SIRT1 protects against urban particulate matter-induced airway inflammation

Abstract

Purpose: Particulate matter (PM) has been implicated as a risk factor for airway injury. However, the molecular mechanisms remain largely unclear. The goal of this study was to determine whether sirtuin1 (SIRT1), an anti-inflammatory and antiaging protein, protects against PM-induced airway inflammation.

Methods: The effect of SIRT1 on PM-induced airway inflammation was assessed by using in vivo models of airway inflammation induced by PM and in vitro culture of human bronchial epithelial (HBE) cells exposed to PM, resveratrol (SIRT1 activator), or both.

Results: PM-stimulated HBE cells showed a significant decrease in SIRT1 but a notable increase in inflammatory cytokines. SIRT1 gene silencing further enhanced PM-induced expression of inflammatory cytokines. In contrast, resveratrol, a SIRT1 activator, reduced the expression of these cytokines compared with the control cells. In vivo, SIRT1 expression was significantly decreased in lung tissues of PM-exposed mice. Interestingly, resveratrol treatment reversed the enhanced total cells, neutrophils and inflammatory cytokines in PM-induced mice. Moreover, SIRT1 mediated PM-induced inflammatory cytokines expression at least partly through MAPK pathways.

Conclusion: These findings suggest that SIRT1 is involved in the pathogenesis of PM-induced airway inflammation and activation of SIRT1 could prevent airway disorders or disease exacerbations induced by airborne particulate pollution.

Keywords: SIRT1; airway inflammation; particulate matter; resveratrol.

Figure 1

PM exposure induces SIRT1 reduction in HBE cells. (A) Cells were exposed to PM at indicated times or concentrations. Western blot analysis for SIRT1 protein expression in HBE cells treated with PM. (B) Immunofluorescence for SIRT1 protein expression in HBE cells treated with PM (Magnification×400). (C) Semi-quantification results of immunofluorescence. (D and E) RT-PCR analysis for a time course (PM at 100 μg/mL for various times) or dose response (various concentrations of PM for 24 h) of SIRT1 protein expression in HBE cells treated with PM. Data are presented as mean ± SD of three independent experiments. *P<0.05, **P<0.01, and ***P<0.001. Abbreviations: PM, Particulate matter; SIRT1, Sirtuin 1; HBE, Human bronchial epithelial.

Figure 2

SIRT1 gene silence increases PM-induced inflammatory cytokines in HBE cells. Cells were transfected with control (CTL) siRNA or SIRT1 siRNA for 24 h, and then were treated with PM (100 μg/mL) for 24 h to measure the levels of IL-6, IL-8, IL-17A, and MUC5AC.(A) SIRT1 expression was measured by Western blot. (B-F) The mRNA levels of IL-6, IL-8, IL-17A, and MUC5AC were measured by RT-PCR. Data are presented as mean ± SD of three independent experiments. **P<0.01 and ***P<0.001. Abbreviations: SIRT1, Sirtuin 1; PM, Particulate matter; HBE, Human bronchial epithelial; MUC5AC, Mucin 5AC.

Figure 3

Resveratrol (Res) inhibits PM-induced inflammatory cytokines in HBE cells. HBE cells were treated with SIRT1 activator resveratrol (Res) together with PM for 24 h. (A-E) The mRNA levels of IL-6, IL-8, IL-17A, and MUC5AC were measured by RT-PCR. Data are presented as Mean ± SD of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Abbreviations: PM, Particulate matter; HBE, Human bronchial epithelial; SIRT1, Sirtuin 1; MUC5A, Mucin 5AC.

Figure 4

Resveratrol (Res) inhibits PM-induced airway inflammation in mice. Mice were instilled intratracheally with normal saline (NS), PM or PM plus resveratrol (Res) for 3 d, and the mice were sacrificed after 24 h. (A) Western blot analysis for SIRT1 protein expression in lung tissues of mice. (B) IHC analysis for representative images of lung tissues of mice stained with SIRT1 protein (red arrows; Magnification×400) and semi-quantified SIRT1 expression of the IHC staining. (C) The total inflammatory cells and the number of neutrophils (D) in the BALF were measured. (E) Representative images of macrophages and neutrophils in the BALF were analyzed (red arrows; Magnification×400) using Giemsa staining. (F) Representative images of lung tissue stained with H&E (Magnification×400) and Semi-quantified inflammation score of the H&E staining (red arrows). Data are representative of three independent studies (n=5–6 mice per group per study). Results are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Abbreviations: M, macrophage; N: neutrophils; PM, Particulate matter; SIRT1, Sirtuin 1; IHC, Immunohistochemistry; BALF, Bronchoalveolar lavage fluid; H&E, Hematoxylin-eosin.

Figure 5

Resveratrol (Res) reduces airway inflammation in response to PM exposure. The levels of IL-6, IL-8, IL-17A, TNF-α, and MUC5AC in lung tissue were analyzed by RT-PCR. Data are representative of three independent studies (n=5–6 mice per group per study). Results are expressed as mean ± SD. *p<0.05, **p<0.01, and ***p<0.001. Abbreviations: PM, Particulate matter; MUC5AC, Mucin 5AC; RT-PCR, Reverse Transcription-Polymerase Chain Reaction.

Figure 6

Resveratrol (Res) suppresses PM-induced inflammation response in HBE cells and mice via MAPK pathways. (A) HBE cells were treated with PM or resveratrol (Res), and the protein levels of MAPK pathways were measured by Western blot. (B) Mice were instilled intratracheally with normal saline (NS), PM or PM plus resveratrol respectively for 3 days, and the mice were sacrificed after 24 h. The protein levels of MAPK pathways were measured by Western blot. Data are representative of three independent studies. Data are representative of three independent studies. Results are expressed as mean ± SD. *p<0.05. Abbreviations: PM, Particulate matter; HBE, Human bronchial epithelial; MAPK, Mitogen-activated protein kinase.

Figure 7

MAPK inhibitors suppress PM-induced inflammation response in HBE cells. (A–I) p38 inhibitor SB203580 and ERK inhibitor U0126 were added 2 h prior PM exposed and cells were collected after 24 h. The levels of TNF-α, IL-6 and, IL-17A were analyzed by RT-PCR. Data are representative of three independent studies. Results are expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Abbreviations: MAPK, Mitogen-activated protein kinase; PM, Particulate matter; HBE, Human bronchial epithelial; RT-PCR, Reverse Transcription-Polymerase Chain Reaction.