Oleamide

Microglial phagocytosis of 6-carboxyfluorescein-labeled Aβ_1–42 (Aβ-FAM, AnaSpec, CA, USA) was evaluated by a plate-based assay. Microglial cells isolated from newborn mice were plated at a density of 50,000 cells per well in a PDL-coated 96-well plate and incubated with 500 nM Aβ-FAM for 24 hours after either oleic acid (Sigma-Aldrich) or oleamide (Sigma-Aldrich) pretreatment for 12 hours. After the medium was removed, extracellular Aβ-FAM was quenched with 0.2% trypan blue, pH 4.4. Cellular fluorescence was measured at 485 nm excitation/535 nm emission using a plate reader (Molecular Device, CA, USA).

Male C57BL/6J mice weighing 22-25 g were purchased from the Laboratory Animal Services Center, The University of Hong Kong. The animals were fed a normal rodent diet ad libitum with free access to water, and were housed in rooms maintained at 22 ± 1°C with a 12 h light/dark cycle (lights on 6:00-18:00). 32 mice were equally divided into 4 groups. oleamide-induced slow intestinal motility model in mice was similar to that as in a previous publication (Capasso et al., 2005 (link)). In brief, mice were intraperitoneally (i.p.) injected with blank solution (5% EtOH), or oleamide (Sigma-Aldrich, United States) (10 mg/kg, dissolved in 5% EtOH) 30 min prior to drug treatment. During treatment, mice in the control and oleamide-treatment groups received saline water orally, one oleamide-treating group, low- (10 g/kg) and high dose (20 g/kg) MZRW were administrated via lavage. Fecal pellet number was counted every 30 minutes after oleamide administration for 2h.

An affinity purified polyclonal anti-Panx1 serum developed in chicken was purchased from Diateva (Roma, Italy). Polyclonal antibody directed against the whole MyoD molecule was acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and monoclonal antibodies anti-LAP2 was purchased from Transduction Laboratories (Louisville, KY, USA). The adenosine 5′-triphosphate bioluminescence assay, ethidium bromide (Etd⁺), suramin, oleamide, oxidized ATP (oATP), carbenoxolone (CBX), 18 β-glycyrrhetinic acid (β-GA), FITC-conjugated goat anti-rabbit IgGs, and TRITC conjugated goat anti-mouse IgGs were obtained from Sigma (St. Louis, MO, USA). Enhanced chemiluminescence (ECL) reagents were from Pierce Biotechnology (Piscataway, NJ, USA). MRS2179 was obtained from TOCRIS (Park Elisville, MO, USA) and pyridoxalphosphate-6-azophenyl-2′,5′-disulphonate (iso-PPADS) was purchased from Cookson (Southampton, UK). Panx1 siRNA and its control (FlexiTube GeneSolution, cat n° ID: 2120593) were obtained from Qiagen (Germantown, MD, USA). Lipofectamine LTX and PLUS Reagent (cat n° 15338100) and Opti-MEM (cat n° 31985-070) were from Life Technologies (Carlsbad, CA, USA). pEGFP-N1 vector was obtained from Clontech Laboratories (Mountain View, CA, USA).

HPLC-grade methanol and acetonitrile (ACN) were purchased from Merk (Darmstadt, Germany). Formic acid was obtained from Fluka (Buchs, Switzerland). LTB4-DMA purchased from Abcam (Cambridge, UK). Sphingosine, decanamide, and oleamide were purchased from Sigma-Aldrich (St. Louis, MO). Hypoxanthine, phenylalanine and L-2-chlorophenylalanine (internal standard) were obtained from Shanghai Jingchun Reagent Co. Ultrapure water was prepared with a Milli-Q water purification system (Millipore, Bedford, MA, USA).

Isolated monocytes (Day 0) were seeded at a density of 4 x 10⁵ cells/well in 12-well plates and differentiated for 6 days in complete medium. Cell culture medium was replaced every 3 days. On day 6, MDMs were stimulated with 100 ng/ml LPS for 3 h. Cells were then stimulated with oleamide (10-40 µg/ml) (Sigma Aldrich, St. Louis, MO, USA) or adenosine triphosphate (ATP) (Sigma-Aldrich, St. Louis, MO, USA) as the positive control for 1 and 3 h. After stimulation, supernatants were collected, and cytokine levels were measured using an ELISA. Cells were collected, and qRT-PCR was used to examine mRNA expression.

All standard chemicals were of analysis grade and purchased form Sigma-Aldrich, Carl Roth, or Fisher Scientific. Oleamide (Sigma-Aldrich, catalog no. O2136) was dissolved at 100 mM in DMSO, erucamide (Sigma-Aldrich; catalog no. 90082) at 50 mM in isopropyl alcohol, and stearamide (Cayman Chemical, catalog no. 21087) at 50 mM in ethanol. di(2-hydroxyethyl)methyldodecylammonium (di-HEMDA) was purchased from Watson International, Ltd. (Kunshan City, Jiangsu, China), and dissolved at 100 mM in Milli-Q water. Peroxidase-coupled goat anti-mouse secondary antibody (catalog no. 115-035-062, batch 134308) was from Jackson ImmunoResearch. The manufacturers of plastic tubes, catalog numbers, and batch numbers are listed in Table 1.

Oleamide, KA, carbamazepine, calpeptin, cresyl violet, hematoxylin and eosin were purchased from Sigma–Aldrich (St. Louis, MO, United States). Fluoro-jade B was purchased from Histo-Chem Inc. (Jefferson, AR, United States). E64d [2S,3S-trans-(ethoxycarbonyloxirane-2-carbonyl)-L-leucine-(3-methyl butyl) amide] was obtained from Enzo Life Sciences, Inc. (Farmingdale, NY, United States). The μ-calpain was purchased from Calbiochem (Darmstadt, Germany). Oleamide was suspended in 0.2% methyl cellulose.