Anti-Inflammatory Effects of Hyperbaric Oxygenation during DSS-Induced Colitis in BALB/c Mice Include Changes in Gene Expression of HIF-1α, Proinflammatory Cytokines, and Antioxidative Enzymes

Figure 6

Lymphocyte H₂O₂ and ONOO⁻ production during colitis and HBO₂ treatment. BALB/c mice at the age of 10–12 weeks were randomly assigned into 4 groups ( min. 4 mice/group/experiment): CTRL: control mice, CTRL + HBO₂: control mice undergoing HBO₂ (60 min/2.4 ATM, 2x/day, days 1–8), DSS: mice receiving dextran sodium sulphate (DSS, 5% w/v, days 1–7), and DSS + HBO₂: DSS treated mice undergoing HBO₂. To assess the basal intracellular H₂O₂ and ONOO⁻ levels, lymphocytes isolated from MLN and spleens were stained with 10 μM DCF DA and analysed by flow cytometry (black bars, (b)). Next, lymphocytes were activated with 100 nM PMA for 30 min and the ROS production measurement repeated (lined white bars, (b)). (a) shows a representative lymphocyte gating strategy panel and a histogram of DCF-DA signal from the negative control (blue), resting lymphocytes (green), and the PMA activated lymphocytes (red). Doublets were excluded by forward scatter area (FSC-A) versus forward scatter height (FSC-H) and the dead cells by 7-AAD staining. Data are presented as mean s.e.m.; ^∗statistically different compared to CTRL, ; ^#statistically different compared to DSS group, ; ^†statistically different from CTRL + HBO₂, ; ^‡statistically different compared to activated lymphocytes of CTRL group, ; ^§statistically different from the activated lymphocytes of DSS group, . DCF DA: dichlorofluorescein diacetate; PMA: phorbol myristate acetate; 7-AAD: 7-aminoactinomycin D; statistically different from nonactivated lymphocytes DSS + HBO₂.

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