P7443

BALB/c mice (6-8 weeks, female) were purchased from Hunan SLAC Experimental Animal Co., Ltd. (Hunan, China) and housed under specific pathogen-free conditions at the Department of Animal Experimental of the Central South University. All animal experiments were conducted by the principles of the Guideline for the Management and Use of Laboratory Animals in China and approved by the Animal Ethics Committee of Central South University. Mice were divided into four groups: 1) blank control group; 2) passive cutaneous anaphylaxis (PCA) model group; 3) PCA +PC intervention group; 4) only PC intervention group. The PCA+PC group and PC treatment group were subcutaneous injections in each ear with 15 ul PC (30 mg/kg/d, P7443, Sigma-Aldrich, USA) for 3 consecutive days and others received an equal volume of saline. DNP-specific IgE (250 ng) was subcutaneously injected on day 4. After 20 h, 100 µl DNP-HSA (1 mg/ml) containing 4% (wt/vol) Evan’s blue dye via into the angular vein. 30 minutes later, ears were collected and incubated with formamide at 63°C for 18 h (23 (link)). The absorbance was measured at the wavelength of 610 nm (BIO-RAD laboratory, USA). The mice were sacrificed 12 h after DNP-HSA injection. The other ear was collected to be stained with toluidine blue, and RT-qPCR was used to detect inflammatory factors.

Pure PCOOH (16:0/18:2-PCOOH) was prepared as previously mentioned.²⁰ (link) Pure 16:0/20:4-PCOOH and 16:0/18:2 PEOOH were prepared by photo-oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (16:0/20:4 PC, Avanti 850459C) and 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (16:0/18:2 PE, Avanti 850802C) respectively, according to previous reports with minor modifications.³⁴ (link)^,35 (link) Crude PCOOH was prepared by photo-oxidation of soybean PC (Sigma Aldrich, Cat#P7443) according to previous reports with minor modifications.³⁴ (link)^,35 (link) Briefly, PC was dissolved in 10 mL of methanol containing 10 μM rose engal. Then, the sample was placed under the LED light and photo-oxidized for 24 h at 4°C. To remove rose engal, the sample was subsequently passed through a Sep-Pak QMA cartridge (360 mg, Waters) followed by evaporation and reconstitution in ethanol.

β-Hexosaminidase (β-hex) release, as a marker of MCs degranulation, was assayed by using a fluorometric assay. The RBL-2H3 cells were plated into 12-well plates (9 × 104 cells per well) and BMMCs were plated into 96-well plates (8 × 103 cells per well). After sensitized with 0.1 µg/mL DNP (dinitrophenylated)-specific IgE (Sigma, USA) overnight, the cells were then treated with or without PC (1, 10, or 100 nmol/mL, P7443, Sigma-Aldrich, USA) for 12 h. Then the PC-containing medium was removed and washed cells with dulbecco’s phosphate buffered saline(D-PBS) twice. To examine degranulation, the cells were stimulated with 1 µg/mL DNP-HSA (dinitrophenylated human serum albumin, Biosearch Technologies, USA) for 30 minutes. Then, cell culture supernatant was collected, and cell pellets were lysed with NP-40 (3 mM). Supernatants and cell lysates, respectively, were incubated with reaction buffer (3 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide, pH 4.4, Sigma, USA) for 1.5 h at 37 °C. The mixture reaction was terminated by 150 µL stop solutions (Na2CO3/NaHCO3, pH 10.6). Then, absorbance at 405 nm was measured with a microplate reader (BIO-RAD Laboratories, USA). The β-hex release was evaluated by using the following formula: β-hex release(%)= [(absorbance of the supernatant)/(optical absorbance of the supernatant+ absorbance of the cell pellet)]×100%.