Ischemic injury promotes Keap1 nitration and disturbance of antioxidative responses in endothelial cells: a potential vasoprotective effect of melatonin.

Clinical epidemiology has indicated that the endothelial injury is a potential contributor to the pathogenesis of ischemic neurovascular damage. In this report, we assessed S-nitrosylation and nitration of Keap1 to identify downstream nitric oxide redox signaling targets into endothelial cells during ischemia. Here, oxygen-glucose deprivation (OGD) exposure initiates the nuclear import of Keap1 in endothelial cells, which interacted with nuclear-localized Nrf2, as demonstrated through co-immunoprecipitation and immunocytochemical assay. Paralleling the ischemia-induced nuclear import of Keap1, increased nitrotyrosine immunoreactivity in endothelial cells was also observed. Consistently, the addition of peroxynitrite provoked nuclear import of Keap1 and a concomitant Nrf2 nuclear import in the endothelial cells. Importantly, pharmacological inhibition of nitrosative stress by melatonin partially inhibited the OGD-induced constitutive nuclear import of Keap1 and subsequently disturbance of Nrf2/Keap1 signaling. Moreover, the effect of melatonin on nitration and S-nitrosylation of keap1 was examined in endothelial cells with 6 hr OGD exposure. Here, we demonstrated that OGD induced tyrosine nitration of Keap1, which was blocked by melatonin treatment, while there were no significant changes in S-nitrosylation of Keap1. The specific amino acid residues of Keap1 involved in tyrosine nitration were identified as Y473 by mass spectrometry. Moreover, the protective role of melatonin against damage to endothelial tight junction integrity was addressed by ZO-1 expression, paralleled with the restored heme oxygenase-1 levels during OGD. Together, our results emphasize that upon nitrosative stress, the protective effect of melatonin on endothelial cells is likely mediated at least in part by inhibition of ischemia-evoked protein nitration of Keap1, hence contributing to relieve the disturbance of Nrf2/Keap1 antioxidative signaling.

[Characteristics of microsomal phase II metabolic enzymes in mouse embryonic stem cell-derived liver tissue].

OBJECTIVE: To investigate the characteristics of phase II metabolic enzymes in mouse embryonic stem (ES) cell-derived liver tissue. METHODS: Mature hepatocytes were differentiated from embryonic stem cells in cultured mouse embryoid bodies (EB) at d18. Western blot was used to detect the expression of uridine 5'-diphosphate glucronosyl transferase (UGT1a1,UGT1a6) and microsomal glutathione S-transferases 1(mGST1) during the differentiation course.The derived liver tissue was incubated with UDPGA and 7-HFC,the formation of 7-HFC glucuronide was detected by HPLC to examine the total activities of UGT1a1 and UGT1a6. Furthermore, the microsomes were incubated with CDNB and GSH,and the mGST1 activity was measured by spectrometry. RESULTS: An increase tendency of UGT1a1 expression was noticed during the differentiation course. UGT1a6 and mGST1 were not detected in the earlier stage until d18 of differentiation. The metabolic activity of mGST1 in the derived hepatocytes was 7.65 nmol/min/mg on d18. CONCLUSION: The ES cell-derived liver tissue possesses partial metabolic function of phase II enzymes on d18 of differentiation,which might be used as a model for in vitro research on hepatic pathophysiology and phase II drug metabolism.

[Establishment of optimized neuronal differentiation-promoting model derived from P19 embryonic carcinoma cells].

OBJECTIVE: To establish an optimized primary drug screen model of neuronal differentiation using P19 embryonal carcinoma cells. METHODS: The final concentration of retinoid acid (RA), days of suspension culture, manner of adherent culture, suitable cell density and adherent culture medium were tested, respectively. Two stages of neuronal differentiation were examined based on morphological changes and immunocytochemistry analysis of neuronal specific protein ß-tubulin III. RESULTS: On d 8 of differentiation culture, neuron-like cells were observed with final concentration of 1 µmol/L RA. Neuron-like network was formed on d 16 of neuronal differentiation. ß-tubulin III was positively stained on both stages, indicating P19 cells were differentiated into neurons. CONCLUSION: The model using RA to induce P19 embryonic carcinoma cells to differentiate into neuron-like cells has been successfully established, which may provide a rapid, phenotypic cell-based platform for primary screening of neurogenesis-promoting drugs.

[Expression of Junctophilin 1 during cardiogenesis of mouse embryonic stem cells and rat embryos].

OBJECTIVE: To investigate the expression of Junctophilin 1 (JP1) in cardiogenesis of mammalian. METHODS: Cardiac differentiation of embryonic stem cells (ESCs) was generated by hanging drop method. Fetal heart was obtained from the rats aged d 14-20 of gestation. The expression of JP1 and JP2 during cardiogenesis of ESCs and rat embryos was analyzed by RT-PCR or Western blotting. Immunofluorescence staining was employed to reveal the distribution of JP1 and JP2 in embryoid body (EB), probing for merging of JP1 and JP2 and cardiac sarcomeric α-Actinin or Troponin-T. Percentage of JP1 and JP2-positive staining cells was analyzed quantitatively by FCS on d17. RESULTS: JP1 mRNA was up-regulated at the early stage (d 5-11) and then decreased. The expression of JP1 protein was up-regulated at the early stage (d 7-9), then decreased gradually and disappeared after d 15. While JP2 gene and protein expression increased in a time-dependent manner during cardiogenesis of rat embryos. The results of immunofluorescence staining showed that there was a parallel co-localization of JP2 with Troponin-T or α-Actinin on d17, while JP1 failed to express in the sarcomeric positive area at the same time point. Furthermore, FCS analysis showed that about 16.59% of cells were JP2-positive, while no cells were stained positively for JP1 in d17 EBs. CONCLUSION: JP1 gene is expressed during the whole process of cardiogenesis, while JP1 protein only appears on the early stage. The expression of JP1 in cardiogenesis of ESCs is consistent with that of rat embryos.

[Phenotype-based primary screening for drugs promoting neuronal subtype differentiation in embryonic stem cells with light microscope].

OBJECTIVE: To set up a platform for phenotype-based primary screening of drug candidates promoting neuronal subtype differentiation in embryonic stem cells (ES) with light microscope. METHODS: Hanging drop culture 4-/4+ method was employed to harvest the cells around embryoid body (EB) at differentiation endpoint. Morphological evaluation for neuron-like cells was performed with light microscope. Axons for more than three times of the length of the cell body were considered as neuron-like cells. The compound(s) that promote neuron-like cells was further evaluated. Icariin (ICA, 10(-6)mol/L) and Isobavachin (IBA, 10(-7)mol/L) were selected to screen the differentiation-promoting activity on ES cells. Immunofluorescence staining with specific antibodies (ChAT, GABA) was used to evaluate the neuron subtypes. RESULTS: The cells treated with IBA showed neuron-like phenotype, but the cells treated with ICA did not exhibit the morphological changes. ES cells treated with IBA was further confirmed to be cholinergic and GABAergic neurons. CONCLUSION: Phenotypic screening with light microscope for molecules promoting neuronal differentiation is an effective method with advantages of less labor and material consuming and time saving, and false-positive results derived from immunofluorescence can be avoided. The method confirms that IBA is able to facilitate ES cells differentiating into neuronal cells, including cholinergic neurons and GABAergic neurons.

[Ginsenoside Rg1 antagonizes ß-amyloid peptide-induced apoptosis in primarily cultured rat neurons via mitochondrial pathway].

OBJECTIVE: To assess the neuroprotective effects of ginsenoside Rg1 against ß-amyloid peptide (Aß(25-35))-induced apoptosis in primarily cultured rat cortical neurons. METHODS: Primarily cultured cortical neurons were obtained from embryonic (E18d) rat fetus and maintained in neurobasal medium for 7d. Primary neurons pretreated with 1 µmol/L, 10 µmol/L or 20 µmol/L Rg1 for 24 h were challenged with 10 µmol/L Aß(25-35) for 72 h. Morphological changes of neurons were evaluated; mitochondrial membrane potential (ΔΨm) was measured; with JC-1 staining and the expression of neural apoptosis-related proteins was detected by Western blot analysis. RESULTS: Exposure to Aß(25-35) for 72 h caused serious neural cell insults. A pretreatment with Rg1 significantly reduced Aß(25-35)induced cell death in a dose-dependent manner, with a maximal effect (-90%) obtained at 20 µmol/L. The JC-1 staining results demonstrated the loss of ΔΨm after Aß(25-35) treatment, while Rg1 maintained the normal level of ΔΨm. A series of mitochondrion-mediated apoptotic events happened after Aß(25-35) treatment, such as decrease of Bcl-2/Bax, release of cytochrome C and activation of caspase 9 and caspase 3, which were all blocked by Rg1 pretreatment. Both estrogen receptor (ER) antagonist ICI182, 780 and glucocorticoid receptor (GR) antagonist RU486 blocked the antiapoptotic effects of Rg1. CONCLUSION: Ginsenoside Rg1 protects primary cultured rat cortical neurons from Aß(25-35)-induced injury, which may be associated with mitochondrion-mediated antiapoptosis pathway.

[Effect of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus].

OBJECTIVE: To investigate the effects of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus. METHODS: SD rats were randomly divided into three groups: control group was given distilled water, lead-exposed groups were given 0.5 g/L (low-dose) or 2.0 g/L(high-dose) lead acetate solution in drinking water. The rat pups started to drink the lead content water until 60 d maturity. The lead contents in blood and brain samples were analyzed by graphite furnace atomic absorption spectrophotometry. The expressions of Beclin 1, LC3, LAMP2 and cathepsin B proteins were detected by Western blot and immunohistochemistry. RESULTS: Compared with control group, the contents of lead were significantly higher in blood and hippocampus samples in chronic lead-exposed rats (P<0.01). Western blot showed that the expression of Beclin 1 and LC3-II/LC3-I increased significantly in high dose lead-exposed group compared with control group (P<0.05 or P<0.001). The confocal laser immunostaining results demonstrated that increased immunofluorescence staining of cathepsin B in hippocampal neurons compared with control animals. CONCLUSION: The disturbance of autophagy-lysosome signaling molecules might be partially contribute to neurotoxicity of chronic lead exposure.

Identification of Crucial Genes and Key Functions in Type 2 Diabetic Hearts by Bioinformatic Analysis.

Type 2 diabetes (T2D) patients with SARS-CoV-2 infection hospitalized develop an acute cardiovascular syndrome. It is urgent to elucidate underlying mechanisms associated with the acute cardiac injury in T2D hearts. We performed bioinformatic analysis on the expression profiles of public datasets to identify the pathogenic and prognostic genes in T2D hearts. Cardiac RNA-sequencing datasets from db/db or BKS mice (GSE161931) were updated to NCBI-Gene Expression Omnibus (NCBI-GEO), and used for the transcriptomics analyses with public datasets from NCBI-GEO of autopsy heart specimens with COVID-19 (5/6 with T2D, GSE150316), or dead healthy persons (GSE133054). Differentially expressed genes (DEGs) and overlapping homologous DEGs among the three datasets were identified using DESeq2. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses were conducted for event enrichment through clusterProfile. The protein-protein interaction (PPI) network of DEGs was established and visualized by Cytoscape. The transcriptions and functions of crucial genes were further validated in db/db hearts. In total, 542 up-regulated and 485 down-regulated DEGs in mice, and 811 up-regulated and 1399 down-regulated DEGs in human were identified, respectively. There were 74 overlapping homologous DEGs among all datasets. Mitochondria inner membrane and serine-type endopeptidase activity were further identified as the top-10 GO events for overlapping DEGs. Cardiac CAPNS1 (calpain small subunit 1) was the unique crucial gene shared by both enriched events. Its transcriptional level significantly increased in T2D mice, but surprisingly decreased in T2D patients with SARS-CoV-2 infection. PPI network was constructed with 30 interactions in overlapping DEGs, including CAPNS1. The substrates Junctophilin2 (Jp2), Tnni3, and Mybpc3 in cardiac calpain/CAPNS1 pathway showed less transcriptional change, although Capns1 increased in transcription in db/db mice. Instead, cytoplasmic JP2 significantly reduced and its hydrolyzed product JP2NT exhibited nuclear translocation in myocardium. This study suggests CAPNS1 is a crucial gene in T2D hearts. Its transcriptional upregulation leads to calpain/CAPNS1-associated JP2 hydrolysis and JP2NT nuclear translocation. Therefore, attenuated cardiac CAPNS1 transcription in T2D patients with SARS-CoV-2 infection highlights a novel target in adverse prognostics and comprehensive therapy. CAPNS1 can also be explored for the molecular signaling involving the onset, progression and prognostic in T2D patients with SARS-CoV-2 infection.

Five-lipoxygenase-activating protein-mediated CYLD attenuation is a candidate driver in hepatic malignant lesion.

Hepatocellular carcinoma (HCC) is an inflammation-associated cancer. However, the lipid pro-inflammatory mediators have only been seldom investigated in HCC pathogenesis. Cylindromatosis (CYLD) attenuation is involved in hepatocarcinogenesis. Here, we aimed to evaluate the significance of hepatic lipid pro-inflammatory metabolites of arachidonate-affected CYLD expression via the 5-lipoxygenase (5-LO) pathway. Resection liver tissues from HCC patients or donors were evaluated for the correlation of 5-LO/cysteinyl leukotrienes (CysLTs) signaling to the expression of CYLD. The impact of functional components in 5-LO/CysLTs cascade on survival of HCC patients was subsequently assessed. Both livers from canines, a preponderant animal for cancer research, and genetic-modified human HCC cells treated with hepatocarcinogen aristolochic acid I (AAI) were further used to reveal the possible relevance between 5-LO pathway activation and CYLD suppression. Five-LO-activating protein (FLAP), an essential partner of 5-LO, was significantly overexpressed and was parallel to CYLD depression, CD34 neovascular localization, and high Ki-67 expression in the resection tissues from HCC patients. Importantly, high hepatic FLAP transcription markedly shortened the median survival time of HCC patients after surgical resection. In the livers of AAI-treated canines, FLAP overexpression was parallel to enhanced CysLTs contents and the simultaneous attenuation of CYLD. Moreover, knock-in FLAP significantly diminished the expression of CYLD in AAI-treated human HCC cells. In summary, the hepatic FLAP/CysLTs axis is a crucial suppressor of CYLD in HCC pathogenesis, which highlights a novel mechanism in hepatocarcinogenesis and progression. FLAP therefore can be explored for the early HCC detection and a target of anti-HCC therapy.

Involvement of ubiquitin-proteasome system in icariin-induced cardiomyocyte differentiation of embryonic stem cells using two-dimensional gel electrophoresis.

Icariin has been shown to significantly facilitate the differentiation of embryonic stem (ES) cells into cardiomyocytes in vitro. However, the mechanism underlying the icariin-induced cardiomyocyte differentiation is still not fully understood. In the present study, 52 differentially displayed proteins selected from two-dimensional electrophoresis gels were identified by MALDI-TOF mass spectrometry analysis. More than half of proteins could be assigned to six main categories: (1) protein synthesis, metabolism, processing and degradation, (2) stress response, (3) cytoskeleton proteins, (4) energy metabolism, (5) carbohydrate metabolism/transport, and (6) RNA/other nucleic acids metabolisms and transport, nuclear proteins. MALDI-TOF/MS showed that icariin treatment resulted in the induction of five ubiquitin-proteasome system (UPS)-related proteins, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), ubiquitin-conjugating enzyme E2N, proteasome 26S, proteasome subunit-alpha type 6, and proteasome subunit-alpha type 2 in the differentiated cardiomyocytes. These results implied that UPS might play an important role in the control of cardiomyocyte differentiation. Epoxomicin (a proteasome inhibitor) significantly reduced the cardiomyocyte differentiation rate of ES cells and proteasome activities, as well as inhibited NF-κB translocation into the nucleus, which were evidently reversed by presence of icariin. Meanwhile, icariin could significantly reverse the reduction of four proteins (proteasome subunit-alpha type 6, proteasome subunit-alpha type 2, UCH-L1, and ubiquitin-conjugating enzyme E2N) expressions owing to application of epoxomicin. These suggest UPS could be a means by which icariin may regulate expressions of key proteins that control cardiomyocyte differentiation. Taken together, these results indicated that UPS played an important role in ES cell differentiate into cardiomyocytes induced by icariin.

Mitochondria are the main target organelle for trivalent monomethylarsonous acid (MMA(III))-induced cytotoxicity.

Excessive generation of reactive oxygen species (ROS) is considered to play an important role in arsenic-induced carcinogenicity in the liver, lungs, and urinary bladder. However, little is known about the mechanism of ROS-based carcinogenicity, including where the ROS are generated, and which arsenic species are the most effective ROS inducers. In order to better understand the mechanism of arsenic toxicity, rat liver RLC-16 cells were exposed to arsenite (iAs(III)) and its intermediate metabolites [i.e., monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III))]. MMA(III) (IC(50) = 1 µM) was found to be the most toxic form, followed by DMA(III) (IC(50) = 2 µM) and iAs(III) (IC(50) = 18 µM). Following exposure to MMA(III), ROS were found to be generated primarily in the mitochondria. DMA(III) exposure resulted in ROS generation in other organelles, while no ROS generation was seen following exposures to low levels of iAs(III). This suggests the mechanisms of induction of ROS are different among the three arsenicals. The effects of iAs(III), MMA(III), and DMA(III) on activities of complexes I-IV in the electron transport chain (ETC) of rat liver submitochondrial particles and on the stimulation of ROS production in intact mitochondria were also studied. Activities of complexes II and IV were significantly inhibited by MMA(III), but only the activity of complexes II was inhibited by DMA(III). Incubation with iAs(III) had no inhibitory effects on any of the four complexes. Generation of ROS in intact mitochondria was significantly increased following incubation with MMA(III), while low levels of ROS generation were observed following incubation with DMA(III). ROS was not produced in mitochondria following exposure to iAs(III). The mechanism underlying cell death is different among As(III), MMA(III), and DMA(III), with mitochondria being one of the primary target organelles for MMA(III)-induced cytotoxicity.

Melatonin ameliorates ischemic-like injury-evoked nitrosative stress: Involvement of HtrA2/PED pathways in endothelial cells.

Peroxynitrite contributes to diverse cellular stresses in the pathogenesis of ischemic complications. Here, we investigate the downstream effector signaling elements of nitrosative stress which regulate ischemia-like cell death in endothelial cells and protective effect of melatonin. When the mitochondrial membrane potential (ΔΨm) of oxygen-glucose deprivation (OGD)-treated cells was assessed using the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol -carbocyanine iodide, we observed spontaneous changes in peroxynitrite formation. Concomitantly, western blot and confocal microscopy analyses indicated that prolonged OGD exposure initiates the release of mitochondrial HtrA2 and dramatically decreases phosphoprotein enriched in astrocytes (PED or PEA-15) protein levels. Consistently, cultured endothelial cells treated with peroxynitrite (1-50 µm) exhibited a concentration-dependent release of mitochondrial HtrA2 and concomitant PED degradation in vitro. Notably, HtrA2 activation coincided with increased nitrotyrosine immunoreactivity in microvessels of rats following microsphere embolism. Additionally, the protective effect of PED overexpression in OGD-induced apoptosis was abolished by transfection with the PED(S104A/S116A) mutant. Furthermore, the effect of melatonin, an potential antioxidant, on endothelial apoptotic cascade was examined in OGD-evoked nitrosative stress. Our data showed that the application of melatonin provided significant protection against OGD-induced peroxynitrite formation and mitochondrial HtrA2 release, accompanied with a decrease in degradation PED and x-linked inhibitor of apoptosis protein, which is associated with activation of the caspase cascade. Taken together, the protective effect of melatonin is likely mediated, in part, by inhibition of peroxynitrate-mediated nitrosative stress, which in turn relieves imbalance of mitochondrial HtrA2-PED signaling and endothelial cell death.

Embryonic stem cell application in drug discovery.

Embryonic stem (ES) cells and their differentiated progeny offer tremendous potential for regenerative medicine, even in the field of drug discovery. There is an urgent need for clinically relevant assays that make use of ES cells because of their rich biological utility. Attention has been focused on small molecules that allow the precise manipulation of cells in vitro, which could allow researchers to obtain homogeneous cell types for cell-based therapies and discover drugs for stimulating the regeneration of endogenous cells. Such therapeutics can act on target cells or their niches in vivo to promote cell survival, proliferation, differentiation, and homing. In the present paper, we reviewed the use of ES cell models for high-throughput/content drug screening and toxicity assessment. In addition, we examined the role of stem cells in large pharmaceutical companies' R&D and discussed a novel subject, nicheology, in stem cell-related research fields.

Promoting effects of isobavachin on neurogenesis of mouse embryonic stem cells were associated with protein prenylation.

AIM: Some small molecules can induce mouse embryonic stem (ES) cells to differentiate into neuronal cells. Here, we explored the effect of isobavachin (IBA), a compound with a prenyl group at position 8 of ring A, on promoting neuronal differentiation and the potential role of its protein prenylation. METHODS: The hanging drop method was employed for embryonic body (EB) formation to mimic embryo development in vivo. The EBs were treated with IBA at a final concentration of 10(-7) mol/L from EB stage (d 4) to d 8+10. Geranylgeranyltransferase I inhibitor GGTI-298 was subsequently used to disrupt protein prenylation. Neuronal subtypes, including neurons and astrocytes, were observed by fluorescence microscopy. Gene and protein expression levels were detected using RT-PCR and Western blot analysis, respectively. RESULTS: With IBA treatment, nestin was highly expressed in the neural progenitors generated from EBs (d 4, d 8+0). EBs then further differentiated into neurons (marked by ß-tubulin III) and astrocytes (marked by GFAP), which were both up-regulated in a time-dependent manner on d 8+5 and d 8+10. Co-treatment with GGTI-298 selectively abolished the IBA-induced neuronal differentiation. Moreover, in the MAPK pathway, p38 and JNK phosphorylation were down-regulated, while ERK phosphorylation was up-regulated after IBA treatment at different neuronal differentiation passages. CONCLUSION: IBA can facilitate mouse ES cells differentiating into neuronal cells. The mechanism involved protein prenylation and, subsequently, phos-ERK activation and the phos-p38 off pathway.

PPAR-beta facilitating maturation of hepatic-like tissue derived from mouse embryonic stem cells accompanied by mitochondriogenesis and membrane potential retention.

Relatively little is known about mitochondria metabolism in differentiating embryonic stem (ES) cells. Present research focused on several elements of cellular energy metabolism in hepatic-like tissue derived from mouse ES cells. We demonstrated that mitochondrial location patterns and mitochondrial membrane potential (DeltaPsi(m)) existed in subsequent differentiation of the tissue. Mitochondriogenesis appeared at the early stage and kept a normal DeltaPsi(m) in differentiated mature hepatocytes. Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) expression was transitorily increased at the beginning, and kept a relatively low level later, which accompanied by expression of PPAR-gamma coactivator (PGC)-1alpha, a master regulator of mitochondrial biogenesis. PPAR-beta expression showed robust up-regulation in the late differentiation course. Enhanced co-expressions of PPAR-beta and albumin with catalysis of UDP-glucuronosyltransferases (UGTs) were observed at mature stage. While PPAR-gamma expression changed little before and after differentiation. Mitochondriogenesis could be accelerated by PPAR-alpha specific agonist WY14643 and abolished by its antagonist GW6471 at the early stage. Neither of them affected mitochondrial DeltaPsi(m) and albumin generation in the differentiated hepatocytes. Furthermore, maturation of hepatic-like tissue and mitochondriogenesis in hepatocyte could be efficiently stimulated by PPAR-beta specific agonist L165041 and abolished by PPAR-beta specific antagonist GSK0660, but not affected by PPAR-gamma specific agonist GW1929. In conclusion, the derived hepatic tissue morphologically possessed cellular energy metabolism features. PPAR-alpha seemed only necessary for early mitochondriogenesis, while less important for DeltaPsi(m) retention in the mature tissue derived. The stimulation of PPAR-beta but not -gamma enhanced hepatogenesis, hepatocytes maturation, and mitochondriogenesis. PPAR-beta took an important role in cellular energy metabolism of hepatogenesis.

The induction of reactive oxygen species and loss of mitochondrial Omi/HtrA2 is associated with S-nitrosoglutathione-induced apoptosis in human endothelial cells.

The pathophysiological relevance of S-nitrosoglutathione (GSNO)-induced endothelial cell injury remains unclear. The main objective of this study was to elucidate the molecular mechanisms of GSNO-induced oxidative stress in endothelial cells. Morphological evaluation through DAPI staining and propidium iodide (PI) flow cytometry was used to detect apoptosis. In cultured EA.hy926 endothelial cells, exposure to GSNO led to a time- and dose-dependent apoptotic cascade. When intracellular reactive oxygen species (ROS) production was measured in GSNO-treated cells with the fluorescent probes 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, we observed elevated ROS levels and a concomitant loss in mitochondrial membrane potential, indicating that GSNO-induced death signaling is mediated through a ROS-mitochondrial pathway. Importantly, we found that peroxynitrite formation and Omi/HtrA2 release from mitochondria were involved in this phenomenon, whereas changes of death-receptor dependent signaling were not detected in the same context. The inhibition of NADPH oxidase activation and Omi/HtrA2 by a pharmacological approach provided significant protection against caspase-3 activation and GSNO-induced cell death, confirming that GSNO triggers the death cascade in endothelial cells in a mitochondria-dependent manner. Taken together, our results indicate that ROS overproduction and loss of mitochondrial Omi/HtrA2 play a pivotal role in reactive nitrogen species-induced cell death, and the modulation of these pathways can be of significant therapeutic benefit.

[4, 8-disubstituted-8, 9-dihydro-pyrazine[2,3-g]quinazoline-7(6H)-ketones: a novel class of antitumor agents].

OBJECTIVE: To evaluate the antitumor activity of a novel class of 4, 8-Disubstituted-8, 9-dihydropyrazine[2, 3-g]quinazoline-7(6H)-ketones in vitro, and to screen potential anticancer compounds for further study. METHODS: Seventeen compounds of 4, 8-Disubstituted-8, 9-dihydropyrazine[2, 3-g]quinazoline-7(6H)-ketones were synthesized with solid-phase method for biological evaluation of EGFR tyrosine kinase. MTT method was used to evaluate the cytotoxic activity in vitro against three human cancer cell lines (human lung carcinoma cell line A549, human leukemia cell lines K562 and human gastric carcinoma cell line SGC7901). RESULTS: Compound 7-13 and 7-14 showed potent antitumor activities against A549 cells, with IC(50) values of 8.10 and 8.12 mol/L, respectively. Eight compounds showed proliferative inhibition effect on K562 cells, especially 7-2, 7-13 and 7-17, with IC(50) values of 2.22,0.57 and 7.20 mol/L,respectively.And compound 7-13 and 7-3 showed potent antitumor activity against SGC7901 cells, with IC(50) values of 4.20 and 9.71 mol/L, respectively. CONCLUSION: The synthesized compounds 4, 8-Disubstituted-8, 9-dihydropyrazine[2, 3-g] quinazoline-7(6H)-ketones show inhibition effects on human cancer cell lines in vitro. Compound 7-13 has anticancer activity in all three cancer cell lines, which might be used as a potential antitumor drug for further study.

[Effect of concanavalin A on expression of MAPEG mRNA in Balb/c mouse brain].

OBJECTIVE: To investigate the gene expression of MAPEG in the cortex of concanavalin A (Con A)-induced mouse immune inflammatory model and the effect of cyclosporine A (Cs A). METHODS: Male Balb/c mouse immune inflammation model was developed by intravenous injection of Con A (20 mg/kg). Cs A (150 mg/kg) was intravenously infected prior to Con A administration. The MAPEG expressions were determined by RT-PCR. RESULT: mGST1, mGST3, LTC(4)S, FLAP and mPGES-1 were detected by RT-PCR but not mGST2. Eight hours after Con A treatment, mGST1 level was up-regulated to 1.2 approximately 1.5 folds of control with or without Cs A treatment. mGST3ìLTC(4)S, FLAP and mPGES-1 mRNA levels were not influenced by Con A administration. CONCLUSION: Immune mechanism may be not involved in mGST1 up-regulation in this model and Con A does not alter arachidonic acid metabolism in cortex.

[Transcription of cytoskeleton protein genes in differentiation of neurons from mouse embryonic stem cells induced by small molecules].

OBJECTIVE: To investigate the transcription of cytoskeleton protein genes in differentiation of neurons from mouse embryonic stem (ES) cells induced by all-trans retinoic acid (RA), and to explore the possibility of setting up a method to screen small molecules with promoting or inhibiting effect. METHODS: The hanging drop method was employed for embryonic body formation to mimic embryo development in vivo. Reverse transcriptase PCR (RT-PCR) was performed to investigate mRNA expression of the neuron-specific cytoskeleton proteins including Mtap2, Nefm and beta-tubulin III which were regarded as the inducing effect indexes of RA. Morphological evaluation and immunocytochemistry staining were conducted to identify the neural derivatives. Moreover, the inducing effects of six synthetic molecules were further evaluated. RESULT: RA up-regulated the mRNA expression of Mtap2 and Nefm, especially Mtap2 increased by 1.27 times, which was consistent with the morphological alteration. However, there was no significant changes of beta-tubulin III expression. With addition of the six synthetic molecules, the transcription of Mtap2 was inhibited, while the Nefm mRNA expression was up-regulated in some degree, especially for molecule 1 and 3 that was increased by 1.4 and 1.2 times, which, however, was not parallel to the morphological changes. CONCLUSION: The transcriptional levels of Mtap2 and Nefm are both up-regulated in the RA-induced differentiation of ES cells towards neurons. The up-regulation of Mtap2 is consistent with the morphological alteration, which might be the key landmark in the RA-induced differentiation of ES cells into neurons.

Reactive oxygen species involved in prenylflavonoids, icariin and icaritin, initiating cardiac differentiation of mouse embryonic stem cells.

The significant promoting effects of some prenylflavonoids on cardiac differentiation of mouse embryonic stem (ES) cells via reactive oxygen species (ROS) signaling pathway were investigated. The most effective differentiation was facilitated by icariin (ICA), followed by icaritin (ICT), while desmethylicaritin (DICT) displayed the weakest but still significant inducible effect. Contrarily, DICT demonstrated the strongest anti-oxidative activity while ICA displayed only little in vitro, which was well matched with the hydroxyl (OH) numbers and the positions in the molecular structures. Therefore, ROS signaling cascades were assumed to be involved in prenylflavonoids induced cardiomyogenesis. Treatment with ICA, intracellular ROS in embryoid bodies was rapidly elevated, which was abolished by the NADPH-oxidase inhibitor apocynin; elimination of intracellular ROS by vitamin E or pyrrolidine dithiocarbamate (PDTC) inhibited ICA induced cardiomyogenesis; ROS-sensitive extracellular-regulated kinase 1, 2 (ERK1, 2) and p38 activation were further observed, the cardiomyogenesis was significantly inhibited in the presence of ERK1, 2 or p38 inhibitor U0126 or SB203580, indicating the roles of NADPH-ROS-MAPKs signaling cascades in prenylflavonoids induced cardiac differentiation. There was no difference in Nox4 NADPH oxidase expression between ICA and ICT treatments, however, ROS concentration in EBs after ICT administration was lower than that after ICA treatment, followed by less activation of ERK1, 2, and p38. These results revealed that the significant promoting effects of prenylflavonoids on cardiac differentiation was at least partly via ROS signaling cascades, and the facilitating abilities preferentially based on the nature of prenylflavonoids themselves, but anti-oxidative activity determined by the OH numbers and the positions in the structures do influence the cardiomyogenesis in vitro.