Glyburide

Primary astrocytes grown in 24-well poly-l-lysine–coated plates for 7 days were loaded with apoE-complexed BODIPY-tocopherol for 16 h, washed with 10% FBS/DMEM to remove the free tocopherol, and imaged by fluorescence microscopy. Secretion was evaluated again after additional 4 h of incubation in 10% FBS/DMEM. To examine the involvement of ABC transporters, 10 μM glyburide (Sigma) was added to the cells 8 h prior to tocopherol loading. Images were taken from 10 fields in triplicate wells on a Leica DM4100B inverted microscope. Quantification of fluorescence was performed as described using ImageJ software (National Institutes of Health). Fluorescence was normalized to protein content in parallel wells using the Bio-Rad protein assay. Each experiment was repeated at least three times.

Diazoxide, Pinacidil, NN-414, Nicorandil, Levcromakalim (gift from Peter Dosa, Department of Medicinal Chemistry, University of Minnesota), Tolbutamide, Gliclazide and Glyburide (Sigma Aldrich or Tocris, Minneapolis, MN, USA) were initially diluted in DMSO and further diluted with saline to a final concentration of 10–100 uM in 5% DMSO in saline (vehicle). Compounds used in this study were used at concentrations found in previous behavioral studies [45 (link),46 (link),47 (link),48 (link),49 (link)]. Intraplantar injections were performed by gently restraining the animal in a Plexiglas restrainer or decapicone. A 27–33 gauge needle was inserted into the hindpaw skin at a 15–30° angle and 10 uL of solution was slowly delivered. A successful injection was determined when a small bleb was seen [50 (link)]. Intraperitoneal injections were delivered into the lower left abdominal quadrant using a 27 gauge needle at a volume of 100 μL. Intrathecal injections (10 μL) were performed by direct lumbar puncture in awake mice as previously described [51 (link),52 (link)]. Most mice were tested with different chemicals, with at least 7 days between successive tests of active chemicals to avoid carryover effects.

PBMCs from SLE patients or healthy controls were isolated and 1 × 10⁶ cells were seeded to 48-well culture plate. Non-adherent cells were washed away and cells were maintained at 37 °C in 5 % CO₂ in a humidified cell culture incubator. Monocytes isolated from PBMCs were stimulated with healthy serum, rheumatoid factor (RF)-positive serum from rheumatoid arthritis (RA) patients, anti-dsDNA antibody-positive serum from SLE patients, or purified anti-dsDNA antibodies for 16 h. In addition, monocytes from SLE patients or healthy controls were stimulated with anti-dsDNA antibodies or control IgG for 16 h. Macrophages differentiated from SLE monocytes were stimulated with anti-dsDNA antibodies or control IgG for 16 h. Unless otherwise stated, drug concentrations were used as the following: Mito-TEMPO ((2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride) (100 μM) (Sigma, Shanghai, China), glyburide (20 μM) (Sigma, Shanghai, China), Ac-YVAD-CMK (50 μM) (Calbiochem, Hong Kong, China) or IκB kinase inhibitor peptide (200 μM) (Calbiochem, Hong Kong, China). Cells were stimulated with 100 ng/ml of LPS for 6 h and 5 μM of ATP were included for the last 2 h, which was used as positive control. Supernatant was collected and frozen at −80 °C until tested.