Hydrogen sulfide attenuates cigarette smoke-induced airway remodeling by upregulating SIRT1 signaling pathway

Abstract

Airway remodeling is one of the characteristics for chronic obstructive pulmonary disease (COPD). The mechanism underlying airway remodeling is associated with epithelial-mesenchymal transition (EMT) in the small airways of smokers and patients with COPD. Sirtuin 1 (SIRT1) is able to reduce oxidative stress, and to modulate EMT. Here, we investigated the effects and mechanisms of hydrogen sulfide (H₂S) on pulmonary EMT in vitro and in vivo. We found that H₂S donor NaHS inhibited cigarette smoke (CS)-induced airway remodeling, EMT and collagen deposition in mouse lungs. In human bronchial epithelial 16HBE cells, NaHS treatment also reduced CS extract (CSE)-induced EMT, collagen deposition and oxidative stress. Mechanistically, NaHS upregulated SIRT1 expression, but inhibited activation of TGF-β1/Smad3 signaling in vivo and in vitro. SIRT1 inhibition by a specific inhibitor EX527 significantly attenuated or abolished the ability of NaHS to reverse the CSE-induced oxidative stress. SIRT1 inhibition also abolished the protection of NaHS against CSE-induced EMT. Moreover, SIRT1 activation attenuated CSE-induced EMT by modifying TGF-β1-mediated Smad3 transactivation. In conclusion, H₂S prevented CS-induced airway remodeling in mice by reversing oxidative stress and EMT, which was partially ameliorated by SIRT1 activation. These findings suggest that H₂S may have therapeutic potential for the prevention and treatment of COPD.

Keywords: Airway remodeling; COPD; Epithelial-mesenchymal transition; Hydrogen sulfide; Oxidative stress; Sirtuin 1.

Fig. 1

NaHS attenuated cigarette smoke (CS)-induced airway remodeling in mice. After CS exposure for 12 weeks, mice were euthanized, and lung tissues were collected. (A) Masson's trichrome staining was performed to determined collagen deposition. Representative images from the lung sections of mice in the four groups. (B) Immunohistochemical staining for α-SMA was performed in the lungs. (C, D) The mRNA levels of collagen 1 and collagen 3 in the lungs were analyzed by Real-time PCR. Data are presented as mean ± SEM of 6 mice/group, **P < 0.01, significantly different from control group; #P < 0.05, ##P < 0.05, significantly different from CS + Saline group.

Fig. 2

NaHS inhibited EMT in CS-exposed mouse lungs. After CS exposure for 12 weeks, mice were euthanized, and lung tissues were collected. (A) Western blot was used to analyze the protein levels of E-cadherin, CK18, Fibronectin, vimentin, FSP-1, α-SMA, Snail, MMP-2, MMP-9 and MMP-12 in the lung tissues. (B) Densitometric analysis of E-cadherin, CK18, Fibronectin, vimentin, FSP-1, α-SMA, Snail, MMP-2, MMP-9 and MMP-12 in the immunoblots using β-actin as the internal reference. (C) The expression of E-cadherin and vimentin in the lungs were visualized using immunofluorescence double staining. (D, E) Western blot was used to analyze the protein levels of TGF-β1, TGFβR1 and p-Smad3 in mouse lungs. Data are presented as mean ± SEM of 6 mice/group, *P < 0.05, **P < 0.01, significantly different from control group; ^#P < 0.05, ^##P < 0.01, significantly different from CS + Saline group.

Fig. 3

NaHS repressed cigarette smoke extract (CSE)-induced EMT and collagen deposition in human bronchial epithelial 16HBE cells. (A) 16HBE cells were treated with 3% CSE and different concentrations of NaHS (100, 200, or 400 μM) for 48 h. The protein levels of E-cadherin, fibronectin and α-SMA was analyzed by Western blot. (B–D) Densitometric analysis of proteins of interest in the immunoblots using β-actin as the internal reference. (E) Immunofluorescence for E-cadherin was performed on human 16HBE cells treated with and without 3% CSE in the presence of 400 μM NaHS for 48 h. (F–H) Western blot was used to detect collagen 1 and collagen 3 levels. (I–K) Western blot was used to analyze the protein levels of TGF-β1 and p-Smad3. Data are presented as mean ± SEM of at least three independent experiments, **P < 0.01, significantly different from untreated cells [3%CSE (-) and NaHS (-)]; #P < 0.05, ##P < 0.01, significantly different from cells treated with CSE only.

Fig. 4

NaHS reduced CSE-induced oxidative stress in human bronchial epithelial 16HBE cells. (A, B) 16HBE cells were incubated with 3% CSE and the ROS scavenger N-Acetyl-l-cysteine (NAC) for 48 h. Western blot was used to detect E-cadherin, fibronectin, Collagen 1 and Collagen 3 protein expressions. Data are presented as mean ± SEM of at least three independent experiments, **P < 0.01, significantly different from control cells [3%CSE (-) and NAC (-)]; ^#P < 0.05, ^##P < 0.01, significantly different from cells treated with CSE only. (C) 16HBE cells treated with and without 3%CSE in the absence or presence of 400 μM NaHS for 48 h. Representative microphotographs showing intracellular ROS. 16HBE cells were treated with 3% CSE and different concentrations of NaHS (100, 200, or 400 μM) for 48 h. (D) MDA levels. (E) Activities of CAT. (F) The GSH/GSSG ratio. Data are presented as mean ± SEM of at least three independent experiments, *P < 0.05, **P < 0.01, significantly different from untreated cells [3%CSE (-) and NaHS (-)]; ^#P < 0.05, ^##P < 0.01, significantly different from cells treated with CSE only.

Fig. 5

Effects of NaHS on SIRT1 signaling in CSE-stimulated 16HBE cells and in CS-exposed mouse lungs. After CS exposure for 12 weeks, lung tissues were collected. (A) The SIRT1 mRNA level was detected by Real-time PCR. (B) The SIRT1 protein level was detected using Western blot. Data are presented as mean ± SEM of 6 mice/group, **P < 0.01, significantly different from control group; ^#P < 0.05, ^##P < 0.01, significantly different from CS + Saline group. 16HBE cells were cultured with and without 3% CSE and/or 100, 200, or 400 μM NaHS for 48 h. (C) The mRNA level of SIRT1 was detected using Real-time PCR. (D) The protein level of SIRT1 was analyzed by Western blot assay. Data are presented as mean ± SEM of at least three independent experiments, *P < 0.05, **P < 0.01, significantly different from control cells [3%CSE (-) and NaHS (-)]; #P < 0.05, ##P < 0.01, significantly different from cells treated with 3%CSE only.

Fig. 6

Effects of SIRT1 on the NaHS-mediated attenuation in CSE-induced oxidative stress and EMT in 16HBE cells. 16HBE cells were cultured with SIRT1 inhibitor (EX527, 20 μM) in the absence and presence of 3% CSE and NaHS (400 μM) for 48 h. (A) The protein level of SOD2 was detected using Western blot. (B) MDA levels. (C) Activities of CAT (D) The GSH/GSSG ratio. (E) Representative microphotographs showing intracellular ROS generation. (F) Protein levels of E-cadherin and α-SMA were detected using Western blot. Data are presented as mean ± SEM of at least three independent experiments, **P < 0.01, significantly different from control cells; ^#P < 0.05, ^##P < 0.01, significantly different from cells treated with 3%CSE only; ^&P < 0.05, ^&&P < 0.01, significantly different from cells treated with CSE and NaHS.

Fig. 7

SIRT1 attenuated canonical TGF-β1 signaling. (A) 16HBE cells were treated with TGF-β1 (5 ng/ml) in the presence or absence of the SIRT1 activator SRT1720. The protein level of p-Smad3 was analyzed by Western blot assay. (B) 16HBE cells were treated with TGF-β1 in the presence or absence of the SIRT1 inhibitor EX 527 (20 μM). The protein level of p-Smad3 was analyzed by Western blot assay. Data are presented as mean ± SEM of at least three independent experiments, **P < 0.01, significantly different from control cells; ^##P < 0.01, significantly different from cells treated with TGF-β1 only.