β-Lapachone suppresses neuroinflammation by modulating the expression of cytokines and matrix metalloproteinases in activated microglia

All reagents for cell culture were purchased from Gibco BRL (Grand Island, NY, USA). β-Lapachone and LPS (Escherichia coli serotype 055:B5) were obtained from Sigma–Aldrich (St. Louis, MO, USA). All reagents and enzymes for reverse transcription polymerase chain reaction (RT-PCR) and oligonucleotides for electrophoretic mobility shift assay (EMSA) were purchased from Promega (Madison, WI, USA). Antibodies against phospho-/total forms of MAPKs, CREB, β-actin, MMPs (MMP-3, MMP-8, MMP-9), and tissue inhibitor of metalloproteinase-2 (TIMP-2) were supplied by Cell Signaling Technology (Beverley, CA, USA), Abcam (Cambridge, UK), or Chemicon (Temecula, CA, USA). Antibodies against HO-1, NQO1, and Iba1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or Novus (Littleton, CO, USA). The antibody for phospho-p47^phox (Ser370) was purchased from Assay Biotechnology Company Inc. (Sunnyvale, CA, USA). All other chemicals were obtained from Sigma–Aldrich, unless otherwise stated.

The immortalized mouse BV2 microglial cell line [24] was grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10 % heat-inactivated fetal bovine serum, streptomycin (10 μg/ml), and penicillin (10 U/ml) at 37 °C under 5 % CO₂. Primary microglial cells were cultured from the cerebral cortices of 1- to 2-day-old Sprague Dawley rat pups as described previously [21]. The purity of microglial cultures was >95 %, as confirmed by Western blot and immunocytochemistry analyses using an antibody specific to ionized calcium-binding adapter protein-1 (IBA-1) staining (data not shown).

Cells (1 × 10⁵ cells per well in a 48-well plate) were pretreated with β-LAP for 1 h and further stimulated with LPS (100 ng/ml) for 16 h. Concentrations of TNF-α, IL-1β, IL-6, and IL-10 in conditioned medium (CM) were measured by ELISA using monoclonal antibodies and procedures recommended by the supplier (PharMingen, San Diego, CA). Accumulated nitrite in CM and intracellular accumulation of reactive oxygen species (ROS) were measured using Griess reagent (Promega) and H₂DCF-DA (Invitrogen, La Jolla, USA), respectively, as previously described [25].

BV2 cells were stimulated with LPS in the presence or absence of β-LAP for 24 h, and the supernatants were collected to measure MMP activity using the SensoLyte® 520 MMP assay system (AnaSpec, San Jose, CA, USA). MMP activity measurements were performed by continuous detection of peptide cleavage using a fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA). MMP activity units were expressed as a change in the fluorescence intensity at an excitation wavelength of 490 nm and an emission wavelength of 520 nm.

C57BL/6 mice (10–11 weeks old) were purchased from the Orient Co., Ltd. (Seoul, Korea). All animal experiments were approved by the Institutional Animal Care and Use Committee at the School of Medicine, Ewha Womans University. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, if available. Systemic inflammation was induced by LPS administration (5 mg/kg, i.p.) to male C57BL/6 mice as previously described [26]. β-LAP (10 mg/kg, i.p.), dissolved in vehicle solution (1 % DMSO and normal saline), was given daily for 4 days before the LPS treatment. Samples were obtained 3 or 6 h after LPS treatment.

Three hours after LPS treatment, the animals were anesthetized with sodium pentobarbital (120 mg/kg i.p.) and perfused transcardially with normal saline containing heparin (5 U/ml), followed by 4 % paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (PBS), pH 7.2. The brains were removed and incubated overnight in fixatives and stored in a 30 % sucrose solution. Serial coronal brain sections of regions containing the hippocampus (20 μm thick, at 600-μm intervals) were collected using a cryostat. Brain sections were incubated in PBS containing 0.1 % Triton X-100, 5 % normal serum, and 1 % bovine serum albumin for 1 h, and then subsequently incubated with primary antibody. On the next day, sections were incubated in a 1:200 dilution of Alexa Fluor 488-labeled donkey anti-rabbit secondary antibody or Alexa Fluor 594-labeled chicken anti-goat antibody (Molecular Probes Inc., Eugene, OR, USA) for 60 min at room temperature, and then washed with 0.05 % Tween 20 in PBS three times, 5 min each. Sections were then stained with a 0.5-μg/ml DAPI staining solution for 20 min at room temperature and washed. The sections were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA), and fluorescence microcopy images were obtained using confocal microscopy (TSC-SP, Leica, Heidelberg, Germany). Iba1-, MMP-3-, MMP-8-, and MMP-9-positive cells were quantified using the Metamorph program (Carl Zeiss, Jena, Germany). Two serial brain sections from each animal were used for further analysis, and quantification of Iba1-, MMP-3-, MMP-8-, and MMP-9-positive cells was performed in three different areas (500 μm² in size) in the lateral cortex and dentate gyrus of the right hemisphere per brain section. The mean cell number from six 500-μm² areas per animal was calculated.

Total RNA (1 μg) isolated from BV2 or primary microglial cells (4.5 × 10⁵ cells on a 6-cm dish), or from the brain tissue of LPS-injected mice, was reverse transcribed, and synthesized cDNA was used as a template for PCR. RT-PCR was performed on a T100 Thermal Cycler (Bio-Rad) with GoTaq polymerase (Promega). The primer sets shown in Table 1 were used to detect specific PCR products, and their values were calculated as fold change relative to control after normalization to the GAPDH gene.

Proteins isolated from total cell lysates, and from CM, were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibodies against MMP-3, MMP-8, and MMP-9; TIMP-2 (1:1000); the phospho- or total form of MAP kinases or CREB; HO-1; NQO1 (1:1000); or p-p47^phox [anti-phospho-(Ser345)-p47phox Ab] (1:1000, Assay Biotechnology). After thorough washing with Tris-buffered saline with Tween 20 (TBST), horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution in TBST; New England Biolabs, Beverly, MA, USA) were applied, and the blots were developed using an enhanced chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL, USA). To detect secreted MMPs, MMP proteins in the conditioned media were enriched using an Amicon® centrifugal filter (Millipore Corp., Billerica, MA, USA).

BV2 cells plated at 50–60 % confluence (2 × 10⁵ cells per well) in 12-well plates were transfected with 1 μg of plasmid DNA (ARE-luc, CRE-luc) using the Convoy™ Platinum transfection reagent (CellTAGen, Seoul, Korea). After 36 h of transfection, cells were treated with β-LAP and LPS (100 ng/ml), or β-LAP only, for 6 h. The luciferase assay was used to determine the effect of β-LAP on ARE or CRE promoter activity. The ARE-luciferase reporter gene was kindly provided by Dr. Young-Joon Surh (Seoul National University, Seoul, Korea). The sequence of the anti-oxidant response element (ARE) construct is as follows: 5′-CTCAGCCTTCCAAATCG CAGTCACAGTGACTCAGCAGAATC-3′ [27, 28]. The CRE-luc vector, which contains four copies of the cyclic AMP response element (CRE, TGACGTCA), was obtained from Stratagene (La Jolla, CA).

Nuclear extracts from treated microglia were prepared as follows. Cells (2 × 10⁷) were treated with 1 ml of lysis buffer (10 mM Tris–HCl, pH 7.9; 10 mM NaCl; 3 mM MgCl₂; 1 % NP-40) on ice for 5 min. After 10 min of centrifugation at 3000 rpm, the pellet was resuspended in 50 μl of extraction buffer (20 mM HEPES, pH 7.9; 20 % glycerol; 1.5 mM MgCl₂; 0.2 mM EDTA; 300 mM NaCl; 1 mM DTT; 1 mM PMSF) and incubated on ice for 30 min. After centrifugation at 13,200 rpm for 15 min, the supernatant was harvested as a nuclear protein extract and stored at −70 °C. Double-stranded DNA oligonucleotides containing the NF-κB, AP-1, ARE, or CRE consensus sequences were end labeled using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) in the presence of [γ-³²P]ATP. Nuclear proteins (5 μg) were incubated with ³²P-labeled probe on ice for 30 min, resolved on a 5 % acrylamide gel, and visualized by autoradiography. We purchased double-stranded DNA oligonucleotides containing the NF-κB, AP-1, or CRE consensus sequences from Promega (Madison, WI, USA) and that containing ARE from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The DNA sequences of the probes are as follows: NF-κB (5′-AGTTGAGGGGAC TTTCCCAGGC-3′), AP-1 (5′-CGCTTGATGAGTCAGCCGGAA-3′), ARE (5′-TGG GGAACCTGTGCTGAGTCACTGGAG-3′), and CRE (5′-AGAGATTGCCTGACGTCAGAGAGCTA-3′).

Unless otherwise stated, all experiments were performed with triplicate samples and repeated at least three times. Data are presented as mean ± SEM, and statistical comparisons among groups were performed using one-way ANOVA followed by Newman–Keuls post hoc tests or t tests. Statistical significance was accepted for P values <0.05.