OBJECTIVE: To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation. METHODS: Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 ^-/-, arhgef10 ^+/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively. RESULTS: WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 ^-/- zebrafish had more severe symptoms than arhgef10 ^+/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis. CONCLUSION Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.
Animals ; Annexin A5 ; Apoptosis ; Blotting, Western ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Cells, Cultured ; Flow Cytometry ; Genotype ; Humans ; In Situ Hybridization ; Larva/physiology* ; Phenotype ; RNA/isolation & purification* ; Real-Time Polymerase Chain Reaction/standards* ; Rho Guanine Nucleotide Exchange Factors/metabolism* ; Sincalide/analysis* ; Spectrophotometry/methods* ; Zebrafish/physiology*

OBJECTIVE: To investigate the expression of miR-126 in diffuse large B-cell lymphoma (DLBCL) tissues and its biological function. METHODS: The lymphoma tissues of 46 DLBCL patients in our hospital were selected as the research object, and the lymph node hyperplasia tissue of 31 patients with reactive hyperplasia were selected as controls. The expression level of miR-126 in the patients' tissues was detected by real-time fluorescent quantitative PCR (RT-qPCR), and the correlation of miR-126 expression with the pathological characteristics and prognosis of the patients was analyzed. The DLBCL cell line SU-DHL-4 was transfected with miR-126 inhibitor and its negative control (NC inhibitor) or miR-126 mimics and its negative control (NC mimics). RT-qPCR assay was used to detect the expression level of miR-126 in cells; MTT method was used to detect cell proliferation activity; single clone formation test was used to detect cells colony-forming ability; Annexin V/PI double staining assay was used to detect cell apoptosis; Transwell test was used to detect cell migration and invasion ability; the expression levels of apoptosis-related proteins cleaved-Caspase-3, Bcl-2 and Bax were detected by Western blot. RESULTS: miR-126 was highly expressed in lymphoma tissues of DLBCL patients, and its expression level was significantly correlated with Hans type, IPI score and Ann-Arbor stage of DLBCL patients (P<0.05). Kaplan-Meier survival analysis showed that the survival rate of DLBCL patients with high expression of miR-126 was significantly lower than that of patients with low expression (P<0.05). Compared with the NC mimics group, the miR-126 expression level, cell proliferation rate, number of colony-forming units, migration and invasion ability, and Bcl-2 protein expression level in the miR-126 mimics group were significantly increased (P<0.05), but the cells apoptotic rate, cleaved-Caspase-3 and Bax protein expression levels were significantly reduced (P<0.05). Compared with the NC inhibitor group, the miR-126 expression level, cell proliferation rate, number of colony-forming units, migration and invasion ability, and Bcl-2 protein expression level in the miR-126 inhibitor group were significantly reduced (P<0.05), but the cells apoptosis rate, cleaved-Caspase-3 and Bax protein expression levels were significantly increased (P<0.05). CONCLUSION miR-126 is highly expressed in lymphoma tissues of DLBCL patients and its expression level is related to the poor prognosis of patients. miR-126 can promote DLBCL cell proliferation, invasion and migration, and inhibit cell apoptosis.
Annexin A5/metabolism* ; Apoptosis ; Apoptosis Regulatory Proteins ; Caspase 3/metabolism* ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Hyperplasia ; Lymphoma, Large B-Cell, Diffuse/genetics* ; MicroRNAs/metabolism* ; bcl-2-Associated X Protein/metabolism*

BACKGROUND: Because of a lack of substances for platelet (PLT) metabolism and preservation, normal saline (NS) washed PLTs can only be stored for short lengths of time. However, the use of platelet additive solutions (PAS) could help solve this problem. In this study, the in vitro quality of NS washed platelets (wPLTs) stored in two types of PAS were compared with those of wPLTs stored in NS. METHODS: Five units of NS washed apheresis platelets were pooled aseptically and separated into five aliquots for storage in NS only as well as T-PAS+ (Terumo BCT, Lakewood, CO, USA) and CompoSol PS (Fenwal, Lake Zurich, IL, USA) with or without 15 mM glucose. The parameters of wPLTs quality were assessed up to 48 hrs after washing and the whole experiment was repeated 10 times independently. RESULTS: wPLTs in two kinds of PAS had better quality than wPLTs in NS, and wPLTs in T-PAS+ showed better quality than those in CompoSol PS. PAS-stored wPLTs with added glucose maintained stable CD62P and Annexin V expression during storage, but exhibited increased lactate accumulation. Evaluation of in vitro quality revealed that all wPLTs had a rating of 4 immediately after washing. However, only T-PAS+-stored wPLTs with glucose maintained a rating of 4 up to 48 hrs of post-washing. CONCLUSION: Using PAS storage for wPLTs may be beneficial compared to NS. The results presented herein suggest that T-PAS+ containing glucose has the potential to extend storage time by up to 48-hours.
Annexin A5 ; Blood Component Removal ; Blood Platelets* ; Blood Preservation ; Glucose ; In Vitro Techniques ; Lactic Acid ; Lakes ; Metabolism

OBJECTIVETo investigate the possible mechanism through which Artemisinin induced apoptosis in pancreatic cell line.

METHODSColumn chromatography, thin layer chromatography (TLC) and proton NMR spectroscopy were used to purify Artemisinin. The flowcytometry was employed to detect apoptosis and reactive oxygen species (ROS).

RESULTSThe results indicated that 50% inhibiting concentration (IC50 value) for pancreatic cell line (RIN) was 45 μmol/L of Artemisinin. Artemisinin had no cytotoxic effect on the growth of peripheral blood lymphocytes. The mechanism of apoptosis was evaluated by measuring intracellular ROS. It was shown that Artemisinin-induced apoptosis occurred independently of the binding of CD95L to CD95 receptor in the RIN cells. Moreover, Artemisinin, in a dose-dependent manner, could significantly increase the level of ROS.

CONCLUSIONArtemisinin can induce apoptosis in the RIN cells via the generation of ROS and triggering the intrinsic pathway of cell death.

Annexin A5 ; metabolism ; Apoptosis ; drug effects ; Artemisinins ; pharmacology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colorimetry ; Flow Cytometry ; Humans ; Iron ; pharmacology ; Pancreatic Neoplasms ; enzymology ; pathology ; Propidium ; metabolism ; Proton Magnetic Resonance Spectroscopy ; Reactive Oxygen Species ; metabolism ; Time Factors ; fas Receptor ; metabolism

BACKGROUNDEndothelial cells derived microRNAs can be detected in plasma and serum and there is evidence that inflammatory disease states may affect the levels of circulating microRNAs. However, there is no direct proof that inflammation induces endothelial cells to release microRNAs into circulation. This study aimed to explore whether inflammation could induce endothelial cells to release microRNAs into circulation and to investigate whether these released microRNAs derived from endothelial cells were transported in microparticles.

METHODSMicroparticles were isolated from human atherosclerotic plaques with an active inflammatory phenotype and normal vascular tissue. Flow cytometry and real-time PCR were used to detect the levels of microparticles and microRNAs. Human umbilical vein endothelial cells (HUVEC) was treated with tumour necrosis factor a (TNF-α, 10 ng/ml) for 24 hours, and then HUVEC and the culture medium were respectively collected.

RESULTSBy comparing microparticles isolated from human atherosclerotic plaques with an active inflammatory phenotype (n = 9) and those from normal vascular tissues (n = 9), we found levels of annexin V(+) microparticles and annexin V(+) CD144(+) microparticles were significantly increased in plaques and angiogenesis associated microRNAs (106b, 25, 92a and 21) were also significantly increased in microparticles from plaques. After exposure to TNF-α at a concentration of 10 ng/ml (TNF-α group, n = 3) or DMEM (control group, n = 3) for 24 hours, counts of microparticles and expressions of microRNAs 106b, 25, 92a and 21 in microparticles isolated from medium significantly increased. However, there were no differences in the intracellular levels of microRNAs 25, 92a or 21 isolated from HUVEC between TNF-α group and control group, while microRNA 106b decreased in TNF-α group.

CONCLUSIONInflammation could induce endothelial cells to release angiogenesis associated microRNAs into circulation, causing higher levels of circulating endothelial cells derived microRNAs in atherosclerosis.

Annexin A5 ; metabolism ; Cell-Derived Microparticles ; metabolism ; Cells, Cultured ; Gene Expression Regulation ; drug effects ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Inflammation ; genetics ; immunology ; MicroRNAs ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo determine the effect of berberine (Ber) on norepinephrine (NE)-induced apoptosis in neonatal rat cardiomyocytes.

METHODSThe cultured neonatal rat cardiomyocytes were treated with NE in the presence or absence of Ber. The activity of lactate dehydrogenase (LDH) in the culture medium was examined, and apoptosis of cardiomyocytes was assessed by Hoechst 33258, isothiocyanate (FITC)-conjugated annexin-V, and propidine iodide (PI) staining. In addition, the activities of caspases-2 and-3 were measured by a fluorescent assay kit. The level of secreted tumor necrosis factor α (TNF-α) and production of intracellular reactive oxygen species (ROS) were also determined.

RESULTSNE at a concentration of 50 μ mol/L induced an obvious increase in the activity of LDH in the culture medium (P<0.05), which was inhibited by coincubation with 0.5, 1.0, or 2.0 μ mol/L Ber (P<0.05). Ber also significantly attenuated NE-induced apoptosis in a dose-dependent manner (P<0.01). Moreover, Ber at a dose of 2 μ mol/L markedly decreased the ROS and TNF-α productions (P <0.05) and inhibited the activation of caspases-2 and -3 in cardiomyocytes exposed to NE (P<0.05)h.

CONCLUSIONThe present study suggested that Ber could reduce NE-induced apoptosis in neonatal rat cardiomyocytes through inhibiting the ROS-TNF-α-caspase signaling pathway.

Animals ; Animals, Newborn ; Annexin A5 ; metabolism ; Apoptosis ; drug effects ; Berberine ; pharmacology ; Caspase 2 ; metabolism ; Caspase 3 ; metabolism ; Caspases ; metabolism ; Cell Nucleus ; drug effects ; metabolism ; Cell Shape ; drug effects ; DNA ; metabolism ; Enzyme Activation ; drug effects ; Flow Cytometry ; Fluorescein-5-isothiocyanate ; metabolism ; Immunohistochemistry ; L-Lactate Dehydrogenase ; metabolism ; Myocytes, Cardiac ; drug effects ; enzymology ; pathology ; Norepinephrine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the effects of endothelial microvesicles (EMVs) induced by calcium ionophore A23187 on H9c2 cardiomyocytes.

METHODSHuman umbilical vein endothelial cells (HUVECs) were treated with 10 micromol/L A23187 for 30 min. EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. EMVs were characterized using 1 and 2 microm latex beads and anti-PE-CD144 antibody by flow cytometry. For functional research, EMVs at different concentrations were cocultured with H9c2 cardiomyocytes for 6 h. Cell viability of H9c2 cells and the activity of LDH leaked from H9c2 cells were tested by colorimetry. Moreover, apoptosis of H9c2 cells was observed through Hoechst 33258 staining and tested by FITC-Annexin V/PI double staining.

RESULTSEMVs were induced by A23187 on HUVECs, and isolated by ultracentrifugation. We identified the membrane vesicles (< 1 microm) induced by A23187 were CD144 positive. In addition, the EMVs could significantly reduce the viability of H9c2 cells, and increase LDH leakage from H9c2 cells in a dose dependent manner (P < 0.05). Condensed nuclei could be observed with the increasing concentrations of EMVs through Hoechst 33258 staining. Furthermore, increased apoptosis rates of H9c2 cells could be assessed through FITC-Annexin V/PI double staining by flow cytometry.

CONCLUSIONMicrovesicles could be released from HUVECs after induced by A23187 through calcium influx, and these EMVs exerted a pro-apoptotic effect on H9c2 cells by induction of apoptosis.

Annexin A5 ; Apoptosis ; Calcimycin ; pharmacology ; Calcium ; metabolism ; Cell Line ; Cell Membrane ; drug effects ; Coculture Techniques ; Flow Cytometry ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; Human Umbilical Vein Endothelial Cells ; Humans ; Myocytes, Cardiac ; drug effects ; Staining and Labeling

AMP-activated protein kinase (AMPK), an important regulator of energy metabolism, is activated in response to cellular stress when intracellular levels of AMP increase. We investigated the neuroprotective effects of AMPK against scopolamine-induced memory impairment in vivo and glutamate-induced cytotoxicity in vitro. An adenovirus expressing AMPK wild type alpha subunit (WT) or a dominant negative form (DN) was injected into the hippocampus of rats using a stereotaxic apparatus. The AMPK WT-injected rats showed significant reversal of the scopolamine induced cognitive deficit as evaluated by escape latency in the Morris water maze. In addition, they showed enhanced acetylcholinesterase (AChE)-reactive neurons in the hippocampus, implying increased cholinergic activity in response to AMPK. We also studied the cellular mechanism by which AMPK protects against glutamate-induced cell death in primary cultured rat hippocampal neurons. We further demonstrated that AMPK WT-infected cells increased cell viability and reduced Annexin V positive hippocampal neurons. Western blot analysis indicated that AMPK WT-infected cells reduced the expression of Bax and had no effects on Bcl-2, which resulted in a decreased Bax/Bcl-2 ratio. These data suggest that AMPK is a useful cognitive impairment treatment target, and that its beneficial effects are mediated via the protective capacity of hippocampal neurons.
Acetylcholinesterase ; Adenoviridae ; AMP-Activated Protein Kinases ; Animals ; Annexin A5 ; Apoptosis ; Blotting, Western ; Cell Death ; Cell Survival ; Energy Metabolism ; Hippocampus ; Memory ; Neurons ; Neuroprotective Agents ; Rats ; Scopolamine Hydrobromide ; United Nations

OBJECTIVETo study the differentially expressed proteins in the process of annexin 5 stimulating testosterone secretion in cultured rat Leydig cells.

METHODSPrimary rat Leydig cells were cultured in vitro and treated with annexin 5 at the concentration of 1 nmol/L for 24 hours, and the cell proteins were extracted to be compared by two-dimensional gel electrophoresis (2-DE). The differentially expressed protein spots were selected to be analyzed by mass spectrometry.

RESULTSWe obtained electrophoresis profiles with high resolution and reproducibility, found 50 differentially expressed protein spots, and identified 36 by mass spectrometry, of which 23 were overexpressed and 13 underexpressed in the Leydig cells treated with annexin 5.

CONCLUSIONDifferentially expressed protein profiles were established in the process of annexin 5 stimulating testosterone secretion in cultured rat Leydig cells, and identified the key role of these proteins in testosterone secretion. Our findings might be helpful to illuminate the mechanism of annexin 5 regulating testosterone secretion in rat Leydig cells.

Animals ; Annexin A5 ; pharmacology ; Cells, Cultured ; Electrophoresis, Gel, Two-Dimensional ; Leydig Cells ; drug effects ; metabolism ; Male ; Mass Spectrometry ; Proteins ; metabolism ; Proteome ; analysis ; Rats ; Rats, Sprague-Dawley ; Testosterone ; secretion

OBJECTIVETo study luteolin-induced non-small cell lung cancer cell line A549 apoptosis and the molecular mechanism for inhibiting its cycle arrest (G2 stage).

METHODMTT assay showed that luteolin had obvious inhibitory effect on A549 and indicated the half inhibition ratio (IC50). Cell cycle and apoptosis were detected by Hoechst 33258 nuclear staining assay, Annexin V-FITC/PI double staining and flow cytometry. Western blotting assay revealed changes in cycle and apoptosis-related proteins induced by luteolin. Possible molecular mechanism was suggested by Western blotting and immunocytochemistry.

RESULTLuteolin had an obvious growth inhibitory effect on A549 cells, with IC50 of 45.2 micromol x L(-1) at 48 h. Flow cytometry showed A549 cells mainly arrested in G2 stage after being treated by luteolin, with low expressions in cyclin A, p-CDC2 and p-Rb. Hoechst 33258 nuclear staining and Annexin V-FITC/PI double staining showed that the luteolin treatment group showed a significant apoptosis rate than the non-treatment group. Western blotting found luteolin can increase phosphorylation of JNK and decrease that of NF-kappaKB (p65). Immunocytochemistry results revealed luteolin can inhibit TNF-alpha-stimulated p65 from nuclear translocation as a transcription factor and thus promoting cell apoptosis.

CONCLUSIONLuteolin can obviously induce apoptosis of human non-small cell lung cancer cell A549 possibly by increasing phosphorylation of JNK to activate mitochondria apoptosis pathway, while inhibiting NF-kappaB from nuclear translocation as a transcription factor.

Annexin A5 ; metabolism ; Apoptosis ; drug effects ; Blotting, Western ; Carcinoma, Non-Small-Cell Lung ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Humans ; Luteolin ; pharmacology ; NF-kappa B ; metabolism