For the preparation of EWCC, chalcanthite was heated and dehydrated. During the heating process, chalcanthite was uniformly heated while carefully being watched for a color change every 3–5 h until it turned completely grey. The heating duration (~10–24 h) varied with the amount and quality of the mineral used. Once the chalcanthite was dehydrated, it was cooled until the remaining heat was completely released. The moisture content of the chalcanthite ranged from 0 to 5%. After cooling, chalcanthite was finely pulverized. The pulverized chalcanthite was stored in a concealed airtight container in a dry place to prevent moisture from accumulating. The prepared chalcanthite powder was mixed with egg whites at ratios of 30:13 to 30:30 for 10 min. The egg whites needed to be homogeneously mixed with a wooden spatula in a ceramic vessel so the utensils would not interfere or cause any chemical reactions during the mixing process. After the chalcanthite and egg whites were sufficiently mixed, the mixture was cooled until the reaction heat was completely released. One hundred milligrams of the obtained substance was then dissolved in 1 liter of deionized water and centrifuged at 6,000 rpm for 10 min. A portion of the upper level concentration (0.80 μm) was filtered by syringe filter after the centrifugation.

The murine BV2 cell line was maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified incubator with 5% CO₂. Confluent cultures were passed by trypsinization. The cells used in the experiments were washed twice with warm DMEM (without phenol red) and treated in a serum-free medium for at least 4 h before treatments. In all experiments, the cells were treated with various concentrations of EWCC at the indicated times before the addition of LPS (0.5 μg/ml).

Cell viability was measured based on the formation of blue formazan that was metabolized from colorless MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO] by mitochondrial dehydrogenases, which are active only in live cells. BV2 cells were plated on 24-well plates at a density of 2×10⁵ cells/well for 24 h and then washed. Cells incubated with various concentrations of EWCC were treated with or without LPS (Escherichia coli 026:B6; Sigma) for 24 h and then incubated in 0.5 mg/ml MTT solution. Three hours later, the supernatant was removed and the formation of formazan was measured at 540 nm using a microplate reader (Dynatech MR-7000; Dynatech Laboratories).

Concentrations of NO in the culture supernatants were determined by measuring nitrite, which is a major stable product of NO, using the Griess reagent (Sigma). Cells (5×10⁵ cells/ml) were stimulated in 24-well plates for 24 h and then 100 μl of each culture medium was mixed with an equal volume of Griess reagent (1% sulfanilamide/0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride/2.5% H₃PO₄). Nitrite levels were determined using an ELISA plate reader at 540 nm and nitrite concentrations were calculated by reference to a standard curve generated by known concentrations of sodium nitrite (29).

BV2 cells were incubated with purpurogallin in either the presence or absence of LPS (0.5 μg/ml) for 24 h. Following the manufacturer’s instructions, a volume of 100 μl of culture-medium supernatant was collected for determination of PGE₂ concentration by ELISA (Cayman, Ann Arbor, MI) (29).

Total RNA was prepared using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) and primed with random hexamers for the synthesis of complementary DNA using M-MLV Reverse Transcriptase (Promega, Madison, WI), according to the manufacturer’s instructions using DNAse I (1 U/μg RNA) pretreated total mRNA. Single stranded cDNA was amplified by a polymerase chain reaction (PCR) with primers for inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, IL-1β, TNF-α and glyceraldhyde-3-phosphate dehydrogenase (GAPDH) (Table I). The following PCR conditions were applied - GAPDH: 18 cycles of denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec and extension at 72°C for 30 sec; iNOS, COX-2, IL-1β and TNF-α: 25 cycles of denaturation at 94°C for 30 sec, annealing at 52°C for a 30-sec extension at 72°C for 30 sec. GAPDH was used as an internal control to evaluate the relative expression of COX-2, iNOS, IL-1β and TNF-α (30).

Cells were washed with PBS three times, placed at a temperature of 4°C and lysed for 30 min in lysis buffer (20 mM sucrose, 1 mM EDTA, 20 μM Tris-Cl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl₂ and 5 μg/ml aprotinin). Lysates were then centrifuged at 12,000 rpm at 4°C. The protein concentration was measured using a Bio-Rad protein assay (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions. Equal amounts of protein (30–50 μg) were separated electrophoretically using 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was then transferred to 0.45-μm polyvinylidene fluoride (PVDF; Millipore, Bedford, MA) membranes. Membranes were soaked in blocking buffer (5% skimmed milk) and then incubated with the primary antibodies (Table II). After thorough washing with PBST, horseradish peroxidase conjugated antibodies were applied and immune complexes were then visualized using the enhanced chemiluminescence (ECL) detection system according to the recommended procedure (Amersham). In a parallel experiment, cells were washed with ice-cold PBS and scraped; and cytoplasmic and nuclear proteins were then extracted using NE-PER^® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Inc., Rockford, IL).

The levels of IL-1β and TNF-α (R&D Systems, Minneapolis, MN) were measured by ELISA kits according to the manufacturer’s instructions. Briefly, BV2 cells (5×10⁵ cells/ml) were plated in 24-well plates and pretreated with indicated concentrations of EWCC for 1 h before treatment of 0.5 μg/ml LPS for 24 h. One hundred microliters of culture-medium supernatants was collected to determine the IL-1β and TNF-α concentrations by ELISA.

For detection of NF-κB p65 translocation, cells were grown on glass coverslips for 24 h and then treated with 0.5 μg/ml LPS, and were either pretreated or not pretreated with EWCC for 60 min. Cells were fixed with 3.7% paraformaldehyde, treated with 0.2% Triton X-100 and blocked with 2% BSA. Cells were then sequentially incubated with the anti-NF-κB p65 antibody, FITC-conjugated donkey anti-rabbit IgG and DAPI solution and examined using a fluorescence microscope (Carl Zeiss, Germany).

Data values represent the means ± SD. Statistical significance was determined using an analysis of variance that was followed by the Student’s t-test. A value of P<0.05 was accepted as statistically significant.

In the present study, we aimed to evaluate the potential anti-inflammatory properties of EWCC regarding the production of two major inflammatory mediators, NO and PGE₂, in LPS-stimulated BV2 microglia. To determine the level of NO production, we measured the nitrite released into the culture medium using Griess reagent. BV2 cells were pretreated with various concentrations of EWCC (25, 50 and 75 μg/ml) for 1 h before being stimulated with LPS (0.5 μg/ml). According to the NO detection assay, LPS alone was able to markedly induce NO production from the cells as compared to those generated by the control. However, pretreatment with EWCC significantly repressed the levels of NO production in LPS-stimulated BV2 microglia in a concentration-dependent manner of up to 75 μg/ml (Fig. 1A).

PGE₂ represents another important inflammatory mediator that is produced from the conversion of arachidonic acid by COXs. Therefore, we next evaluated the inhibitory effects of EWCC on PGE₂ levels present in the supernatant. To assess whether EWCC inhibits production of LPS-induced PGE₂ in BV2 microglia, cells were pretreated with various concentrations of EWCC for 1 h and then stimulated with 0.5 μg/ml LPS. After incubation for 24 h, the cell culture medium was harvested and the production of PGE₂ was measured using an ELISA. As shown in Fig. 1B, the amount of PGE₂ present in the culture medium increased from initial levels after 24 h of exposure to LPS alone, however a marked repression was observed after administration of EWCC in a concentration-dependent fashion.

In order to exclude the cytotoxic effect of EWCC on the cell growth of BV2 cells, the cells were exposed to various concentrations of EWCC for 24 h in the presence or absence of LPS, and cell viability was then measured by an MTT assay. Under the same condition, we also analyzed whether chromatin condensation, a hallmark of apoptosis, was induced by EWCC treatment. As indicated in Fig. 2, the concentrations of EWCC (25–75 μg/ml) used to inhibit NO and PGE₂ production did not affect cell viability. These results clearly indicated that the inhibition of NO and PGE₂ production in LPS-stimulated BV2 cells was not due to the cytotoxic action of EWCC.

To ascertain whether the inhibition of NO and the PGE₂ production by EWCC treatment is associated with decreased levels of iNOS and COX-2, we performed RT-PCR and western blot analysis for detection of mRNA and protein levels. The levels of iNOS and COX-2 protein were markedly upregulated after 24 h of LPS (0.5 μg/ml) treatment, and EWCC significantly inhibited iNOS and COX-2 protein expression in LPS-stimulated BV2 microglia in a concentration-dependent manner (Fig. 3A). Next, to investigate whether or not EWCC suppresses the LPS-mediated induction of iNOS and COX-2 via a pretranslational mechanism, the effects of EWCC on iNOS and COX-2 mRNA expression were evaluated (Fig. 3B). RT-PCR analysis showed that the reduction in iNOS and COX-2 mRNA correlated with the reduction in the corresponding protein levels. The results suggest that EWCC-induced reduction in the expression of iNOS and COX-2 resulted in the inhibition of NO and PGE₂ production.

We next investigated whether EWCC suppresses the production of pro-inflammatory cytokines, IL-1β and TNF-α, in LPS-stimulated BV2 cells. BV2 microglia were incubated with EWCC in the absence or presence of 0.5 μg/ml of LPS for 24 h and the cytokine levels were evaluated in the culture supernatants. As indicated in Fig. 4A and B, induction of IL-1β and TNF-α by LPS treatment was significantly decreased by EWCC in cell culture media. In a parallel experiment, using RT-PCR, we studied the effects of EWCC on LPS-induced IL-1β and TNF-α mRNA expression. As shown in Fig. 5, IL-1β and TNF-α mRNA transcription also decreased following EWCC treatment. These results suggest that EWCC is effective in the suppression of pro-inflammatory cytokine production through the alteration of the transcription levels of IL-1β and TNF-α in activated microglia.

NF-κB is one of the important transcription factors that regulate gene expression of pro-inflammatory mediators including iNOS, COX-2, TNF-α and IL-1β in activated BV2 microglia (24–26); therefore, to further characterize the mechanism through which EWCC inhibits pro-inflammatory and/or cytotoxic factor expression, we examined whether EWCC prevents the translocation of the p65 subunit of NF-κB to the nucleus. Western blot analysis showed that the amount of NF-κB p65 in the nucleus was markedly increased after exposure to LPS alone, but the LPS-induced p65 level in the nuclear fractions was reduced by EWCC pretreatment (Fig. 6). Similar results were also found using immunofluorescence microscopy (Fig. 7). In addition, we investigated whether EWCC blocks the LPS-stimulated degradation of IκB-α using western blotting. As shown in Fig. 6, IκB-α was markedly degraded at 30 min after LPS treatment. However, this LPS-induced IκB-α degradation was significantly reversed by EWCC. These results suggest that EWCC inhibits NF-κB activation in BV2 microglia cells by suppression of IκB degradation and nuclear translocation of NF-κB. Taken together the above findings show the anti-inflammatory effect of EWCC in the LPS-stimulated BV2 cells involved the NF-κB pathway.

Findings from recent studies have indicated that the phosphoinositide 3-kinase (PI3K)/Akt signaling molecule is able to trigger NF-κB activation through IκB degradation (31,32). Therefore, we examined the effect of EWCC on LPS-induced PI3K/Akt activation. As shown in Fig. 8, phosphorylation of PI3K and Akt showed a marked increase within 15 min after LPS stimulation; however, EWCC pretreatment resulted in a significant blockage of LPS-induced PI3K and Akt phosphorylation. The results show that inhibition of pro-inflammatory mediator expression by EWCC in LPS-stimulated BV2 microglia was associated with the inactivation of the PI3K/Akt signaling pathway.

Mitogen-activated protein kinases (MAPKs) are the most important signaling molecules with involvement in activated microglia (33,34). Therefore, we determined the activation levels of the MAPK pathway at various times after LPS stimulation of BV2 cells. Stimulation of BV2 cells with LPS led to the rapid activation of p38MAPK, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), with peak levels of each phospho-MAPK occurring 15–60 min after the addition of LPS. However, EWCC pretreatment significantly inhibited phosphorylation of MAPKs in LPS-stimulated BV2 microglia (Fig. 9). This finding suggests that EWCC is capable of disrupting key signal transduction pathways activated by LPS in BV2 microglia, which subsequently prevents the production of pro-inflammatory mediators.