[Treatment of ornithine transcarbamylase deficiency in a child with glyceryl phenylbutyrate]. / è‹¯ä¸é ¸ç”˜æ²¹é ¯æ²»ç–—å„¿ç«¥é¸Ÿæ°¨é ¸æ°¨ç”²é °åŸºè½¬ç§»é ¶ç¼ºä¹ç—‡1例.

Glyceryl phenylbutyrate (GPB) serves as a long-term management medication for Ornithine transcarbamylase deficiency (OTCD), effectively controlling hyperammonemia, but there is a lack of experience in using this medicine in China. This article retrospectively analyzes the case of a child diagnosed with OTCD at Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, including a review of related literature. After diagnosis, the patient was treated with GPB, followed by efficacy follow-up and pharmacological monitoring. The 6-year and 6-month-old male patient exhibited poor speech development, disobedience, temper tantrums, and aggressive behavior. Blood ammonia levels peaked at 327 µmol/L; urine organic acid analysis indicated elevated uracil levels; cranial MRI showed extensive abnormal signals in both cerebral hemispheres. Genetic testing revealed de novo mutation in the OTC gene (c.241T>C, p.S81P). Blood ammonia levels were approximately 43, 80, and 56 µmol/L at 1, 2, and 3 months after starting GPB treatment, respectively. During treatment, blood ammonia was well-controlled without drug-related adverse effects. The patient showed improvement in developmental delays, obedience, temperament, and absence of aggressive behavior.

Gut microbiota-derived cholic acid mediates neonatal brain immaturity and white matter injury under chronic hypoxia.

Chronic hypoxia, common in neonates, disrupts gut microbiota balance, which is crucial for brain development. This study utilized cyanotic congenital heart disease (CCHD) patients and a neonatal hypoxic rat model to explore the association. Both hypoxic rats and CCHD infants exhibited brain immaturity, white matter injury (WMI), brain inflammation, and motor/learning deficits. Through 16s rRNA sequencing and metabolomic analysis, a reduction in B. thetaiotaomicron and P. distasonis was identified, leading to cholic acid accumulation. This accumulation triggered M1 microglial activation and inflammation-induced WMI. Administration of these bacteria rescued cholic acid-induced WMI in hypoxic rats. These findings suggest that gut microbiota-derived cholic acid mediates neonatal WMI and brain inflammation, contributing to brain immaturity under chronic hypoxia. Therapeutic targeting of these bacteria provides a non-invasive intervention for chronic hypoxia patients.

Insulin-like Growth Factor 1 Promotes Cell Proliferation by Downregulation of G-Protein-Coupled Receptor 17 Expression via PI3K/Akt/FoxO1 Signaling in SK-N-SH Cells.

Insulin-like growth factor 1 (IGF-1) not only regulates neuronal function and development but also is neuroprotective in the setting of acute ischemic stroke. G-protein-coupled receptor 17 (GPR17) expression in brain tissue serves as an indicator of brain damage. As whether IGF-1 regulates GPR17 expression remains unknown, the aim of this study is to investigate how IGF-1 regulates GPR17 expression in vitro. Human neuroblastoma SK-N-SH cells were used. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to mediate the silencing of FoxO1, while adenoviral vectors were used for its overexpression. Verification of the relevant signaling cascade was performed using a FoxO1 inhibitor (AS1842856), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), and a GPR17 antagonist (cangrelor). Cell proliferation was analyzed using EdU staining; immunofluorescence staining was used to detect the expression and subcellular localization of FoxO1. Chromatin immunoprecipitation was used to analyze the binding of FoxO1 to the GPR17 promoter in SK-N-SH cells. The expression of FoxO1, GPR17, and protein kinase B (also known as Akt) mRNA and protein as well as the levels of FoxO1 and Akt phosphorylation were investigated in this study. IGF-1 was found to downregulate FoxO1 and GPR17 expression in SK-N-SH cells while promoting cell viability and proliferation. Inhibition of FoxO1 and antagonism of GPR17 were found to play a role similar to that of IGF-1. Silencing of FoxO1 by lentivirus-mediated shRNA resulted in the downregulation of FoxO1 and GPR17 expression. The overexpression of FoxO1 via adenoviral vectors resulted in the upregulation of FoxO1 and GPR17 expression. Blocking of PI3K signaling by LY294002 inhibited the effect of IGF-1 on GPR17 suppression. Results from chromatin immunoprecipitation revealed that IGF-1 promotes FoxO1 nuclear export and reduces FoxO1 binding to the GPR17 promoter in SK-N-SH cells. Here, we conclude that IGF-1 enhances cell viability and proliferation in SK-N-SH cells via the promotion of FoxO1 nuclear export and reduction of FoxO1 binding to the GPR17 promoter via PI3K/Akt signaling. Our findings suggest that the enhancement of IGF-1 signaling to antagonize GPR17 serves as a potential therapeutic strategy in the management of acute ischemic stroke.

Integrin-dependent microgliosis mediates ketamine-induced neuronal apoptosis during postnatal rat retinal development.

PURPOSE: Remodeling of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) plays a pivotal role for microglia in developing retina. We tested whether integrin-dependent microgliosis mediates ketamine-induced neuronal apoptosis in the developing rat retina. METHODS: We performed immunofluorescence assays to investigate the role of integrin receptors expressed in the microglia in ketamine-induced neuronal apoptosis. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to investigate the protein and mRNA levels of cytokines (TNF-α, IL-1ß) and/or chemokines (CCL2, CXCL6, CXCL10, and CXCL12). Experiments were performed using whole-mount retinas dissected from P7 Sprague-Dawley rats. RESULTS: Integrin receptors expressed in microglia were upregulated in ketamine-induced neuronal apoptosis in the early developing rat retina. Downregulating integrin receptors with RGD peptide ameliorated ketamine-induced microgliosis through: 1) ameliorating the change in microglia morphology from immature ramified microglia to an amoeboid state; 2) decreasing the number of microglia and intensity of activated microglia in the retinal ganglion cell layer (GCL); and 3) decreasing cytokine (TNF-α and IL-1ß) and chemokine (CCL2, CXCL10) levels in the retinal tissue. Inhibition of activated microglia with minocycline or the blockade of cytokines (TNF-α and IL-1ß) with a receptor antagonist (RA) attenuated neuronal apoptosis after exposure to ketamine. CONCLUSIONS: The upregulation of integrin ß1 receptors in the microglia acts as a signaling molecule, triggering microgliosis to aggravate ketamine-induced neuronal apoptosis via the release of TNF-α and IL-1ß in the early developing rat retina.

Grape seed proanthocyanidin extract induces apoptosis of HL-60/ADR cells via the Bax/Bcl-2 caspase-3/9 signaling pathway.

BACKGROUND: Our previous study detailed the direct induction of apoptosis by grape seed proanthocyanidin extract (GSPE) in a multidrug resistant human acute myeloid leukemia (AML) HL-60/adriamycin (HL-60/ADR) cell line, although the mechanism of this effect was not detailed. This study aims to elucidate the mechanism underlying GSPE-induced cell apoptosis in HL-60/ADR cells. METHODS: HL-60/ADR cells were studied to evaluate effects of GSPE (0-100 µg/mL); a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to identify the cytotoxic effect of varying GSPE concentrations. Trypan blue staining was used to observe changes in cell viability; flow cytometry assays were used to verify apoptosis. Expression of Bax and Bcl-2 mRNA was analyzed using real-time polymerase chain reaction (PCR). Activity of caspase-3 and caspase-9 was also detected. RESULTS: Here, GSPE was found to inhibit HL-60/ADR cell growth and induce cell apoptosis in a dose-dependent manner. Real-time PCR findings revealed that GSPE concentrations above 75 µg/mL significantly increase expression of Bax mRNA (P<0.001). GSPE concentrations above 25 µg/mL were found to significantly decrease expression of Bcl-2 mRNA (P<0.01), while concentrations above 50 µg/mL were found to significantly increase caspase-3 activity after 6, 12 and 24 h (P<0.01). However, only 100 µg/mL GSPE was found to significantly increase caspase-9 activity (P<0.001 at 6 and 12 h; P<0.05 at 24 h). CONCLUSIONS: GSPE inhibits the proliferation of HL-60/ADR cells by the induction of apoptosis in a dose-dependent manner via the Bax/Bcl-2 caspase-3/9 signaling pathway.

Grape seed proanthocyanidin extract reverses multidrug resistance in HL-60/ADR cells via inhibition of the PI3K/Akt signaling pathway.

BACKGROUND AND PURPOSE: Multidrug resistance (MDR) is a great challenge and obstacle in cancer treatment. It is a common problem in the treatment of acute myeloid leukemia (AML). Whether grape seed proanthocyanidin extract (GSPE) could reverse MDR in patients with AML is still unknown. The aim of this study was to investigate the MDR reverse ability of GSPE and its possible mechanism in vitro. MATERIALS AND METHODS: Human leukemia cell line HL-60 cells and HL-ADR cells were used. MTT assay were employed to identify the cytotoxic effects of different chemotherapeutic drugs and reverse ability of GSPE. Flow cytometry assays were used to verify the cell apoptosis induced by GSPE. MDR-related genes expression was tested by real-time polymerase chain reaction (Q-PCR). MDR-related protein expression was assessed by Western blotting assays. The genes and their related protein expression of multidrug resistance-associated protein 1 (MRP1), multidrug resistance protein 1 (MDR1) and lung resistance-related protein (LRP) were tested in this study. KEY RESULTS: We found that HL-60/ADR cells were resistant to a variety of chemotherapeutic drugs, including cytarabine (Ara-C), adriamycin (ADR), vincristine (VCR), daunorubicin (DNR), mitoxantrone (MTZ), pirarubicin (THP), homoharringtonine (HHT) and etoposide (VP16). Co-treatment with GSPE could significant lower the IC50 of Ara-C and ADR in HL-60/ADR cells (P < 0.01). MDR related mRNA and their protein expression of MRP1 and MDR1 were significant highly expressed in HL-60/ADR cells than HL-60 cells (P < 0.01). But only protein expression of LRP was higher in HL-60/ADR cells than HL-60 cells (P < 0.05). GSPE could induce a higher intracellular level of ADR in HL-60/ADR cells. It could also inhibit Akt phosphorylation resulted in the down regulation of MRP1, MDR1 and LRP and induce cell apoptosis. 25.0 µg/mL GSPE significant inhibited the Akt phosphorylation (P < 0.05). CONCLUSION AND IMPLICATIONS: GSPE-reversed MDR of HL-60/ADR cells might be associated with the inhibition of the PI3K/Akt signaling pathway, which resulted in the down-regulation the expression of MRP1, MDR1 and LRP. These results provide that GSPE may serve as a combination therapy in AML chemotherapy for future study.

[G protein-coupled receptor 17 is involved in CoCl2-induced hypoxic injury in RGC-5 cells].

OBJECTIVE: To investigate the effect of G protein-coupled receptor 17 (GPR17) on hypoxia injury in retinal ganglion cells in vitro. METHODS: CoCl2 (400 µmol/L) was used to induce hypoxic injury in RGC-5 cells. The expression of GPR17 and the effect of GPR17 ligands were investigated, and the role of GPR17 in hypoxia injury was further studied by transfection of RGC-5 cells with GPR17 small interfering RNA (siRNA). The cell viability was determined by MTT and the cell apoptosis rate was detected by flow cytometry analysis. The expression of GPR17 mRNA was determined with RT-PCR. RESULTS: mRNA expressions of GPR17 in RGC-5 cells with and without CoCl2 treatment were 0.36±0.05 and 0.26±0.08(P<0.01). Compared with hypoxia without any treatment, pretreatment with GPR17 agonists (LTD4, UDP, UDP-G) significantly reduced cell viability (the survival rates of cells decreased by 29.6%, 31.8% and 33.9%, all P<0.01), while the effect of GPR17 antagonist (cangrelor) was the opposite (the survival rates of cells increased by 33.2%, P<0.01). Transfection with GPR17 SiRNA inhibited hypoxia-induced up-expression of GPR17 mRNA (P<0.01)and reduced cell apoptosis[rates of cell apoptosis were(39.73±2.06)%,(42.50±3.64)% and (24.98±2.16)% for blank control, NC siRNA and GPR17 siRNA groups, P<0.01]. CONCLUSIONS: GPR17 may mediate hypoxia injury in RGC-5 cells, while the knockdown of GPR17 can reduce the hypoxia injury.

[Protective effect of grape seed proanthocyanidin on cultured RGC-5 cells against CoCl2-induced hypoxic injury].

OBJECTIVE: To investigate the protective effects of grape seed proanthocyanidin extracts (GSPE) against CoCl2-induced hypoxic injury in cultured RGC-5 cells. METHODS: CoCl2(400 µmol/L) was used to induce hypoxic injury in cultured RGC-5 cells; the cells were pretreated with 0,100,200,400 and 800µmol/L GSPE for 24h. The cell viability was assayed by MTT; the apoptosis was detected by Hoechst 33342 staining; the intracellular reactive oxygen species (ROS) was measured by H2DCFDA oxidative reaction. The mRNA expression of Bcl-2, caspase 9 and caspase 3 was determined by real-time PCR. RESULTS: Compared to hypoxic control group, pretreatment with GSPE significantly increased viability of RGC-5 cells (P<0.001), reduced cell apoptosis (P<0 .001) and intracellular ROS(P <0 .001). In addition, GSPE significantly increased the mRNA expression of Bcl-2(P<0 .001) and decreased mRNA expression of caspase 9(P<0 .001) and caspase 3(P<0 .001) compared to hypoxic control group. CONCLUSION: GSPE may have a protective effect against CoCl2-induced hypoxic injury in cultured RGC-5 cells. The decrease of intercellular ROS, up-regulation of Bcl-2 and down-regulation of caspase 9 and caspase 3 may be involved in the mechanism of the protective effect of GSPE.

Endostar inhibits hypoxia-induced cell proliferation and migration via the hypoxia-inducible factor-1α/vascular endothelial growth factor pathway in vitro.

Endostar, a recombinant human endostatin, is recognized as one of the most effective angiogenesis inhibitors. The angiogenesis inhibitory effects of Endostar suggest a possible beneficial role of Endostar in choroidal neovascularization (CNV), which is predominantly induced by hypoxia. In our previous study, it was reported that Endostar may inhibit the proliferation and migration of RF/6A choroid­retinal endothelial cells. However, the inhibitory effect of Endostar on hypoxia­induced cell proliferation and migration in RF/6A cells has not yet been elucidated. Therefore, the present study investigated the effect of Endostar on hypoxia­induced cell proliferation and migration in RF/6A cells and the possible mechanisms underlying this effect. Under chemical hypoxia conditions, cell viability was increased to 114.9±10.1 and 123.6±9.6% in cells treated with 100 and 200 µm CoCl2, respectively, compared with the control (P<0.01). Pretreatment with 10­100 µg/ml Endostar significantly inhibited CoCl2­induced cell proliferation (P<0.05), and pre­treatment with 10 µg/ml Endostar for 24, 48 and 96 h attenuated CoCl2­promoted cell migration by 60.5, 48.3 and 39.6%, respectively, compared with the control (P<0.001). In addition, pretreatment with 10 µg/ml Endostar reversed the cell cycle arrest at S phase and the increased expression of hypoxia­inducible factor­1α (HIF­1α) and vascular endothelial growth factor (VEGF) mRNA in RF/6A cells treated with 200 µM CoCl2. These data indicate that Endostar inhibited CoCl2­induced hypoxic proliferation and migration, and limited cell cycle progression in vitro possibly through the HIF­1α/VEGF pathway.

Suppression of choroidal neovascularization by Endostar in rats.

Choroidal neovascularization (CNV) is common in various retinal and choroidal diseases, and may result in severe and irreversible loss of vision. Our previous studies suggested that Endostar, a novel recombinant endostatin, is able to inhibit the proliferation and migration of choroid­retinal endothelial cells. To further evaluate the effect of Endostar on the formation of CNV in vivo, a rat model of laser­induced CNV was constructed and Endostar or phosphate­buffered saline treatment was administered intravitreally every other day. Using fluorescein angiography (FA), reduced CNV incidence and leakage grade was observed in the Endostar group. In addition, CNV area and maximal thickness were prominently reduced in the Endostar group measured by choroid flat mounts and sections. Furthermore, vascular endothelial growth factor (VEGF), hypoxia­inducible factor 1α and chemokine C­X­C motif ligand 1 were markedly reduced in the Endostar group as determined by quantitative polymerase chain reaction and downregulation of VEGF was also verified by western blot analysis at the protein level. This study demonstrates that Endostar suppressed CNV in a rat model, which may be largely mediated by the downregulation of VEGF and other angiogenic molecules.

ERK/Egr-1 signaling pathway is involved in CysLT2 receptor-mediated IL-8 production in HEK293 cells.

The CysLT2 receptor is involved in myocardial ischemia/reperfusion injury, differentiation of colorectal cancers, bleomycin-induced pulmonary inflammation and fibrosis. However, the signal transduction of cysteinyl leukotriene receptor 2 (CysLT2) in inflammatory responses remains to be clarified. In HEK293 cells stably expressing hCysLT1, hCysLT2 and rGPR17, we determined the signaling pathways for interleukin-8 (IL-8) production after CysLT2 receptor activation. HEK293 cells were stably transfected with the recombinant plasmids of pcDNA3.1(+)-hCysLT1, pcDNA3.1(+)-hCysLT2 and pcDNA3.1-rGPR17. Leukotriene C4 (LTC4) and LTD4 were used as the agonists to induce IL-8 production and the related changes in signal molecules. We found that LTC4 and LTD4 significantly induced IL-8 promoter activation in the HEK293 cells stably expressing hCysLT2, but not in those expressing hCysLT1 and rGPR17. In hCysLT2-HEK293 cells, LTC4 induced elevation of intracellular calcium, ERK1/2 phosphorylation and Egr-1 expression, and stimulated IL-8 expression and release. These responses were blocked by the selective CysLT2 receptor antagonist HAMI3379. The ERK1/2 inhibitor U0126 inhibited Egr-1 and IL-8 expression as well as IL-8 release, but the JNK and p38 inhibitors did not have the inhibitory effects. Down-regulation of Egr-1 by RNA interference with its siRNA inhibited the LTC4-induced IL-8 expression and release. In conclusion, these findings indicate the ERK-Egr-1 pathway of CysLT2 receptors mediates IL-8 production induced by the pro-inflammatory mediators LTC4 and LTD4.

[(-)-Epigallocatechin gallate regulates expression of apoptotic genes and protects cultured human lens epithelial cells under hyperglycemia].

(-)-Epigallocatechin gallate (EGCG), the most abundant component in green tea, has a potent anti-apoptotic activity. The purpose of this study was to investigate the protective effects of EGCG and their molecular mechanisms on high glucose-induced apoptosis of human lens epithelial cells (HLEB-3). HLEB-3 cells were exposed to various concentrations of glucose and EGCG. Cell death was assessed by MTT assay and flow cytometry using annexin V and propidium iodide. The expression of the Bcl-2 family, c-fos, c-myc and p53 was measured by real-time PCR. EGCG decreased the Bcl-2/Bax expression stimulated by a high glucose. Moreover, EGCG suppressed the high glucose-induced expression of c-fos, c-myc and p53. These findings suggest that EGCG protects HLEB-3 cells from high glucose-induced apoptosis by regulating the gene expression of the Bcl-2 family, c-fos, c-myc and p53. Thus, EGCG may have a potential protective effect against diabetic cataract formation.

Structure-based drug design using GPCR homology modeling: toward the discovery of novel selective CysLT2 antagonists.

3D structure of CysLT2 receptor was constructed by using homology modeling and molecular simulations. The binding pocket of CysLT2 receptor and the proposition of the interaction mode between CysLT2 and HAMI3379 were identified. A series of dicarboxylated chalcones was then virtually evaluated through molecular docking studies. A total of six compounds 13a-f with preferable scores was further synthesized and tested for CysLT2 antagonistic activities by determination of the cytosolic free Ca(2+) levels in HEK293 cells. Compounds 13e and 13f exhibited potent and selective CysLT2 antagonistic activities with IC50 values being 7.5 and 0.25 µM, respectively.

Nuclear translocation of cysteinyl leukotriene receptor 1 is involved in oxygen-glucose deprivation-induced damage to endothelial cells.

AIM: Cysteinyl leukotriene receptor 1 (CysLT(1) receptor) is located in epithelial cells, and translocates from the plasma membrane to the nucleus in a ligand-dependent manner. Here, we investigated whether CysLT(1) receptors translocated to the nucleus in endothelial cells after ischemic insult in vitro and whether it was involved in ischemic injury to endothelial cells. METHODS: EA.hy926 cell line, derived from human umbilical vein endothelial cells, was subjected to oxygen-glucose deprivation (OGD). The expression and distribution of CysLT(1) receptors were detected by immunofluorescent staining, immunogold labeling and immunoblotting analyses. Cell viability was evaluated using MTT reduction assay. Necrosis and apoptosis were determined by double fluorescent staining with propidium iodide and Hoechst 33342. RESULTS: CysLT(1) receptors were primarily distributed in the cytoplasm and nucleus in EA.hy926 cells, and few was found in the cell membrane. OGD induced the translocation of CysLT(1) receptors from the cytoplasm to the nucleus in a time-depen dent manner, with a peak reached at 6 h. OGD-induced nuclear translocation of CysLT(1) receptors was inhibited by pretreatment with the CysLT(1) receptor antagonist pranlukast (10 µmol/L), or by preincubation with NLS-pep, a peptide corresponding to the nuclear localization sequence of CysLT(1) receptor (10 µg/mL). However, zileuton, an inhibitor of 5-lipoxygenase that was a key enzyme in cysteinyl leukotriene generation, did not inhibit the nuclear translocation of CysLT(1) receptors. Moreover, preincubation with NLS-pep (0.4 µg/mL) significantly ameliorated OGD-induced cell viability reduction and necrosis. CONCLUSION: CysLT(1) receptors in endothelial cells translocate to the nucleus in a ligand-independent manner after ischemic insult in vitro, and it is involved in the ischemic injury.

[Construction of HEK293 cell lines expressing hCysLT2 receptor and its application in screening of antagonists].

OBJECTIVE: To construct HEK293 cell lines stably expressing hCysLT(2) receptor, and to evaluate its application in screening of synthetic compounds with antagonist activity. METHODS: The recombinant plasmid pcDNA3.1(+)-hCysLT(2) was transfected into HEK293 cells using Lipofectamin 2000. The transfected HEK293 cells were selected in 96 well plates by limiting dilution with 600 µg/ml C418 for 8 weeks. The expression of human CysLT(2) receptor was detected by RT-PCR and immunofluorescence staining. In HEK293 cells stably transfected with hCysLT(2), the agonist LTD(4)-induced elevation of intracellular calcium concentration ([Ca2(+)]i) was measured as the index for screening compounds with antagonist activity. RESULT: After selection in 96 well plates by limiting dilution, 12 monoclones were obtained and 11 of them highly expressed hCysLT(2) receptor. The positive control ATP at 50 µmol/L and LTD(4) at 100 nmol/L elevated [Ca2(+)]i in hCysLT(2)-HEK293 cells. AP-2100984 inhibited LTD(4)-induced [Ca2(+)]i elevation, but selective CysLT(1) receptor antagonists did not exert such an effect. The newly synthesized compounds DXW2, DXW3, DXW4, DXW5, DXW9, DXW25, DXW26, DXW29 and DXW35 at 1 µmol/L significantly inhibited LTD(4)-induced [Ca2(+)]i elevation. The IC(50) values of DXW4 and DXW5 were 0.25 µmol/L and 7.5 µmol/L. CONCLUSION: HEK293 cell lines stably expressing hCysLT(2) receptor have been successfully constructed, and can be used to screen compounds with CysLT(2) receptor antagonist activity.

Pranlukast attenuates hydrogen peroxide-induced necrosis in endothelial cells by inhibiting oxygen reactive species-mediated collapse of mitochondrial membrane potential.

OBJECTIVE: Recently, we reported that pranlukast, an antagonist of cysteinyl leukotriene receptor 1, attenuates ischemic injury in endothelial cells by decreasing reactive oxygen species (ROS) production and inhibiting nuclear factor-κB activation in a leukotriene-independent manner. In this study, we investigated the effect of pranlukast on oxidative stress injury induced by hydrogen peroxide (H2O2) in EA.hy926 cells, a human endothelial cell line, and the possible mechanisms. METHODS AND RESULTS: We found that H2O2 reduced cell viability and increased lactate dehydrogenase release in a concentration- and time-dependent manner. Necrosis was the main death mode, and the necrotic rate increased 32% after exposure to 220 µM H2O2 for 4 hours. Pretreatment with pranlukast significantly ameliorated the reduced viability and the increased lactate dehydrogenase release and necrosis after exposure to H2O2. We next examined the mechanisms underlying the antinecrotic effects of pranlukast. The results showed that pranlukast attenuated excessive ROS production and ameliorated the reduced superoxide dismuase and glutathione peroxidase activity in EA.hy926 cells exposed to H2O2. Pranlukast also inhibited the collapse of mitochondrial membrane potential (MMP) induced by H2O2. Inhibition of ROS production by N-acetyl-l-cysteine, a powerful antioxidant, reduced MMP collapse and necrosis. Inhibition of MMP collapse by cyclosporine A, a mitochondrial permeability transition inhibitor, attenuated necrosis but failed to reduce ROS production. In addition, we found no expression of 5-lipoxygenase in EA.hy926 cells and zileuton, a 5-lipoxygenase inhibitor, did not affect the cellular injury induced by H2O2. CONCLUSION: Pranlukast protects endothelial cells from H2O2-induced necrosis by inhibiting ROS-mediated collapse of mitochondrial membrane potential, and this is leukotriene-independent.

[Construction and identification of eukaryotic expression vector of rat GPR17 gene].

OBJECTIVE: To construct the eukaryotic expression vector of rat GPR17 (rGPR17) cDNA,and to identify its function in HEK293 cells. METHODS: Total RNA was extracted from rat brain tissue; full-length GPR17 cDNA was prepared by RT-PCR, and cloned into pcDNA3.1(+) plasmid. The recombinant plasmid was converted into E.coli DH5alpha and confirmed by PCR, double enzyme digestion analysis and DNA sequencing. The recombinant plasmid pcDNA3.1(+)-rGPR17 was transiently transfected into HEK293 cells using Lipofectamin 2000. Expression of rGPR17 gene was confirmed by RT-PCR and immunofluorescence staining. The exogenous LTD(4) enhanced intracellular calcium was measured using Fluo-4. RESULT: RT-PCR, double enzyme digestion analysis and sequencing showed that the rGPR17 gene was cloned into recombinant vector, and the recombinant rGPR17 was expressed after transfection in HKE293 cells. LTD(4) increased intracellular calcium release in the transfected HEK293 cells. CONCLUSION: The eukaryotic expression vector of rGPR17 cDNA has been constructed; it is functionally expressed in HEK293 cells. This work provides a basis for further research of the GPR17 receptor and its antagonists.