Involvement of sirtuin 1 in airway inflammation and hyperresponsiveness of allergic airway disease

Levels of SIRT1 protein in lung tissues of OVA-sensitized and OVA-challenged mice. A, Western blot analyses of SIRT1. B, Densitometric analyses are presented as the relative ratio of SIRT1 to actin. The relative ratio of SIRT1 in the lung tissues of control mice is arbitrarily presented as 1. Data represent means ± SEMs from 8 mice per group. One, 6, 24, 48, and 72 hours are the time periods of the sampling after the last challenge in mice sensitized and challenged with OVA or saline. Control, No treatment (mice with no sensitization and no challenge); Pre, 1 hour before the first challenge (OVA-sensitized mice without OVA challenge or saline-sensitized mice without saline challenge). #P < .05 versus the Pre group. ^∗P < 0.05 versus the saline group.

Effect of sirtinol on SIRT1 expression in nuclear protein extracts from lung tissues (A and B) and from primary cultured tracheal epithelial cells (C and D) of OVA-sensitized and OVA-challenged mice. Fig 2, A, Western blot analysis of SIRT1 in lung tissues. Fig 2, B, Densitometric analyses are presented as the relative ratio of SIRT1 to actin. Sampling was performed at 72 hours after the last challenge in saline-inhaled mice administered saline (SAL+SAL), OVA-inhaled mice administered saline (OVA+SAL), OVA-inhaled mice administered drug vehicle (OVA+VEH), OVA-inhaled mice administered 0.1 mg/kg sirtinol (OVA+sirtinol 0.1 mg/kg), and OVA-inhaled mice administered 0.5 mg/kg of sirtinol (OVA+sirtinol 0.5 mg/kg). The relative ratio of SIRT1 in the lung tissues of SAL+SAL mice is arbitrarily presented as 1. Bars represent means ± SEMs from 8 mice per group. #P < .05 versus the SAL+SAL group. ^∗P < .05 versus the OVA+SAL group. Fig 2, C, Western blot analysis of SIRT1 in airway epithelial cells. Fig 2, D, Densitometric analyses. The relative ratio of SIRT1 in the tracheal epithelial cells of control mice is arbitrarily presented as 1. Bars represent means ± SEMs from 6 independent experiments. Control, Epithelial cells isolated from saline-sensitized and saline-challenged mice; DMSO, dimethyl sulfoxide. #P < .05 versus control mice. ^∗P < 0.05 versus OVA-inhaled mice treated with drug vehicle only.

Effect of sirtinol on HIF-1α and HIF-1β levels in nuclear protein extracts from lung tissues (A and B) and from primary cultured tracheal epithelial cells (C and D) of OVA-sensitized and OVA-challenged mice. The groups are defined as in the legend for Fig 2. Fig 3, A, Western blot analysis of HIF-1α and HIF-1β protein in lung tissues. Fig 3, B, Densitometric analyses are presented as the relative ratio of HIF-1α to HIF-1β. The relative ratio of HIF-1α in the lung tissues of SAL+SAL mice is arbitrarily presented as 1. Bars represent means ± SEMs from 8 mice per group. #P < .05 versus the SAL+SAL group. ^∗P < .05 versus the OVA+SAL group. Fig 3, C, Western blot analysis of HIF-1α and HIF-1β in airway epithelial cells. Fig 3, D, Densitometric analyses. The relative ratio of HIF-1α in the tracheal epithelial cells of control mice is arbitrarily presented as 1. Bars represent means ± SEMs from 6 independent experiments. Control, Epithelial cells isolated from saline-sensitized and saline-challenged mice; DMSO, dimethyl sulfoxide. #P < 0.05 versus control mice. ^∗P < 0.05 versus OVA-inhaled mice treated with drug vehicle only.

Effect of sirtinol, 2ME2, or CBO-P11 on VEGF expression and plasma exudation in OVA-sensitized and OVA-challenged mice. The groups are defined as in the legend for Fig 2. A, Western blot analysis of VEGF. B, Densitometric analyses are presented as the relative ratio of VEGF to actin. The relative ratio of VEGF in the lung tissues of SAL+SAL mice is arbitrarily presented as 1. C, Enzyme immunoassay of VEGF in BAL fluids. D, Plasma exudation was quantified by means of EBD assay. Bars represent means ± SEMs from 8 mice per group. #P < .05 versus the SAL+SAL group. ^∗P < .05 versus the OVA+SAL group.

Effect of sirtinol, 2ME2, or CBO-P11 on IL-4, IL-5, and IL-13 levels in lungs of OVA-sensitized and OVA-challenged mice. The groups are defined as in the legend for Fig 2. A, Western blotting of IL-4, IL-5, and IL-13 in lung tissues. B, Densitometric analyses are presented as the relative ratio of each molecule to actin. The relative ratio of each molecule in the lung tissues of SAL+SAL mice is arbitrarily presented as 1. C, Enzyme immunoassay of IL-4, IL-5, and IL-13 in BAL fluids. Bars represent means ± SEMs from 8 mice per group. #P < .05 versus SAL+SAL mice. ^∗P < 0.05 versus OVA+SAL mice.

Total cells and differential cellular components in BAL fluids, inflammation in lung tissues, airway mucus expression, and airway responsiveness of OVA-sensitized and OVA-challenged mice. The groups are defined as in the legend for Fig 2. A, The numbers of total and differential cellular components of BAL fluids. B, Inflammation scores. Total lung inflammation was defined as the average of the peribronchial and perivascular inflammation scores. C, Quantitation of airway mucus expression. D-F, Representative PAS-stained sections of the lungs. Sampling was performed in saline-inhaled mice administered saline (Fig 6, D), OVA-inhaled mice administered saline (Fig 6, E), and OVA-inhaled mice administered 0.5 mg/kg sirtinol (Fig 6, F). The violet color indicates PAS-positive mucus expression. Bars indicate 50 μm. G, Airway responsiveness in OVA-sensitized and OVA-challenged mice. Bars represent means ± SEMs from 8 mice per group. #P < .05 versus SAL+SAL mice. ^∗P < .05 versus OVA+SAL mice.

Levels of HIF-1α protein in lung tissues of OVA-sensitized and OVA-challenged mice. A, Western blot analyses of HIF-1α protein. B, Densitometric analyses are presented as the relative ratio of HIF-1α to HIF-1β. The relative ratio of HIF-1α in the lung tissues of control mice is arbitrarily presented as 1. Data represent means ± SEMs from 8 mice per group. One, 6, 24, 48, and 72 hours are the time periods of the sampling after the last challenge in mice sensitized and challenged with OVA or saline. Control, No treatment (mice with no sensitization and no challenge); Pre, 1 hour before the first challenge (OVA-sensitized mice without OVA challenge or saline-sensitized mice without saline challenge). #P < .05 versus the Pre group. ^∗P < .05 versus the saline group.

Effect of sirtinol on SIRT1 enzyme activity in OVA-sensitized and OVA-challenged mice. A, The fluorometric assay for SIRT1 deacetylase in lung tissues. Sampling was performed at 72 hours after the last challenge in saline-inhaled mice administered drug vehicle (SAL+VEH), OVA-inhaled mice administered drug vehicle (OVA+VEH), OVA-inhaled mice administered 0.1 mg/kg sirtinol (OVA+sirtinol 0.1 mg/kg), and OVA-inhaled mice administered 0.5 mg/kg sirtinol (OVA+sirtinol 0.5 mg/kg). Bars represent means ± SEMs from 6 mice per group. #P < .05 versus SAL+VEH mice. ^∗P < .05 versus OVA+VEH mice. B, Fluorometric assay for SIRT1 deacetylase in primary tracheal epithelial cells. Bars represent means ± SEMs from 6 independent experiments. Control, Epithelial cells isolated from saline-sensitized and saline-challenged mice; DMSO, dimethyl sulfoxide; AFU, arbitrary fluorescence units. #P < .05 versus control mice. ^∗P < .05 versus OVA-inhaled mice treated with drug vehicle only.

Kinetics of SIRT1 enzyme activity and SIRT1 and HIF-1α protein expression after treatment with sirtinol in nuclear extracts of primary tracheal epithelial cells of OVA-sensitized and OVA-challenged mice. A, Western blot analyses of SIRT1. B, Densitometric analyses are presented as the relative ratio of SIRT1 to actin. C, The fluorometric assay for SIRT1 deacetylase. AFU, Arbitrary fluorescence units. D, Western blot analyses of HIF-1α and HIF-1β protein. E, Densitometric analyses are presented as the relative ratio of HIF-1α to HIF-1β. The relative ratio of SIRT1 or HIF-1α in airway epithelial cells of the mice before treatment with sirtinol (0h) is arbitrarily presented as 1. Each sample was incubated with sirtinol (10 μmol/L) for 0, 8, 11, 14, and 18 hours, respectively. Bars represent means ± SEMs from 6 independent experiments. #P < .05 versus 0h.

Effect of sirtinol on IFN-γ protein levels in OVA-sensitized and OVA-challenged mice. Groups are defined as in the legend for Fig 2. A, Western blotting of IFN-γ protein in lung tissues. B, Densitometric analyses are presented as the relative ratio of IFN-γ to actin. The relative ratio of IFN-γ in the lung tissues of SAL+VEH mice is arbitrarily presented as 1. Bars represent means ± SEMs from 6 mice per group. #P < 0.05 versus SAL+VEH mice.

Effect of sirtinol on nuclear translocation of NF-κB in OVA-sensitized and OVA-challenged mice. Groups are defined as in the legend for Fig E2. A, Western blot analyses of NF-κB p65 levels in nuclear (Nuc) and cytosolic (Cyt) protein extracts of lung tissues. Densitometric analyses are presented as the relative ratio of NF-κB p65 levels in all groups to those in SAL+VEH mice. The relative ratio of NF-κB in nuclear protein extracts from the lung tissues of SAL+VEH mice is arbitrarily presented as 1. Bars represent means ± SEMs from 6 mice per group. #P < .05 versus SAL+VEH mice. B, Western blot analysis of NF-κB p65 levels in nuclear (Nuc) and cytosolic (Cyt) protein extracts of primary tracheal epithelial cells. Densitometric analyses are presented as the relative ratio of NF-κB p65 levels in OVA-inhaled mice compared with those in control mice. The relative ratio of NF-κB p65 in the tracheal epithelial cells of control mice is arbitrarily presented as 1. Bars represent means ± SEMs from 6 independent experiments. Control, Epithelial cells isolated from saline-sensitized and saline-challenged mice; DMSO, dimethyl sulfoxide. #P < .05 versus control mice.

Effect of sirtinol on p-Akt and Akt protein levels in OVA-sensitized and OVA-challenged mice. Groups are defined as in the legend for Fig 2. A, Western blotting of p-Akt and Akt in lung tissues. B, Densitometric analyses are presented as the relative ratio of p-Akt to Akt. The relative ratio of p-Akt in the lung tissues of SAL+SAL mice is arbitrarily presented as 1. Bars represent means ± SEMs from 8 mice per group. #P < 0.05 versus SAL+SAL mice. ^∗P < 0.05 versus OVA+SAL mice.

Effect of LY294002 or wortmannin on HIF-1α levels in nuclear protein extracts of lung tissues from OVA-sensitized and OVA-challenged mice. Groups are defined as in the legend for Fig 2. A, Representative Western blot of HIF-1α and HIF-1β in lung tissues administered LY294002 or wortmannin. B, Densitometric analyses are presented as the relative ratio of HIF-1α to HIF-1β. The relative ratio of HIF-1α in nuclear protein extracts of the lung tissues of SAL+VEH mice is arbitrarily presented as 1. Data represent means ± SEMs from 6 mice per group. #P < .05 versus SAL+VEH mice. ^∗P < 0.05 versus OVA+VEH mice.

Effect of LY294002 or wortmannin on VEGF protein levels in lungs of OVA-sensitized and OVA-challenged mice. Groups are defined as in the legend for Fig 2. A, Western blot analyses of VEGF levels in lung tissues. B, Densitometric analyses are presented as the relative ratio of VEGF to actin. The relative ratio of VEGF in lung tissues of SAL+VEH mice is arbitrarily presented as 1. C, Enzyme immunoassay of VEGF in BAL fluids. Data represent means ± SEMs from 6 mice per group. #P < .05 versus SAL+VEH mice. ^∗P < .05 versus OVA+VEH mice.

Effect of LY294002 or wortmannin on SIRT1 in nuclear extracts of lung tissues from OVA-sensitized and OVA-challenged mice. Groups are defined as in the legend for Fig 2. A, Western blot analyses of nuclear SIRT1 levels in lung tissues. B, Densitometric analyses are presented as the relative ratio of SIRT1 to actin. The relative ratio of SIRT1 in lung tissues of SAL+VEH mice is arbitrarily presented as 1. Data represent means ± SEMs from 6 mice per group. #P < 0.05 versus SAL+VEH mice. ^∗P < 0.05 versus OVA+VEH mice.

SIRT 1 and HIF-1α levels in nuclear extracts of lung tissues of mice challenged with OVA or saline. Sampling was performed at 72 hours after the last challenge in saline-sensitized and saline-challenged mice (group I), OVA-sensitized and OVA-challenged mice (group II), and OVA-challenged mice without prior sensitization (group III). A, Western blotting of SIRT1 in nuclear extracts of lung tissues. B, Densitometric analyses are presented as the relative ratio of SIRT1 to actin. C, Western blotting of HIF-1α and HIF-1β in nuclear extracts of lung tissues. D, Densitometric analyses are presented as the relative ratio of HIF-1α to HIF-1β. The relative ratio of SIRT1 or HIF-1α in the lung tissues of saline-sensitized and saline-challenged mice is arbitrarily presented as 1. Bars represent means ± SEMs from 6 mice per group. #P < .05 versus group I. ^∗P < 0.05 versus group II.

A proposed mechanism for the involvement of SIRT1 in allergic airway disease.