart failure patients, it may be a viable therapeutic to reduce infarct size in STEMI patients.

α-Tocotrienol Protects Neurons by Preventing Tau Hyperphosphorylation via Inhibiting Microtubule Affinity-Regulating Kinase Activation.

In the pathological process of Alzheimer's disease, neuronal cell death is closely related to the accumulation of reactive oxygen species. Our previous studies have found that oxidative stress can activate microtubule affinity-regulating kinases, resulting in elevated phosphorylation levels of tau protein specifically at the Ser262 residue in N1E-115 cells that have been subjected to exposure to hydrogen peroxide. This process may be one of the pathogenic mechanisms of Alzheimer's disease. Vitamin E is a fat-soluble, naturally occurring antioxidant that plays a crucial role in biological systems. This study aimed to examine the probable processes that contribute to the inhibiting effect on the abnormal phosphorylation of tau protein and the neuroprotective activity of a particular type of vitamin E, α-tocotrienol. The experimental analysis revealed that α-tocotrienol showed significant neuroprotective effects in the N1E-115 cell line. Our data further suggest that one of the mechanisms underlying the neuroprotective effects of α-tocotrienol may be through the inhibition of microtubule affinity-regulated kinase activation, which significantly reduces the oxidative stress-induced aberrant elevation of p-Tau (Ser262) levels. These results indicate that α-tocotrienol may represent an intriguing strategy for treating or preventing Alzheimer's disease.

Oxidative stress induces tau hyperphosphorylation via MARK activation in neuroblastoma N1E-115 cells.

Reactive oxygen species are considered a cause of neuronal cell death in Alzheimer's disease (AD). Abnormal tau phosphorylation is a proven pathological hallmark of AD. Microtubule affinity-regulating kinases (MARKs) regulate tau-microtubule binding and play a crucial role in neuronal survival. In this study, we hypothesized that oxidative stress increases the phosphorylation of Ser262 of tau protein through activation of MARKs, which is the main reason for the development of AD. We investigated the relationship between tau hyperphosphorylation on Ser262 and MARKs in N1E-115 cells subjected to oxidative stress by exposure to a low concentration of hydrogen peroxide. This work builds on the observation that hyperphosphorylation of tau is significantly increased by oxidative stress. MARKs activation correlated with tau hyperphosphorylation at Ser262, a site that is essential to maintain microtubule stability and is the initial phosphorylation site in AD. These results indicated that MARKs inhibitors might serve a role as therapeutic tools for the treatment of AD.

Filtering blue light mitigates the deleterious effects induced by the oxidative stress in human retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a major cause of blindness in elderly. It is characterized by the loss of central vision due to damaged retinal pigment epithelial (RPE) cells and photoreceptors. Blue Light (BL) exposure was proposed as a risk factor for AMD progression. We undertook this study to determine the effects of BL on the behaviour of RPE cells and their potential mitigation by BL-filtering intraocular lenses (IOL). Human RPE cells were exposed or not to BL, with the absence or presence of either a clear ultraviolet (UV)-filtering IOL (CIOL), or a yellow UV- and BL-filtering IOL (YIOL). Cells were analyzed for their oxidative stress by measuring the levels of reactive oxygen species (ROS), and their viability. BL exposure significantly increased the levels of both total cellular and mitochondrial ROS. While this increase was not affected by placing the CIOL in the BL beam, YIOL decreased the levels of both ROS reservoirs. Increased ROS production was accompanied by increased cell death which was similarly decreased when cells were protected with the YIOL. Pre-treatment of cells with N-acetylcycteine (NAC) abolished the increased cell death, suggesting that the effects of BL on cell viability were mainly due to increased levels of ROS. BL is deleterious to RPE cells due to increased oxidative stress and cell death. These effects were mitigated by filtering these radiations. The use of BL-filtering devices may represent a strategy to reduce these effects on RPE cells and delay the onset of AMD.

Anticancer effects of mifepristone on human uveal melanoma cells.

BACKGROUND: Uveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds. METHODS: The effects of incremental concentrations of MF (0, 5, 10, 20, or 40 µM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR. RESULTS: MF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor. CONCLUSION: This study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

The role of hydrogen in the prevention and treatment of coronary atherosclerotic heart disease.

Coronary atherosclerotic heart disease (CHD) is a primary cardiovascular disease caused by atherosclerosis (AS), which is characterized by chronic inflammation and lipid oxidative deposition. Molecular hydrogen (H2) is an effective anti-inflammatory agent and has potential to ameliorate glycolipid metabolism disorders, which is believed to exert beneficial effects on the prevention and treatment of CHD. It is suggested that H2 reduces inflammation in CHD by regulating multiple pathways, including NF-κB inflammatory pathway, pyroptosis, mitophagy, endoplasmic reticulum (ER) stress, and Nrf2 antioxidant pathway. Additionally, H2 may improve glycolipid metabolism by mediation of PI3K and AMPK signalling pathways, contributing to inhibition of the occurrence and development of CHD. This review elaborates pathogenesis of CHD and evaluates the role of H2 in CHD. Moreover, possible molecular mechanisms have been discussed and speculated, aiming to provide more strategies and directions for subsequent studies of H2 in CHD.

Toralactone alleviates cisplatin-induced acute kidney injury by modulating the gut microbiota-renal axis.

BACKGROUND: Gut microbiota has been reported to be perturbed by cisplatin and to modulate the nephrotoxicity of chemotherapeutic agents. However, the critical role of toralactone, a bioactive components of Cassia obtusifolia L. seeds, in modulating the gut microbiota in the pathogenesis of cisplatin-induced nephrotoxicity remains to be elucidated. METHODS: In this study, we verified the reno-protective effects of toralactone and compared the composition and function of the gut microbiota in the normal, cisplatin-treated and low or high dose of toralactone-treated mice using 16S rDNA gene sequencing. We also investigated the gut microbiota related LPS/TLR4/NF-κB/TNF-α pathway in renal tissue. To elucidate the causal relationship between gut dysbiosis and cisplatin nephrotoxicity, an antibiotic cocktail was administered to deplete the gut microbiota and fecal microbiota transplantation (FMT) was performed prior to cisplatin treatment. RESULTS: The renal histopathology showed that toralactone significantly alleviated cisplatin-induced renal injury. 16S rDNA gene sequencing analysis demonstrated that toralactone treatment effectively reversed cisplatin-induced gut microbiota dysbiosis in mice. FMT from toralactone-treated mice to cisplatin-induced kidney injury mice was observed to have the reno-protective effects, and deletion of gut microbiota by antibiotics was found to negate the reno-protective effect of toralactone. Interestingly, the renal tissue of cisplatin-associated kidney injury mice showed activation of the LPS/TLR4/NF-κB pathway and increase in TNF-α within the renal tissue, whereas toralactone treatment was observed to inhibit the LPS/TLR4/NF-κB/TNF-α pathway. CONCLUSION: This study elucidated the reno-protective effects for the first time, demonstrating that it exerts its beneficial effects through the gut microbiota, which mediate the LPS/TLR4/NF-κB/TNF-α inflammatory pathway. It may help to develop therapeutic approaches using toralactone and targeted restoration of the gut microbiota.

Hydrogen gas inhalation ameliorates LPS-induced BPD by inhibiting inflammation via regulating the TLR4-NFκB-IL6/NLRP3 signaling pathway in the placenta.

INTRODUCTION: Hydrogen (H2) is regarded as a novel therapeutic agent against several diseases owing to its inherent biosafety. Bronchopulmonary dysplasia (BPD) has been widely considered among adverse pregnancy outcomes, without effective treatment. Placenta plays a role in defense, synthesis, and immunity, which provides a new perspective for the treatment of BPD. This study aimed to investigate if H2 reduced the placental inflammation to protect the neonatal rat against BPD damage and potential mechanisms. METHODS: We induced neonatal BPD model by injecting lipopolysaccharide (LPS, 1 µg) into the amniotic fluid at embryonic day 16.5 as LPS group. LPS + H2 group inhaled 42% H2 gas (4 h/day) until the samples were collected. We primarily analyzed the neonatal outcomes and then compared inflammatory levels from the control group (CON), LPS group and LPS + H2 group. HE staining was performed to evaluate inflammatory levels. RNA sequencing revealed dominant differentially expressed genes. Bioinformatics analysis (GO and KEGG) of RNA-seq was applied to mine the signaling pathways involved in protective effect of H2 on the development of LPS-induced BPD. We further used qRT-PCR, Western blot and ELISA methods to verify differential expression of mRNA and proteins. Moreover, we verified the correlation between the upstream signaling pathways and the downstream targets in LPS-induced BPD model. RESULTS: Upon administration of H2, the inflammatory infiltration degree of the LPS-induced placenta was reduced, and infiltration significantly narrowed. Hydrogen normalized LPS-induced perturbed lung development and reduced the death ratio of the fetus and neonate. RNA-seq results revealed the importance of inflammatory response biological processes and Toll-like receptor signaling pathway in protective effect of hydrogen on BPD. The over-activated upstream signals [Toll-like receptor 4 (TLR4), nuclear factor kappa-B p65 (NF-κB p65), Caspase1 (Casp1) and NLR family pyrin domain containing 3 (NLRP3) inflammasome] in LPS placenta were attenuated by H2 inhalation. The downstream targets, inflammatory cytokines/chemokines [interleukin (IL)-6, IL-18, IL-1ß, C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 1 (CXCL1)], were decreased both in mRNA and protein levels by H2 inhalation in LPS-induced placentas to rescue them from BPD. Correlation analysis displayed a positive association of TLR4-mediated signaling pathway both proinflammatory cytokines and chemokines in placenta. CONCLUSION: H2 inhalation ameliorates LPS-induced BPD by inhibiting excessive inflammatory cytokines and chemokines via the TLR4-NFκB-IL6/NLRP3 signaling pathway in placenta and may be a potential therapeutic strategy for BPD.

Clinical features and GCDH gene variants in three Chinese families with glutaric aciduria type 1: A case series and literature review.

Aim: To analyze the clinical phenotype and genetic etiology of three cases of glutaric aciduria type 1 (GA1) in Chinese children. Methods: We performed genetic and metabolic testing using tandem mass spectrometry (MS/MS) and gas chromatography-mass spectrometry (GC/MS), followed by trio whole-exome sequencing (trio-WES) and Sanger sequencing. A literature review on glutaric aciduria type 1 (GA1) in Chinese patients was also conducted. Results: Sequencing results showed each case had compound heterozygous variants in GCDH(NM_000159.4): c.214C > G (p.Arg72Gly) and c.411C > G (p.Tyr137Term) (Case 1), c.214C > G (p.Arg72Gly) and c.1204C > T (p.Arg402Trp) (Case 2), and c.1228G > T (p.Val410Leu) and c.395G > A (p.Arg132Gln) (Case 3). These variants were inherited from their respective parents. Notably, the c.214C > G variant found in two children was a novel variant not previously reported. A review of the literature revealed that, clinically, the majority of patients experienced onset in infancy and early childhood (82%). Additionally, 38.36% were diagnosed through newborn screening, with the primary reasons for the initial visit being delayed development (32.43%) and infections (21.61%). The most common clinical manifestations included increased head circumference (77.19%) and motor developmental delay (65.15%). Biochemically, patients exhibited significant elevations in C5DC (98.51%) and C5DC/C8 (94.87%) in blood, as well as GA (94.37%) and 3OHGA (69.39%) in urine. Radiographically, patients showed a high prevalence of abnormalities in cranial MRI (86.15%) and EEG (73.33%). Genetically, 67 distinct GCDH gene variants were identified among 73 patients, with missense variants being the most prevalent type (73.97%). The most frequent variant was c.1244-2 A > C, observed in 17.12% of cases. Additionally, the majority of variant sites were located in exons 11 (25.37%) and 6 (22.39%). Conclusion: GCDH variants were identified as the causative factors in the three children. The discovery of the novel variant (c.214C > G) expands the spectrum of pathogenic GCDH variants. These findings facilitate the diagnosis and treatment of affected children and provide a basis for genetic counseling and prenatal diagnosis for their families.

Identification of hub genes, signaling pathways and immune infiltration of recurrent spontaneous abortion based on bioinformatics analysis with clinical verification.

OBJECTIVE: The present study was aimed to identify hub genes associated with recurrent spontaneous abortion (RSA) via both bioinformatics analysis and clinical verification, also to evaluate the related pathways and immune infiltration situation of RSA, for exploring its underlying mechanism. MATERIALS AND METHODS: We screened candidate hub genes associated with RSA via bioinformatic analysis in the microarray datasets GSE22490 downloaded from the Gene Expression Omnibus (GEO) database. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to validate these hub genes. Several kinds of enrichment analysis were carried out to find out the pathways related to RSA. Additionally, CIBERSORT was used for evaluation of local immune Infiltration status of RSA. RESULTS: There were 536 differentially expressed genes (DEGs) including 301 upregulated and 235 downregulated genes in RSA group compared with healthy control group. Four hub genes (STAT3, TLR2, TLR4 and CD86) were finally screened out according to the protein-protein interaction (PPI) network analysis, RT-qPCR and Western blotting. Enrichment analysis showed that Toll-like receptor signaling pathway, neutrophil chemotaxis, chemokine signaling pathway and Fc gamma receptor-mediated phagocytosis were strongly associated with RSA. And in immune infiltration analysis, RSA tissue was found containing a higher proportion of monocytes and eosinophils. CONCLUSION: This study screened out four hub genes and several important pathways changed in the trophoblastic tissue of RSA patients. We also found that monocytes and eosinophils may be involved in RSA. These findings provide theoretical basis for further studies on the mechanisms of RSA.

Effects of Keemun and Dianhong Black Tea in Alleviating Excess Lipid Accumulation in the Liver of Obese Mice: A Comparative Study.

The chemical compositions of black teas differ greatly and may have different health benefits; however, systematic investigations into such benefits are lacking. Here, the chemical profiles of Keemun black tea (KBT) and Dianhong black tea (DBT), two common categories of tea in China, were analyzed, and their lipid-lowering effects in male C57BL/6 mice fed a high-fat diet (60% energy from fat) or the diet supplemented with 2% black tea powder for 15 weeks were investigated. The compounds most crucial in differentiating KBT and DBT were determined to be phenolic compounds, theanine, and D-psicose. DBT was more effective than KBT in preventing excess hepatic fat accumulation. Both black teas effectively and comparably altered the mRNA levels of hepatic lipid-metabolizing genes. DBT had more favorable effects in stimulating fecal fat excretion than did KBT. The differentiating compounds with the higher values of variable importance in the projection (VIP) might predominantly contribute to the different health benefits; however, the most essential compound or combination of compounds requires clarification.

EV-ADD, a database for EV-associated DNA in human liquid biopsy samples.

Extracellular vesicles (EVs) play a key role in cellular communication both in physiological conditions and in pathologies such as cancer. Emerging evidence has shown that EVs are active carriers of molecular cargo (e.g. protein and nucleic acids) and a powerful source of biomarkers and targets. While recent studies on EV-associated DNA (EV-DNA) in human biofluids have generated a large amount of data, there is currently no database that catalogues information on EV-DNA. To fill this gap, we have manually curated a database of EV-DNA data derived from human biofluids (liquid biopsy) and in-vitro studies, called the Extracellular Vesicle-Associated DNA Database (EV-ADD). This database contains validated experimental details and data extracted from peer-reviewed published literature. It can be easily queried to search for EV isolation methods and characterization, EV-DNA isolation techniques, quality validation, DNA fragment size, volume of starting material, gene names and disease context. Currently, our database contains samples representing 23 diseases, with 13 different types of EV isolation techniques applied on eight different human biofluids (e.g. blood, saliva). In addition, EV-ADD encompasses EV-DNA data both representing the whole genome and specifically including oncogenes, such as KRAS, EGFR, BRAF, MYC, and mitochondrial DNA (mtDNA). An EV-ADD data metric system was also integrated to assign a compliancy score to the MISEV guidelines based on experimental parameters reported in each study. While currently available databases document the presence of proteins, lipids, RNA and metabolites in EVs (e.g. Vesiclepedia, ExoCarta, ExoBCD, EVpedia, and EV-TRACK), to the best of our knowledge, EV-ADD is the first of its kind to compile all available EV-DNA datasets derived from human biofluid samples. We believe that this database provides an important reference resource on EV-DNA-based liquid biopsy research, serving as a learning tool and to showcase the latest developments in the EV-DNA field. EV-ADD will be updated yearly as newly published EV-DNA data becomes available and it is freely available at www.evdnadatabase.com.

Activation of Autophagic Flux Maintains Mitochondrial Homeostasis during Cardiac Ischemia/Reperfusion Injury.

Reperfusion injury after extended ischemia accounts for approximately 50% of myocardial infarct size, and there is no standard therapy. HDAC inhibition reduces infarct size and enhances cardiomyocyte autophagy and PGC1α-mediated mitochondrial biogenesis when administered at the time of reperfusion. Furthermore, a specific autophagy-inducing peptide, Tat-Beclin 1 (TB), reduces infarct size when administered at the time of reperfusion. However, since SAHA affects multiple pathways in addition to inducing autophagy, whether autophagic flux induced by TB maintains mitochondrial homeostasis during ischemia/reperfusion (I/R) injury is unknown. We tested whether the augmentation of autophagic flux by TB has cardioprotection by preserving mitochondrial homeostasis both in vitro and in vivo. Wild-type mice were randomized into two groups: Tat-Scrambled (TS) peptide as the control and TB as the experimental group. Mice were subjected to I/R surgery (45 min coronary ligation, 24 h reperfusion). Autophagic flux, mitochondrial DNA (mtDNA), mitochondrial morphology, and mitochondrial dynamic genes were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were treated with a simulated I/R injury to verify cardiomyocyte specificity. The essential autophagy gene, ATG7, conditional cardiomyocyte-specific knockout (ATG7 cKO) mice, and isolated adult mouse ventricular myocytes (AMVMs) were used to evaluate the dependency of autophagy in adult cardiomyocytes. In NRVMs subjected to I/R, TB increased autophagic flux, mtDNA content, mitochondrial function, reduced reactive oxygen species (ROS), and mtDNA damage. Similarly, in the infarct border zone of the mouse heart, TB induced autophagy, increased mitochondrial size and mtDNA content, and promoted the expression of PGC1α and mitochondrial dynamic genes. Conversely, loss of ATG7 in AMVMs and in the myocardium of ATG7 cKO mice abolished the beneficial effects of TB on mitochondrial homeostasis. Thus, autophagic flux is a sufficient and essential process to mitigate myocardial reperfusion injury by maintaining mitochondrial homeostasis and partly by inducing PGC1α-mediated mitochondrial biogenesis.

Effects of different dietary polyphenols on conformational changes and functional properties of protein-polyphenol covalent complexes.

In this study, conjugates of whey protein isolate (WPI) and four polyphenols (epigallocatechin gallate [EGCG], quercetin [QC], apigenin [AG], and naringenin [NG]) were prepared through free-radical grafting. The results for polyphenol binding equivalents and content of free amino and sulfhydryl groups as well as those from sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the covalent interaction between WPI and the polyphenols. Fourier transform infrared spectroscopy and fluorescence spectrum analysis identified the potential binding sites of the complexes and determined changes in the protein structure. The particle size distribution and scanning electron microscopy data demonstrated increases in conjugate particle sizes and surface changes in the complexes. The conjugation process significantly increased the polyphenols' antioxidant properties and thermal stabilities, whereas surface hydrophobicity was substantially reduced. WPI-EGCG had the best functional properties, followed by WPI-QC, WPI-AG, and WPI-NG.

Hydrogen, a Novel Therapeutic Molecule, Regulates Oxidative Stress, Inflammation, and Apoptosis.

Molecular hydrogen (H2) is a colorless and odorless gas. Studies have shown that H2 inhalation has the therapeutic effects in many animal studies and clinical trials, and its application is recommended in the novel coronavirus pneumonia treatment guidelines in China recently. H2 has a relatively small molecular mass, which helps it quickly spread and penetrate cell membranes to exert a wide range of biological effects. It may play a role in the treatment and prevention of a variety of acute and chronic inflammatory diseases, such as acute pancreatitis, sepsis, respiratory disease, ischemia reperfusion injury diseases, autoimmunity diseases, etc.. H2 is primarily administered via inhalation, drinking H2-rich water, or injection of H2 saline. It may participate in the anti-inflammatory and antioxidant activity (mitochondrial energy metabolism), immune system regulation, and cell death (apoptosis, autophagy, and pyroptosis) through annihilating excess reactive oxygen species production and modulating nuclear transcription factor. However, the underlying mechanism of H2 has not yet been fully revealed. Owing to its safety and potential efficacy, H2 has a promising potential for clinical use against many diseases. This review will demonstrate the role of H2 in antioxidative, anti-inflammatory, and antiapoptotic effects and its underlying mechanism, particularly in coronavirus disease-2019 (COVID-19), providing strategies for the medical application of H2 for various diseases.

Intrauterine endogenous high glucocorticoids program ovarian dysfunction in female offspring secondary to prenatal caffeine exposure.

Ovarian dysfunction has an intrauterine origin, and prenatal caffeine exposure (PCE) could lead to abnormal follicle counts in offspring after birth. However, the effect of PCE on offspring ovarian function and its mechanism of intrauterine programming have not been reported thus far. In this study, pregnant Wistar rats were intragastrically administered caffeine (30 and 120 mg/kg·d) at gestational days 9-20 (GD9-20). Certain tests were performed on the blood, ovaries and hypothalamus of female offspring at different time points. PCE female offspring had ovarian dysfunction in adulthood compared with the control. Further results showed that in utero ovarian morphological development and estradiol synthesis were inhibited but rapidly increased during puberty in the PCE group. The histone 3 lysine 27 acetylation (H3K27ac) level of the insulin-like growth factor 1 (IGF1) promoter region and its expression were decreased in the ovary, which was due to exposure to high levels of fetal blood corticosterone, and the H3K27ac level of IGF1 and its expression shifted to increase after birth with a decrease in serum corticosterone levels. Chronic stress led to increased serum corticosterone levels in adult offspring, whereas ovarian morphological development, the H3K27ac level of IGF1 and its expression, and estradiol synthesis were significantly inhibited. Moreover, the activity of the hypothalamic-pituitary-ovarian (HPO) axis was increased in the early postnatal period of PCE offspring, and chronic stress reversed these changes. In the KGN cell line, it was found that cortisol could promote the translocation of the glucocorticoid receptor (GR) into the nucleus and upregulate histone deacetylase 10 (HDAC10) to inhibit the H3K27ac level of IGF1 and its expression and estradiol synthesis. In summary, PCE is associated with ovarian dysfunction in female adult offspring, and the potential mechanism is related to intrauterine high glucocorticoid exposure by activating the GR and recruiting HDAC10 to affect ovarian glucocorticoid-IGF1 axis programming and to inhibit estradiol synthesis.

Benzophenones from Guignardia sp. IFB-E028, an endophyte on Hopea hainanensis.

The first natural S-containing benzophenone dimer, named guignasulfide (3), was isolated from the culture of Guignardia sp. IFB-E028, an endophytic fungus residing in healthy leaves of Hopea hainanensis. Its structure was determined through correlative analyses of its MS, 1D- and 2D-NMR spectroscopic data. Two other known benzophenone derivatives, monomethylsulochrin and rhizoctonic acid (1 and 2, resp.) were also isolated. Guignasulfide (3) was more active against the human liver cancer cell line HepG2 (IC(50) value: 5.2+/-0.4 microM) than metabolites 1 and 2 (IC(50) values: 63.5+/-0.6 and 60.2+/-0.5 microM); compounds 1-3 showed also moderately inhibitory effects on the human bacterial pathogen Helicobacter pylori with MIC values of 28.9+/-0.1, 60.2+/-0.4, and 42.9+/-0.5 microM, respectively.

Decreased levels of H3K9ac and H3K27ac in the promotor region of ovarian P450 aromatase mediated low estradiol synthesis in female offspring rats induced by prenatal nicotine exposure as well as in human granulosa cells after nicotine treatment.

Prenatal nicotine exposure (PNE) could induce ovarian dysplasia in offspring. This study aimed to confirm its intrauterine origin and explore a programming mechanism of ovarian dysplasia caused by PNE. Pregnant Wistar rats were injected subcutaneously with nicotine (2 mg/kg.d) from gestation day (GD) 9 to GD20. Serum of female offspring was obtained for hormone assays and ovarian tissues were collected. The results showed that PNE impaired ovarian development, and inhibited estradiol production and cytochrome P450 aromatase (P450arom) expression before and after birth. Moreover, the nicotinic acetylcholine receptors (nAChRs) expression was increased in utero, while histone 3 lysine 9 acetylation (H3K9ac) and H3K27ac levels in the P450arom promoter region were decreased persistently in PNE group before and after birth. In vitro, nicotine decreased P450arom expression and estradiol production in human granulosa cell line KGN. Furthermore, nicotine treatment up-regulated nAChRα6 and α9 expression and down-regulated the H3K9ac and H3K27ac levels of the P450arom promoter region. Non-specific nAChRs inhibitor vecuronium bromide reversed these effects. These results suggest that PNE could induce ovarian dysplasia and inhibit estradiol synthesis in the female offspring rats, which was related to the decreased H3K9ac and H3K27ac levels in the promotor region of the P450arom via the nAChRs.

Prenatal ethanol exposure induces susceptibility to premature ovarian insufficiency.

Prenatal ethanol exposure (PEE) adversely affects the offspring reproductive system. We aimed to confirm the susceptibility to premature ovarian insufficiency (POI) in female PEE offspring and elucidate its intrauterine programming mechanism. The pregnant Wistar female rats were intragastrically administered with 4 g/kg×d of ethanol from gestational day (GD) 9 to 20. Offspring reproductive parameters were detected on GD20, postnatal week (PW) 6, and PW12. The PEE foetuses showed a decreased number of oocytes, increased ovarian cell apoptosis, and upregulated expression levels of ovarian insulin-like growth factor 1 (IGF1) signaling pathway and steroidogenic enzymes. The proportion of atretic follicles in adult rats was increased, while the number of anti-Müllerian hormone-positive antral follicles was decreased. The serum oestradiol (E2) levels were decreased, but the follicle stimulation hormone levels were elevated. The ovarian Igf1 signaling pathway was transformed from activation during puberty to relative inhibition in adulthood, and the expression levels of ovarian steroidogenic enzymes were inhibited in adulthood. Furthermore, we treated the human granulosa cell line KGN with different ethanol concentrations (15, 30, 60, 120 mM) and found that the expression of IGF1 signaling pathway components, 3ß-HSD, and P450arom, as well as the production of E2, was increased. After IGF1 siRNA transfection, P450arom expression and E2 production were downregulated. These results suggest that PEE induces POI susceptibility in adult females, which may be caused by over-activation of the foetal ovarian Igf1 signaling pathway and steroidogenesis under PEE, resulting in accelerated early development of folliculogenesis and depletion of primordial follicles.

Inhibition of soluble epoxide hydrolase by extracts derived from inflammation-treating Chinese medicinal herbs.

Soluble epoxide hydrolase (sEH) has been proved to be a key enzyme involved in inflammation progression, and inhibition of sEH is therefore very helpful or crucial for the treatment of inflammation-related diseases. In order to uncover new clues suggesting the presence of phytochemical-based sEH inhibitors, and to rationalize the utility of the inflammation-treating Chinese medicinal herbs, the ethanol extracts derived from 46 medicinal herbs, traditionally used for the treatment of inflammation-associated diseases in China, were tested for sEH-inhibition activity using a recently developed sensitive fluorescence-based assay. Screened at 10 microg/mL, four extracts showed substantial inhibitions of sEH (inhibition rates >50%). The ethanol extract of Sophora flavescens root (Fabaceae) possessed the strongest inhibitory activity against sEH (IC(50): 2.07 microg/mL). These preliminary findings highlighted the presence of sEH inhibitor(s) in the plant kingdom, and the possibility that the inflammation-treating herbal medicines could be an untapped reservoir for sEH-inhibition agents.