Fig 1: ELISA analysis showed altered levels of circadian molecular clock proteins in mice exposed to acute WPS.C57BL/6J mice were exposed to air or WPS for 10 days and euthanized 24 hrs post-last exposure. Levels of BMAL1, NR1D1 (REV-ERBa), and PER2 were measured in lung homogenates by ELISA as per manufacturer’s instructions. Data are shown as mean ± SEM, n = 4–7 per group except for BMAL1 ELISA we used n = 84 for air-exposed mice. Statistical significance was calculated using the unpaired Student’s t-test. **P < 0.01 vs. air controls.
Fig 2: Schematic representation of waterpipe smoke (WPS) and e-cigarette vapor containing nicotine-induced alterations in the expression and abundance of circadian molecular clock genes.Exposure to alternative tobacco products, such as (A) WPS and (B) e-cig vapors containing propylene glycol with nicotine affects the expression of circadian molecular clock genes in mouse lungs. Molecular clock consists of a heterodimer of transcription factors BMAL1and CLOCK. The BMAL1:CLOCK heterodimer starts transcription of Per2 and Cry2 by binding to specific DNA elements (E-box) located in the promoter region. Altered expression of circadian clock genes may be a result of acute WPS and/or e-cig exposure induced oxidative stress and lung inflammatory response. This can negatively affect pulmonary function during chronic exposures and thus may contribute to the pathobiology of chronic airway diseases. BMAL1, brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1; CLOCK, circadian locomotor output cycles protein kaput; nuclear receptor subfamily 1 group D member 1 (Nr1d1 or Rev-erba); nuclear receptor subfamily 1, group D, member 2 (Nr1d2 or Rev-erbß); Rora, retinoic acid receptor-related orphan receptor a; period circadian regulator 2 (Per2); Cryptochrome 2 (Cry2).
Fig 3: ELISA analysis showed altered levels of circadian molecular clock proteins in mice exposed to inhaled e-cig vapor containing nicotine.C57BL/6J mice were exposed to e-cig vapor containing nicotine (25 mg/ml), propylene glycol (PG) alone and air as a control (2 hrs/day) and euthanized 2 hrs post-last exposure on day 3. The levels of BMAL1, NR1D1 (REV-ERBa), and PER2, were measured in lung homogenates by ELISA as per manufacturer’s instructions. Data are shown as mean ± SEM, n = 5–6 per group.
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