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Cigarette smoking is an independent risk factor for cardiovascular disease. Nicotine, the major component in cigarette smoke, has been identified to promote atherosclerosis. However, the underlying mechanism of nicotine induced atherogenesis has not been fully elucidated. Recently mast cell degranulation has been reported to play an important role in high fat diet induced atherosclerosis by interacting with macrophages. Here we would like to study whether mast cell act a key mediator in macrophage function and foam cell function in an in vitro model.
Primary cultured murine bone marrow-derived mast cells (BMCMCs) and murine abdominal perfusion-derived macrophages were used in this study. After 4 weeks’culturing in medium with recombinant murineIL-3, greater than 95% of the cell population was consisted of mast cells as proved by toluidine blue stain and immunostaining of mast cell specific chymase and tryptase. Mature mast cells were harvested and divided into 5 groups to be treated with PBS as a negative control, mast cell degranulation stimulator compound 48/80 as a positive control, 100μg/ml nicotine,100μg/ml nicotine with 100mM mast cell stabilizer disodium cromoglicate pretreatment and nicotine 100μg/ml with nicotine receptor nAChR blocker 10μg/ml mecamylamine pretreatment. At 0.5hr, 1hr, 2hrs after nicotine treatment, conditioned supernatants were harvested for β-hexosaminidase activity test to analyze the mast cell degranulation level, then the total supernatants were harvested and used to treat the murine peritoneal macrophages for 24hrs. Migration ability of macrophages was tested by transwell assay, while foam cell formation ability was tested by oil red O staining to show the OxLDL uptake.
We found that 100μg/ml nicotine significantly induced mast cell degranulation, and this phenomenon has been suppressed when pretreated by mast cell stabilizer disodium cromoglicate or nAChR blocker mecamylamine pretreated mast cell. Foam cell formation ratio in the compound 48/80 (71.0±9.9%)and nicotine 100μg/ml conditional treatment group (70.4±14.7%)were significantly higher when comparing to the negative control (47.3±4.2%)and mast cell stablizersodium cromoglicate pretreatment (44.6±10.3%) and nAChR blocker mecamylamine pretreatment (50.1±11.0%) inhibited the foam cell formation. Migration ability in the compound 48/80 (104±23/field) and nicotine conditional treatment group (102±16/field) were also significantly higher comparing with the negative (33±14/field) and mast cell stablizersodium cromoglicate 100mM pretreatment (59±16/field) and nicotine 100μg/ml with nAChR blocker mecamylamine treatment conditional supernatant treatment group (32±11/field).
Nicotine might induce mast cell degranulation through nAChR and then activate mast cell to release a range of proinflammatory mediators to increase the migration ability of macrophages as well as the foam cell formation. Administration of mast cell stabilizer and nAChR blocker revealed the potential of applying mast cell stabilizer in preventing nicotine induced atherogenesis.