Upregulation of GPR133 expression impaired the phagocytosis of macrophages in recurrent spontaneous miscarriage.

Decidual macrophages are the second-largest immune cell group at the maternal-foetal interface. They participate in apoptotic cell removal, and protect the foetus from microorganisms or pathogens. Dysfunction of decidual macrophages gives rise to pregnancy complications such as preeclampsia and recurrent spontaneous miscarriage (RSM). However, the mechanisms by which decidual macrophages are involved in the occurrence of adverse pregnancy outcomes have not been elucidated. Here we integrated DNA methylation and gene expression data from decidua macrophages to identify potential risk factors related to RSM. GPR133 was significantly hypomethylated and upregulated in decidual macrophages from RSM patients. Further demethylation analysis demonstrated that GPR133 expression in decidual macrophages was significantly increased by 5-Aza-dC treatment. In addition, the influence of GPR133 on the phagocytic ability of macrophages was explored. Phagocytosis was impaired in the decidual macrophages of RSM patients with increased GPR133 expression. Increased GPR133 expression induced by demethylation treatment in the decidual macrophages of healthy control patients led to a significant decrease in phagocytic function. Importantly, knockdown of GPR133 resulted in a significant improvement in the phagocytic function of THP-1 macrophages. In conclusion, the existing studies have shown the influence of GPR133 on the phagocytic function of decidual macrophages and pregnancy outcomes, providing new data and ideas for future research on the role of decidual macrophages in RSM.

Dysregulated Gln-Glu-α-ketoglutarate axis impairs maternal decidualization and increases the risk of recurrent spontaneous miscarriage.

Recurrent spontaneous miscarriage (RSM) affects 1%-2% of fertile women worldwide and poses a risk of future pregnancy complications. Increasing evidence has indicated that defective endometrial stromal decidualization is a potential cause of RSM. Here, we perform liquid chromatography with mass spectrometry (LC-MS)-based metabolite profiling in human endometrial stromal cells (ESCs) and differentiated ESCs (DESCs) and find that accumulated α-ketoglutarate (αKG) derived from activated glutaminolysis contributes to maternal decidualization. Contrarily, ESCs obtained from patients with RSM show glutaminolysis blockade and aberrant decidualization. We further find that enhanced Gln-Glu-αKG flux decreases histone methylation and supports ATP production during decidualization. In vivo, feeding mice a Glu-free diet leads to a reduction of αKG, impaired decidualization, and an increase of fetal loss rate. Isotopic tracing approaches demonstrate Gln-dependent oxidative metabolism as a prevalent direction during decidualization. Our results demonstrate an essential prerequisite of Gln-Glu-αKG flux to regulate maternal decidualization, suggesting αKG supplementation as a putative strategy to rectify deficient decidualization in patients with RSM.

Effects of Metabolism on Macrophage Polarization Under Different Disease Backgrounds.

Macrophages are versatile immune cells associated with various diseases, and their phenotypes and functions change on the basis of the surrounding environments. Reprogramming of metabolism is required for the proper polarization of macrophages. This review will focus on basic metabolic pathways, the effects of key enzymes and specific products, relationships between cellular metabolism and macrophage polarization in different diseases and the potential prospect of therapy targeted key metabolic enzymes. In particular, the types and characteristics of macrophages at the maternal-fetal interface and their effects on a successful conception will be discussed.

Decreased nitric oxide content mediated by asymmetrical dimethylarginine and protein l-arginine methyltransferase 3 in macrophages induces trophoblast apoptosis: a potential cause of recurrent miscarriage.

STUDY QUESTION: Is the protein l-arginine methyltransferase 3 (PRMT3)/asymmetrical dimethylarginine (ADMA)/nitric oxide (NO) pathway involved in the development of recurrent miscarriage (RM), and what is the potential mechanism? SUMMARY ANSWER: Elevated levels of PRMT3 and ADMA inhibit NO formation in the decidua, thereby impairing the functions of trophoblast cells at the maternal-foetal interface. WHAT IS KNOWN ALREADY: Decreased NO bioavailability is associated with RM. ADMA, an endogenous inhibitor of nitric oxide synthase (NOS), is derived from the methylation of protein arginine residues by PRMTs and serves as a predictor of mortality in critical illness. STUDY DESIGN, SIZE, DURATION: A total of 145 women with RM and 149 healthy women undergoing elective termination of an early normal pregnancy were enrolled. Ninety-six female CBA/J, 24 male DBA/2 and 24 male BALB/c mice were included. CBA/J × DBA/2 matings represent the abortion group, while CBA/J × BALB/c matings represent the normal control group. The CBA/J pregnant mice were then categorised into four groups: (i) normal + vehicle group (n = 28), (ii) abortion + vehicle group (n = 28), (iii) normal + SGC707 (a PRMT3 inhibitor) group (n = 20) and (iv) abortion + SGC707 group (n = 20). All injections were made intraperitoneally on Days 0.5, 3.5 and 6.5 of pregnancy. Decidual tissues were collected on Days 8.5, 9.5 and 10.5 of gestation. The embryo resorption rates were calculated on Day 9.5 and Day 10.5 of gestation. PARTICIPANTS/MATERIALS, SETTING, METHODS: NO concentration, ADMA content, NOS activity, expression levels of NOS and PRMTs in decidual tissues were determined using conventional assay kits or western blotting. PRMT3 expression was further analysed in decidual stromal cells, macrophages and natural killer cells. A co-culture system between decidual macrophages (DMs) and HTR-8/SVneo trophoblasts was constructed to study the roles of the PRMT3/ADMA/NO signalling pathway. Trophoblast apoptosis was analysed via Annexin V-fluorescein isothiocyanate/propidium iodide staining. CBA/J × DBA/2 mouse models were used to investigate the effects of SGC707 on embryo resorption rates. MAIN RESULTS AND THE ROLE OF CHANCE: Our results show that NO concentration and NOS activity were decreased, but ADMA content and PRMT3 expression were increased in the decidua of RM patients. Moreover, compared with the normal control subjects, PRMT3 expression was significantly up-regulated in the macrophages but not in the natural killer cells or stromal cells of the decidua from RM patients. The inhibition of PRMT3 results in a significant decrease in ADMA accumulation and an increase in NO concentration in macrophages. When co-cultured with DMs, which were treated with SGC707 and ADMA, trophoblast apoptosis was suppressed and induced, respectively. In vivo experiments revealed that the administration of SGC707 reduced the embryo resorption rate of CBA/J × DBA/2 mice. LIMITATIONS, REASONS FOR CAUTION: All sets of experiments were not performed with the same samples. The main reason is that each tissue needs to be reserved for clinical diagnosis and only a small piece of each tissue can be cut and collected for this study. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that the PRMT3/ADMA/NO pathway is a potential marker and target for the clinical diagnosis and therapy of RM. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Key Research and Development Program of China (2017YFC1001401), National Natural Science Foundation of China (81730039, 82071653, 81671460, 81971384 and 82171657) and Shanghai Municipal Medical and Health Discipline Construction Projects (2017ZZ02015). The authors have declared no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Regulation of the innate immune cells during pregnancy: An immune checkpoint perspective.

The foetus can be regarded as a half-allograft implanted into the maternal body. In a successful pregnancy, the mother does not reject the foetus because of the immune tolerance mechanism at the maternal-foetal interface. The innate immune cells are a large part of the decidual leukocytes contributing significantly to a successful pregnancy. Although the contributions have been recognized, their role in human pregnancy has not been completely elucidated. Additionally, the accumulated evidence demonstrates that the immune checkpoint molecules expressed on the immune cells are co-inhibitory receptors regulating their activation and biological function. Therefore, it is critical to understand the immune microenvironment and explore the function of the innate immune cells during pregnancy. This review summarizes the classic immune checkpoints such as PD-1, CTLA-4 and some novel molecules recently identified, including TIM-3, CD200, TIGIT and the Siglecs family on the decidual and peripheral innate immune cells during pregnancy. Furthermore, it emphasizes the role of the immune checkpoint molecules in pregnancy-associated complications and reproductive immunotherapy.

Upregulation of miR-29a suppressed the migration and invasion of trophoblasts by directly targeting LOXL2 in preeclampsia.

OBJECTIVE: Preeclampsia is a pregnancy-specific disorder that is a major cause of maternal and foetal morbidity and mortality, with a prevalence of 6-8% of pregnancies. Although the downregulation of lysyl oxidase (LOX) and LOX-like protein 2 (LOXL2), which leads to reduced trophoblast cell migration and invasion through activation of the TGF-ß1/Smad3/collagen pathway, is relevant to preeclampsia, the mechanisms regulating differences in the gene expression of LOX and LOXL2 in placentas are not yet understood. This study aimed to investigate the mechanisms regulating differences in the gene expression of LOX and LOXL2 in placentas. METHODS: The expression of miRNAs, LOX and LOXL2 in preeclamptic placentas and control placentas was analysed by qPCR. Localisation of miR29a and LOXL2 in preeclamptic placentas was performed by RNA-Fluorescence in-situ hybridization assay. The direct regulation of LOXL2 by miR-29a was assessed by dual-luciferase reporter assays in human extravillous trophoblast cells (HTR8/SVneo). Cell migration and invasion were evaluated by Transwell assays in HTR8/SVneo cells. RESULTS: miR-29a expression was upregulated in preeclamptic placentas and negatively correlated with LOXL2 mRNA expression levels. RNA-Fluorescence in-situ hybridization assay revealed a clear overlap between miR-29a and LOXL2 in the placentas of preeclampic women. LOXL2 was a direct target gene of miR-29a, as confirmed by a dual-luciferase reporter assay in HTR8/SVneo trophoblast cells. miR-29a suppressed HTR8/SVneo trophoblast cell migration and invasion. LOXL2 overexpression reversed the inhibitory effects of miR-29a on HTR8/SVneo trophoblast cell migration and invasion. CONCLUSION: Our results suggest that the upregulation of miR-29a suppresses the migration and invasion of HTR8/SVneo trophoblast cells by directly targeting LOXL2 in preeclampsia.

Downregulation of HDAC8 expression decreases CD163 levels and promotes the apoptosis of macrophages by activating the ERK signaling pathway in recurrent spontaneous miscarriage.

Recurrent spontaneous miscarriage (RSM) is a systemic disorder that has been defined as two or more pregnancies lost before the 20th week of gestation. Although the impaired function of macrophages at the maternal-fetal interface has been reported to be associated with RSM, the underlying mechanisms have not been fully elucidated. Here, we revealed that HDAC8 plays a critical role in RSM. Our results show that the mRNA and protein expression of HDAC8 was decreased in decidual macrophages from RSM patients. Moreover, the knockdown of HDAC8 resulted in a significant decrease in CD163 expression and an increase in apoptosis in dTHP-1 macrophages. Mechanistically, the ERK signaling pathway was activated in HDAC8-knockdown macrophages. When HDAC8-knockdown cells were pretreated with the ERK inhibitor U0126, expression levels of CD163, activated caspases 3, 7 and 9, and the apoptosis rate, were rescued. Taken together, our current results suggest that HDAC8 plays an important role in macrophage activation and apoptosis and may contribute to maintaining normal pregnancy by increasing the expression of M2 marker genes and inhibiting the apoptosis of macrophages at the maternal-fetal interface.

Molecular characteristics and possible functions of innate lymphoid cells in the uterus and gut.

With the discovery of innate lymphoid cells (ILCs), which are especially enriched in barrier surfaces, the family of innate lymphocytes has grown. A unique characterization of these cells can provide a phenotypical definition of ILCs and their specific functions in different tissue environments. Although ILCs are part of the innate immune system, they are derived from lymphoid lineages lacking rearranged antigen-specific and pattern-recognition receptors. The International Union of Immunological Societies (IUIS) favors the notion that ILCs can be generally divided into five main groups, namely, NK cells, ILC1s, ILC2s, ILC3s and LTi cells. These cells can be specifically stimulated by environmental and pathogen-derived signals. Upon stimulation, ILCs can rapidly secrete a wide range of soluble cytokines that can modulate the functions of effector cells. Over the last decade, ILCs, especially helper ILCs, which do not include NK cells, have been recognized to be a crucial cell type involved in integrating diverse host immune responses. Recently, emerging research has shown that helper ILCs also play a critical role in promoting tissue restoration and immune responses at barrier surfaces. Notably, helper ILCs act as a double-edged sword, being involved in the inflammatory and reparative responses during homeostasis and disease. Therefore, in this review, we summarize the current findings regarding the molecular characteristics and tissue-specific effector functions of helper ILCs in the uterus during physiological and pathological pregnancy and in the intestine during homeostasis and inflammation.

BODIPY-based selenides as fluorescent probes for rapid, sensitive and mitochondria-specific detection of hypochlorous acid.

Hypochlorous acid (HClO) is a powerful microbicidal agent in the innate immue system; however, abnormal HClO levels can cause tissue damage and many diseases. Thus, it is vitally important to develop facile, rapid and accurate analytical methods for the detection of HClO/ClO-in vitro and in vivo. In this work, we have constructed three meso-substituted BODIPY selenides with different hydrocarbyl groups (ethyl for BSe-Et, benzyl for BSe-Bz and phenyl for BSe-Ph) as fluorescent probes for the detection of HClO/ClO-. All three non-fluorescent probes can sense HClO/ClO- to form fluorescent selenoxides by blocking the photo-induced electron transfer process. Their sensing properties display a clear relationship with the structure of the hydrocarbyl. The sensing reactivity is heavily dependent on the electron-donating ability of hydrocarbyls, with the order of the response time as BSe-Et (2 s) < BSe-Bz (5 s) ≪ BSe-Ph (>100 s). Both BSe-Et and BSe-Bz afford a large fluorescence response and very low detection limits (0.3 nM and 0.8 nM), and BSe-Bz displays a higher selectivity over BSe-Et. Finally, as a representative, BSe-Bz was successfully applied to the detection of exo- and endogenous HClO in living cells, and demonstrated to be a mitochondria-localized fluorescent probe.

Macrophage Polarization in Physiological and Pathological Pregnancy.

The immunology of pregnancy is complex and poorly defined. During the complex process of pregnancy, macrophages secrete many cytokines/chemokines and play pivotal roles in the maintenance of maternal-fetal tolerance. Here, we summarized the current knowledge of macrophage polarization and the mechanisms involved in physiological or pathological pregnancy processes, including miscarriage, preeclampsia, and preterm birth. Although current evidence provides a compelling argument that macrophages are important in pregnancy, our understanding of the roles and mechanisms of macrophages in pregnancy is still rudimentary. Since macrophages exhibit functional plasticity, they may be ideal targets for therapeutic manipulation during pathological pregnancy. Additional studies are needed to better define the functions and mechanisms of various macrophage subsets in both normal and pathological pregnancy.

A C21-Steroidal Glycoside from Cynanchum atratum Attenuates Concanavalin A-Induced Liver Injury in Mice.

Cynatratoside A (CyA) is a C21 Steroidal glycoside with pregnane skeleton isolated from the root of Cynanchum atratum Bunge (Asclepiadaceae). This study aimed to investigate the effects of CyA on concanavalin A (Con A)-induced autoimmune hepatitis (AIH) and the underlying mechanism. CyA was orally administered to mice at 10 and 40 mg/kg 8 h before and 1 h after Con A treatment. The effects of CyA on Con A-induced spleen and liver in mice were assessed via histopathological changes, T lymphocyte amounts and the expressions of IL-1ß and ICAM-1. Con A-induced L-02 hepatocytes were used to evaluate whether CyA (0.1⁻10 µM) can directly protect hepatocytes from cytotoxicity and the possible mechanism. The results revealed that CyA treatment could significantly improve the histopathological changes of spleen and liver, reduce the proliferation of splenic T lymphocytes, and decrease the expressions of IL-1ß and ICAM-1 in liver. The experiment in vitro showed that CyA inhibited Con A-induced hepatotoxicity in a concentration-dependent manner. CyA (10 µM) significantly increased/decreased the expression of Bcl-2/Bax and reduced the levels of cleaved caspases-9 and -3. Our study demonstrated for the first time that CyA has a significant protective effect on Con A-induced AIH by inhibiting the activation and adhesion of T lymphocytes and blocking hepatocyte apoptosis.

Downregulation of lysyl oxidase and lysyl oxidase-like protein 2 suppressed the migration and invasion of trophoblasts by activating the TGF-ß/collagen pathway in preeclampsia.

Preeclampsia is a pregnancy-specific disorder that is a major cause of maternal and fetal morbidity and mortality with a prevalence of 6-8% of pregnancies. Although impaired trophoblast invasion in early pregnancy is known to be closely associated with preeclampsia, the underlying mechanisms remain elusive. Here we revealed that lysyl oxidase (LOX) and LOX-like protein 2 (LOXL2) play a critical role in preeclampsia. Our results demonstrated that LOX and LOXL2 expression decreased in preeclamptic placentas. Moreover, knockdown of LOX or LOXL2 suppressed trophoblast cell migration and invasion. Mechanistically, collagen production was induced in LOX- or LOXL2-downregulated trophoblast cells through activation of the TGF-ß1/Smad3 pathway. Notably, inhibition of the TGF-ß1/Smad3 pathway could rescue the defects caused by LOX or LOXL2 knockdown, thereby underlining the significance of the TGF-ß1/Smad3 pathway downstream of LOX and LOXL2 in trophoblast cells. Additionally, induced collagen production and activated TGF-ß1/Smad3 were observed in clinical samples from preeclamptic placentas. Collectively, our study suggests that the downregulation of LOX and LOXL2 leading to reduced trophoblast cell migration and invasion through activation of the TGF-ß1/Smad3/collagen pathway is relevant to preeclampsia. Thus, we proposed that LOX, LOXL2, and the TGF-ß1/Smad3/collagen pathway can serve as potential markers and targets for clinical diagnosis and therapy for preeclampsia.

Efficacy Comparison of Preprandial and Postprandial Prandilin 25 Administration in Patients with Newly Diagnosed Type 2 Diabetes Using a Continuous Glucose Monitoring System.

INTRODUCTION: The aim of this study was to determine the clinical efficacy of preprandial and postprandial Prandilin 25 (premixed insulin lispro 25) administration in patients with newly diagnosed type 2 diabetes mellitus (T2DM) using a continuous glucose monitoring (CGM) system. METHODS: This was a single-center, self-controlled comparative clinical trial. Newly diagnosed T2DM patients with hemoglobin A1c > 8.0% were hospitalized and received Prandilin 25 plus metformin treatment. Glycemic control was reached after a 7-to-8-day run-in period. Patients underwent 2 days of treatment consisting of preprandial Prandilin 25 on day 1 and postprandial Prandilin 25 on day 2 at the same dosage. The primary outcome was the 24-h mean amplitude of glycemic excursion (24 hMAGE); secondary outcomes were other daily glycemic variability parameters, including 24-h mean blood glucose (24hMBG), 24-h standard deviation of blood glucose (24hSDBG), large amplitude of glycemic excursion (LAGE), incremental area under the curve (AUC) values for different glucose levels, postprandial glucose excursion, and incidence of hypoglycemia, which were assessed using a CGM system. RESULTS: Eighty-five patients completed this study. There was no statistically significant difference in 24hMAGE, 24hMBG, 24hSDBG, or LAGE between the preprandial injection group and the postprandial injection group. Similarly, there was no between-treatment difference in the AUC for a blood glucose level below 3.9 mmol/L, in the AUC for a blood glucose level above 10.0 mmol/L, or in the percentages of time that the blood glucose level was below 3.9 mmol/L or above 10.0 mmol/L. Further analysis showed that the pre-meal glucose, peak height, and time to peak after each meal, the relative areas under the CGM curve at 1-4 h after each meal, as well as the incidence of hypoglycemia, were similar for the preprandial and postprandial Prandilin 25 groups. CONCLUSION: In patients with T2DM managed with premixed insulin lispro 25, postprandial injection (within 30 min of meal onset) may be an acceptable alternative to preprandial injection when the regular preprandial insulin dose is omitted. TRIAL REGISTRATION: Chinese Clinical Trial Register identifier: ChiCTR1800015828.

Trophoblast-Derived CXCL16 Decreased Granzyme B Production of Decidual γδ T Cells and Promoted Bcl-xL Expression of Trophoblasts.

BACKGROUND: Decidual γδ T cells are known to regulate the function of trophoblasts at the maternal-fetal interface; however, little is known about the molecular mechanisms of cross talk between trophoblast cells and decidual γδ T cells. METHODS: Expression of chemokine C-X-C motif ligand 6 (CXCL16) and its receptor CXCR6 was evaluated in first-trimester human villus and decidual tissues by immunohistochemistry. γδ T cells were isolated from first-trimester human deciduae and cocultured with JEG3 trophoblast cells. Cell proliferation and apoptosis-related molecules, together with cytotoxicity factor and cytokine production, were measured by flow cytometry analysis. RESULTS: Expression of CXCL16 and CXCR6 was reduced at the maternal-fetal interface in patients who experienced unexplained recurrent spontaneous abortion as compared to healthy pregnancy women. With the administration of pregnancy-related hormones or coculture with JEG3 cells, CXCR6 expression was upregulated on decidual γδ T cells. CXCL16 derived from JEG3 cells caused a decrease in granzyme B production of decidual γδ T cells. In addition, decidual γδ T cells educated by JEG3-derived CXCL16 upregulated the expression of Bcl-xL in JEG3 cells. CONCLUSION: This study suggested that the CXCL16/CXCR6 axis may contribute to maintaining normal pregnancy by reducing the secretion of cytotoxic factor granzyme B of decidual γδ T cells and promoting the expression of antiapoptotic marker Bcl-xL of trophoblasts.

Comparison of Efficacy and Economic Value of Prandilin 25 and Humalog Mix 25 in Patients with Newly Diagnosed Type 2 Diabetes by a Continuous Glucose Monitoring System.

INTRODUCTION: To determine the clinical efficacy and economic value of insulin lispro 25-Prandilin 25 vs. insulin lispro 25-Humalog mix 25 in treatment of newly diagnosed type 2 diabetes mellitus (T2DM) by a continuous glucose monitoring system (CGMS). METHODS: This was a single-center, randomized, case-crossover clinical trial. Participants were randomly allocated to two groups and underwent two kinds of insulin lispro 25 treatment separated by a 1-day washout period. In total, 81 patients with newly diagnosed T2DM with hemoglobin A1c (HbA1c) above 9% were hospitalized and randomly divided to receive Prandilin 25/Humalog mix 25 or Humalog mix 25/Prandilin 25 treatment. All participants were subjected to metformin therapy simultaneously. Glycemic control was reached after 7-8 days Prandilin 25 or Humalog mix 25 treatment; each patient received continuous glucose monitoring (CGM) for 5 consecutive days (from day 1 to day 5). On day 3 of CGM performance, Prandilin 25 treatment was switched to Humalog mix 25 treatment at the same dosage or vice versa. Parameters representing glycemic variability (GV) and postprandial glucose excursions, including 24-h mean blood glucose (24hMBG), 24-h standard deviation of blood glucose (24hSDBG), 24-h mean amplitude of glycemic excursion (24hMAGE), large amplitude of glycemic excursion (LAGE), incremental area under the curve (AUC) for different glucose levels, and postmeal relative areas under the CGM curve (AUCpp) for 1-4 h of each meal, were calculated for each patient. RESULTS: No significant differences were found in the 24hMAGE, 24hMBG, 24hSDBG, LAGE, mean 1-h preprandial blood glucose and the incidence of hypoglycemia between the Prandilin 25 treatment group and Humalog mix 25 treatment group. Similarly, there were no between-treatment differences for AUC and time when blood glucose was below 3.9 mmol/l, between 3.9 mmol/l and 10.0 mmol/l, or above 10.0 mmol/l. Further analysis showed the AUCpp for 1-4 h of each meal for two kinds of treatments were similar. However, the mean estimated cost of Prandilin 25 was only 85% of Humalog mix 25 in one treatment course. CONCLUSION: Prandilin 25 is non-inferior in clinical efficacy compared with Humalog mix 25. In view of the significant difference in the cost of the two kinds of insulin lispro 25, Prandilin 25 is a much more cost-effective anti-diabetes drug for management of T2DM. TRIAL REGISTRATION: Chinese Clinical Trial Register identifier, ChiCTR1800015829.

Hypoxic-stabilized EPAS1 proteins transactivate DNMT1 and cause promoter hypermethylation and transcription inhibition of EPAS1 in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality globally. Although cigarette smoking is by far the most important risk factor for lung cancer, the aberrant expression of oncogenes and tumor suppressor genes contributes a great deal to tumorigenesis. Here, we reveal that aberrant expression of endothelial PAS domain-containing protein 1 ( EPAS1) gene, which encodes hypoxia inducible factor 2α, has a critical role in NSCLC. Our results showed EPAS1 mRNA was down-regulated in 82.5% of NSCLC tissues, and a new region of EPAS1 promoter was found to be highly methylated in lung cancer cell lines and NSCLC tissues. Moreover, the methylation rates were negatively correlated to EPAS1 mRNA expression in lung tissues. Further, demethylation analysis demonstrated EPAS1 was regulated by DNA methyltransferases (DNMTs) in NSCLC. In contrast, DNMT1 was verified as an EPAS1 target gene by chromatin immunoprecipitation assay and could be transactivated by stabilized EPAS1 proteins in hypoxic lung cells, thereby decreasing EPAS1 mRNA expression by methylation regulation. Collectively, our study suggests there might be a mechanism of negative-feedback regulation for EPAS1 in NSCLC. That is, hypoxic-stabilized EPAS1 proteins transactivated DNMT1, which further promoted the hypermethylation of EPAS1 promoter and decreased EPAS1 mRNA expression levels in NSCLC.-Xu, X.-H., Bao, Y., Wang, X., Yan, F., Guo, S., Ma, Y., Xu, D., Jin, L., Xu, J., Wang, J. Hypoxic-stabilized EPAS1 proteins transactivate DNMT1 and cause promoter hypermethylation and transcription inhibition of EPAS1 in non-small cell lung cancer.

Neotuberostemonine inhibits the differentiation of lung fibroblasts into myofibroblasts in mice by regulating HIF-1α signaling.

Pulmonary fibrosis may be partially the result of deregulated tissue repair in response to chronic hypoxia. In this study we explored the effects of hypoxia on lung fibroblasts and the effects of neotuberostemonine (NTS), a natural alkaloid isolated from Stemona tuberosa, on activation of fibroblasts in vitro and in vivo. PLFs (primary mouse lung fibroblasts) were activated and differentiated after exposure to 1% O2 or treatment with CoCl2 (100 µmol/L), evidenced by markedly increased protein or mRNA expression of HIF-1α, TGF-ß, FGF2, α-SMA and Col-1α/3α, which was blocked after silencing HIF-1α, suggesting that the activation of fibroblasts was HIF-1α-dependent. NTS (0.1-10 µmol/L) dose-dependently suppressed hypoxia-induced activation and differentiation of PLFs, whereas the inhibitory effect of NTS was abolished by co-treatment with MG132, a proteasome inhibitor. Since prolyl hydroxylation is a critical step in initiation of HIF-1α degradation, we further showed that NTS treatment reversed hypoxia- or CoCl2-induced reduction in expression of prolyl hydroxylated-HIF-1α. With hypoxyprobe immunofiuorescence staining, we showed that NTS treatment directly reversed the lower oxygen tension in hypoxia-exposed PLFs. In a mouse model of lung fibrosis, oral administration of NTS (30 mg·kg-1·d-1, for 1 or 2 weeks) effectively attenuated bleomycin-induced pulmonary fibrosis by inhibiting the levels of HIF-1α and its downstream profibrotic factors (TGF-ß, FGF2 and α-SMA). Taken together, these results demonstrate that NTS inhibits the protein expression of HIF-1α and its downstream factors TGF-ß, FGF2 and α-SMA both in hypoxia-exposed fibroblasts and in lung tissues of BLM-treated mice. NTS with anti-HIF-1α activity may be a promising pharmacological agent for the treatment of pulmonary fibrosis.

Fluorescent Chemosensor for Selective Detection of Phosgene in Solutions and in Gas Phase.

The detection of highly toxic chemicals in a convenient, fast, and reliable manner is essential for coping with serious threats to humankind and public security caused by unexpected terrorist attacks and industrial accidents. In this paper, a highly selective fluorescent probe has been constructed through o-phenylenediamine covalently linking to coumarin (o-Pac), which can respond to phosgene in turn-on fluorescence mode. The response time is less than 0.5 min and the detection limit is as low as 3 nM in solutions. More importantly, the sensor exhibits good selectivity to phosgene over triphosgene and various acyl chlorides. Furthermore, a portable test paper has been fabricated with polystyrene membrane containing o-Pac for real-time selective monitoring of phosgene in gas phase.

BODIPY-Based Fluorescent Sensor for the Recognization of Phosgene in Solutions and in Gas Phase.

As a highly toxic and widely used chemical, phosgene has become a serious threat to humankind and public security because of its potential use by terrorists and unexpected release during industrial accidents. For this reason, it is an urgent need to develop facile, fast, and selective detection methods of phosgene. In this Article, we have constructed a highly selective fluorescent sensor o-Pab for phosgene with a BODIPY unit as a fluorophore and o-phenylenediamine as a reactive site. The sensor o-Pab exhibits rapid response (∼15 s) in both colorimetric and turn-on fluorescence modes, high selectivity for phosgene over nerve agent mimics and various acyl chlorides and a low detection limit (2.7 nM) in solutions. In contrast to most undistinguishable sensors reported, o-Pab can react with phosgene but not with its substitutes, triphosgene and biphosgene. The excellent discrimination of o-Pab has been demonstrated to be due to the difference in highly reactive and bifunctional phosgene relative to its substitutes. Furthermore, a facile testing paper has been fabricated with poly(ethylene oxide) immobilizing o-Pab on a filter paper for real-time selective monitoring of phosgene in gaseous phase.

Antitussive, expectorant, and anti-inflammatory activities of four caffeoylquinic acids isolated from Tussilago farfara.

CONTEXT: The flower bud of Tussilago farfara L. (Compositae) (FTF) is one of the traditional Chinese medicinal herbs used to treat cough, phlegm, bronchitic, and asthmatic conditions. OBJECTIVE: The objective of this study is to isolate four caffeoylquinic acids from the ethyl acetate extract (EtE) of FTF and to evaluate their antitussive, expectorant, and anti-inflammatory activities. MATERIALS AND METHODS: The structures of compounds 1-4 isolated from EtE were determined by spectral analysis. Mice were orally treated with these compounds and their mixture (in a ratio of 5:28:41:26 as in EtE) at doses of 10 and 20 mg/kg once daily for 3 d. The antitussive and expectorant activities were evaluated separately with the ammonia liquor-induced model and the phenol red secretion model. The anti-inflammation activity was evaluated using leukocyte count in the bronchoalveolar lavage fluid after ammonia liquor-induced acute airway inflammation. RESULTS: The four compounds were identified as chlorogenic acid (1), 3,5-dicaffeoylquinic acid (2), 3,4-dicaffeoylquinic acid (3), and 4,5-dicaffeoylquinic acid (4). All compounds, especially compound 4 (58.0% inhibition in cough frequency), showed a significant antitussive effect. However, the mixture was the most effective to inhibit the cough frequency by 61.7%. All compounds also showed a significant expectorant effect, while compound 2 was the most potent to enhance the phenol red secretion by 35.7%. All compounds significantly alleviated inflammation, but compound 4 showed the strongest effect to inhibit the leukocytosis by 49.7%. DISCUSSION AND CONCLUSION: The caffeoylquinic acids and their mixture, exhibiting significant antitussive, expectorant, and anti-inflammatory effects, could be considered as the main effective ingredients of FTF, and they may act in a collective and synergistic way.