All cell and tissue culture products were purchased from Invitrogen (Carlsbad, CA, USA). HS (product no. 082–0461) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Escherichia coli lipopolysaccharide (LPS) and other chemicals were purchased from Sigma (St. Louis, MO, USA). Antibodies against phospho-p44/42 MAPK, p44/42 MAPK, phospho-SAPK/JNK, SAPK/JNK, phospho-p38, p38, phospho-Akt and Akt were purchased from Cell Signaling Technology (Beverly, MA, USA).

Rats were maintained in accordance with the Institutional Animal Care and Use Committee guidelines of Kyung Hee University. All animal protocols were approved by the Animal Ethics Committee of Kyung Hee University in accordance with the 14th article of the Korean Animal Protection Law.

Organotypic hippocampal slice cultures were prepared from male Sprague-Dawley rats (seven days old; Orient, Kyunggido, Korea) using the methods previously described by Stoppini and others (16,17). Briefly, the hippocampus was isolated and cut transversely at a thickness of 350 μm with a McIlwain Tissue Chopper (Mickle Laboratory Engineering, Surrey, UK). The slices were placed on membrane inserts (Millicell-CM; Millipore, Bedford, MA, USA) in six-well plates. Each well contained 1 ml of culture medium composed of 50% MEM, 25% Hank’s Balanced Salt Solution and 25% horse serum. The slices were cultured at 36°C in an incubator in the presence of 5% CO₂ for 12–14 days and the medium was changed every 2–3 days.

Neurotoxicity was evaluated by the uptake of the fluorescent dye propidium iodide (PI) as previously described (17,18). Briefly, LPS (10 μg/ml) was applied to hippocampal cultures with or without pretreatment with HS. Following LPS treatment, the culture medium was collected and subjected to the nitrite assay prior to being replaced with fresh serum-free medium containing 5 μg/ml PI. Neuronal death was observed within 30–60 min of PI addition. PI-stained images were captured using a laser scanning microscope (LSM 510; Carl Zeiss, Cambridge, UK) and the observed PI-uptake areas were measured using confocal microscopy with LSM 510 software (release 3.2; Carl Zeiss). All the data were background subtracted using the fluorescence emission originated from a region on the insert containing no tissue. For immunofluorescent staining of neurons, hippocampal slices were fixed in 4% paraformaldehyde and stained with Alexa fluor 488-conjugated mouse anti-NeuN monoclonal antibody (Chemicon International, Temecula, CA, USA). The immunostained images were observed under a Carl Zeiss LSM 510 microscope.

Primary microglial cells were prepared from cerebral cortices of one-day-old rat pups (Orient) as described previously (19,20). Cells reached confluence at 12–14 days and flasks were agitated to remove the microglia. The detached cells were incubated for 1 h and the non-adherent cells were removed. The adherent microglial cells were cultured for 24 h and the purity of the cultures was routinely >95%, as judged by immunostaining with an anti-OX-42 antibody (Chemicon). The cells were pretreated with HS in fresh medium containing 0.1% fetal bovine serum for 30 min prior to the addition of LPS.

Nitrite in culture supernatants was measured as an indicator of NO production. An aliquot of the culture supernatant was mixed with a volume of Griess reagent (Molecular Probes, Eugene, OR, USA) and the absorbance at 570 nm was determined using a microplate reader. Sodium nitrite (0–100 μM) was used as a standard to assess nitrite concentrations.

For the cell viability assay, cultures were incubated in MTT solution (1 mg/ml; Sigma) in two volumes of culture medium for 1 h at 37°C. The MTT solution was then removed, the cells were dissolved in dimethyl sulfoxide (150 μl) and optical density of the samples was measured at 570 nm using a microplate reader.

Following each treatment, culture medium was collected in microcentrifuge tubes and centrifuged at 10,000 × g for 10 min. The supernatants were assayed for secreted mediators using PGE₂ and rat IL-1β immunoassay kits (R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.

Presence of intracellular ROS was measured using a non-fluorescent 2′,7′-dichlorofluorescein (DCFH-DA; Molecular Probes) dye as described previously (21). DCF fluorescence was measured using a Wallac 1420 fluorometer (Perkin Elmer, Waltham, MA, USA) at 485 nm for excitation and 530 nm for emission.

Cells were lysed on ice in lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail; Sigma]. Following centrifugation, the supernatant was collected and assayed for protein concentration using a DC Protein Assay kit (Bio-Rad, Hercules, CA, USA). Lysate samples containing 30 μg of protein were fractionated by SDS-10% polyacrylamide gel electrophoresis and then electroblotted onto nitrocellulose membranes. The membranes were probed with primary antibodies and immunoreactivity was detected with ECL Reagent (Amersham Biosciences, Piscataway, NJ, USA).

For statistical analysis, data were expressed as the mean ± SEM from three independent experiments. The Student’s paired t-test was used for statistical analyses which were performed using SPSS software (version 13.0, SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

In an effort to develop neuroprotective drugs, strategies to ameliorate the inflammatory microenvironment, which indirectly damages neurons via glial cell mediators, are promising (4). We previously established an experimental condition to determine the effect of LPS exposure on neuronal damage in organotypic hippocampal slice cultures (20). In the present study, slice cultures exposed to LPS for 72 h exhibited marked PI uptake in the hippocampus in comparison to untreated control slices (Fig. 1A). The increased PI uptake was markedly blocked by treatment with HS (Fig. 1A and B), in parallel with the inhibition of LPS-induced production of various proinflammatory mediators, including NO, PGE₂ and IL-1β (Fig. 1D-F). Reduced immunoreactivity of NeuN, a neuronal-specific marker, accompanied with elevated PI fluorescence indicated that loss of neurons resulted from the LPS insult (Fig. 1C). Treatment with HS restored immunoreactivity of NeuN and simultaneously decreased PI fluorescence (Fig. 1C). Together, these results indicate that HS has protective effects against inflammation-induced neurotoxicity.

To determine the mechanisms of the anti-inflammatory effects of HS in more detail, various mediators of microglial activation were measured. The effects of treatment with HS on secretion of proinflammatory mediators from microglial cells were tested. HS suppressed LPS-induced nitrite release from microglial cells in a dose-dependent manner (Fig. 2A). Cell viability, as measured using the MTT assay, was not significantly affected at ≤20 μM (Fig. 2B). HS appeared to decrease cell viability of activated microglia to a degree at 30 μM (Fig. 2B), however, it alone had no effect on basal NO release at <30 μM (Fig. 2C).

HS was found to reduce LPS-induced production of PGE₂ (Fig. 3A). Intracellular ROS act as second messengers in the regulation of the LPS-stimulated production of neurotoxic factors in microglia (22). The ROS levels measured using DCFH-DA revealed that pretreatment with HS decreased LPS-induced ROS production in microglia (Fig. 3B).

Multiple signaling pathways, including those involving mitogen-activated protein kinases (MAPKs) and Akt, have been reported to be involved in LPS-induced signal transduction, which results in the induction of proinflammatory gene expression (23–26). The present study demonstrates that HS markedly inhibited the LPS-enhanced phosphorylation of p44/42 MAPK, p38, SAPK/JNK (Fig. 4). Among the kinases, phosphorylation of SAPK/JNK was reduced to the greatest extent by HS (Fig. 4). Taken together, the present data indicate that the anti-inflammatory action of HS in microglia is, at least in part, mediated by the inhibition of these signaling pathways.