Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, trypsin/EDTA and glutamine were supplied by Gibco-BRL (Rockville, MD, USA). ECL western blotting detection reagents and PRO-PREP protein extract solution were supplied by iNtRON Biotechnology (Houston, TX, USA). Anti-p-Akt antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin antibody, Glucose oxidase (GOX) and other chemicals were purchased from Sigma (St. Louis, MO, USA).

B35 neuroblastoma cells were purchased from American Type Culture Collection (Manassas, VA, USA). For all experiments, cells were grown in DMEM supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin and 10% heat inactivated FBS in culture plates. The cells were incubated in a 37℃ incubator under 5% CO₂ in a humidified atmosphere. After reaching confluence, cells were pretreated with various drugs depending on the experimental design.

Cell viability changes due to GOX and culture conditions were determined in terms of LDH released into media of cell cultures. Cells in the exponential phase of growth were seeded at 1 × 10⁴ cells/well in 24-well plates. After different treatments, 50 ml of standards or 50 ml of culture supernatant (controls and treated cells) and 50 ml of substrate mix were transferred into a 96-well-plate. After incubation for 30 min at room temperature, plates were read using a SUNRISE ELISA-plate Reader (Tecan, Crailsheim, Germany) at 490 nm. LDH released from cells is expressed as a percentage of total cellular LDH.

Whole cell lysates were isolated using PRO-PREP protein extract solution. The whole cell lysates were centrifuged at 100,000 g for 20 min at 4℃. The protein concentration in each lysate was determined by the Bradford assay. To detect Akt-phosphorylation, 30 micrograms of protein were subjected to 10% SDS-polyacrylamide gel electrophoresis for 90 min at 120 V depending on the molecular size of the phospho-Akt protein. The separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes for 2 h at 20 mA using SD Semidry Transfer Cells (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were stained with Ponceau S solution to determine efficiency of transfer and/or protein loading levels. Then the PVDF membranes were blocked for 2 h at room temperature with 5% nonfat milk in Tris-buffed saline (TBS, pH 7.0) containing 0.05% Tween 20 (TBS-T). The membranes were incubated with an anti-phospho-Akt antibody overnight at 4℃, using a dilution of 1 : 500 in 5% bovine serum albumin (BSA) in TBS-T. For the detection of bound antibody, horseradish peroxidase conjugated anti-goat or anti-rabbit IgG was used. The signals were detected by ECL using a western blotting luminol reagent system (iNtRON Biotechnology, Houston, TX, USA) and autoradiography.

A total of 1 × 10⁷ cells were seeded in 100 mm culture dishes, incubated at 37℃, and harvested at the indicated times after mitosis. Cells were stained with propidium iodide (PI) using a previously described method [8]. The harvested cells were trypsinized, washed twice in PBS, and fixed in 70% ethanol on ice for a minimum of 2 h. Cells were washed 2 additional times with PBS and stained for 30 min at 37℃ in 50 µg/ml PI solution containing 200 µg/ml RNase A and 0.1% Triton X-100. After incubation of samples in the dark for 30 minutes, cell cycle profiles were determined using a Beckton-Dickinson flow cytometer (Becton Dickenson Biosciences, San Jose, CA, USA) with CellQuest® Software.

Values are expressed as means ± SD. Differences between data sets were assessed by one-way analysis of variance (ANOVA) followed by the Newman-Keuls test. P < 0.05 was regarded as statistically significant.

The effects of midazolam on cell viability during ROS injury were analyzed by LDH assay. As expected, 10 h of incubation with 20 mU/ml of GOX significantly decreased cell viability. Fig. 1A shows that GOX derived ROS injury decreased cell viability about 1.6-2 times compared to the control. However, treatment with midazolam (5 and 10 µg/ml) concentration-dependently reduced the decreased cell viability due to ROS injury, and this difference was statistically significant. To determine the effect of an Akt-phosphorylation inhibitor on cell viability, 5 µM concentrations of LY294002 were used to treat cells for 1 h prior to treatment with midazolam. As shown in Fig. 1B, LY294002 inhibited the protective effect of midazolam. For example, pretreatment with 5 µM LY294002 significantly decreased cell viability that had been induced by midazolam.

We next asked whether midazolam can protect B35 cells from oxidative stress induced injury by regulating Akt-phosphorylation. Akt phosphorylation plays important roles in general cellular defense systems against oxidative stress in mammalian cells. As shown in Fig. 2, western blot analysis indicated that midazolam-induced cytoprotective effects in ROS injury may be mediated by induction of Akt-phosphorylation through PI3 kinase activation. The effect of midazolam on Akt-phosphorylation was dose dependent, but the induction was abolished by pretreatment with LY294002, a PI3 kinase inhibitor.

It is generally known that ROS induces apoptosis in many mammalian cells [9] and GOX is frequently used as a chronic ROS producing agent [10]. Therefore, we investigated whether midazolam protects cells from GOX-induced apoptosis. Fig. 3 shows that GOX-induced apoptosis was diminished by midazolam pretreatment. The effect is mediated through Akt-phosphorylation because the diminished apoptosis due to addition of midazolam became obvious again as the concentration of LY294002, an inhibitor of PI3 kinase, increased. FACS analysis (Fig. 3) showed that about 61% of cells underwent apoptosis after GOX (20 U/ml), but pretreatment with midazolam significantly reduced the fraction of dead cells to 21%. Co-treatment with LY294002 significantly reduced the anti-apoptotic effect of midazolam.