5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine and diacetyldichlorofluorescein

Patulin (PAT) is one of the most widely disseminated mycotoxins found in agricultural products and could cause renal damage. However, the mechanism of cell damage remains obscure. In this study, the human embryonic kidney cells (HEK293) were treated with PAT (2.5-15 μM). The cytotoxicity was assessed with MTT assay and apoptotic cells were detected by flow cytometry, and further identified by chromatin condensation and nuclear fragmentation with Hoechst 33342 under fluorescence microscope. Reactive oxygen species (ROS) with DCFH-DA was analyzed by fluorometry. The activities of superoxide dismutase (SOD), glutathione (GSH) and catalase (CAT) and malondialdehyde (MDA) content were determined to reveal the potential mechanism of PAT induced apoptosis. The mitochondrial membrane potential was measured by JC-1 probe. The results showed that PAT dose-dependently inhibited the growth of HEK293 cells and resulted in apoptosis in HEK293 cells. Treatment with PAT could induce ROS and MDA accumulation, accompanied by the losses of SOD, CAT, GSH and disruption of the mitochondrial membrane potential. These data suggest that PAT may induce apoptosis in HEK293 cells, in which oxidative stress is involved.

Topics: Antioxidants; Apoptosis; Benzimidazoles; Carbocyanines; Catalase; Fluoresceins; Glutathione; HEK293 Cells; Humans; Membrane Potential, Mitochondrial; Mycotoxins; Oxidative Stress; Patulin; Reactive Oxygen Species; Superoxide Dismutase

Recent studies have suggested that intrinsic apoptotic signaling cascade is involved in endothelial barrier dysfunction following hemorrhagic shock (HS), which results in vascular hyperpermeability. Our previous study demonstrated that ulinastatin (UTI) inhibits oxidant-induced endothelial hyperpermeability and apoptotic signaling. In present study, we hypothesized that UTI would improve HS-induced vascular hyperpermeability by regulating the intrinsic apoptotic signaling cascade.. Hemorrhagic shock was induced in rats by withdrawing blood to reduce the mean arterial pressure to 40-45 mmHg for 60 min, followed by reperfusion. Mesenteric postcapillary venules were examined for changes in hyperpermeability by intravital microscopy. In vitro, Rat lung microvascular endothelial cells (RLMVECs) were exposed in hemorrhagic shock serum for 120 min, followed by transendothelial electrical resistance (TER) estimation. Mitochondrial release of cytochrome c and caspase-3 activation was estimated in vivo. In vitro, ratio of cell apoptosis was evaluated by Annexin-V/PI double stain assay; mitochondrial membrane potential (∆Ψm) was determined with JC-1; intracellular ATP content was assayed by a commercial kit; reactive oxygen species (ROS) was measured by DCFH-DA; adherens junction protein β-catenin was detected by immunofluorescense staining.. In vivo, UTI attenuated HS-induced vascular hyperpermeability versus the HS group (P < 0.05); In vitro, UTI attenuated shock serum induced RLMEC monolayer hyperpermeability (P < 0.05). In vivo, UTI inhibited HS-induced cytochrome c release and caspase-3 activation (P < 0.05). In vitro, shock serum induced cell apoptosis, low ATP level, ∆Ψm depolarization, ROS increase were improved by UTI pre-treatment (P < 0.05). UTI improved shock serum induced disruption of endothelial cell adherens junction.. UTI inhibits vascular hyperpermeability following HS. UTI regulates oxidative stress and intrinsic apoptotic signaling following HS.

Topics: Animals; Apoptosis; Benzimidazoles; beta Catenin; Capillary Permeability; Carbocyanines; Caspase 3; Cytochromes c; Endothelial Cells; Fluoresceins; Glycoproteins; Humans; Male; Membrane Potential, Mitochondrial; Mesentery; Mitochondria; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Shock, Hemorrhagic; Signal Transduction; Trypsin Inhibitors

The mechanisms of cytokine-induced beta-cell death are poorly characterised. In rat insulin-producing RINm5F cells, the combination of interleukin-1beta, interferon-gamma and tumour necrosis factor-alpha presently induced disruption of the mitochondrial membrane potential (Deltapsi(m)) as demonstrated by reduced JC-1 fluorescence. The reduction of Deltapsi(m) was maximal after 8 h and was preceded by increased formation of reactive oxygen species (ROS), as assessed by dichlorofluorescein-diacetate (DCFH-DA) fluorescence. A nitric oxide synthase-, but not a ROS-inhibitor, prevented cytokine-induced loss of Deltapsi(m). Overexpression of the anti-apoptotic protein Bcl-2 increased both JC-1 and DCFH-DA fluorescence, which was paralleled by protection against cytokine-induced apoptosis and necrosis. It is concluded that cytokines induce a nitric oxide-dependent disruption of Deltapsi(m) and that this may be a necessary event for both beta-cell apoptosis and necrosis. Bcl-2 may prevent beta-cell death by counteracting mitochondrial permeability transition.

Topics: Animals; Apoptosis; Benzimidazoles; Carbocyanines; Catalase; Cell Line; Enzyme Inhibitors; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Interferon-gamma; Interleukin-1; Islets of Langerhans; Membrane Potentials; Mitochondria; Necrosis; Nitric Oxide Synthase; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species; Tumor Necrosis Factor-alpha