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. 2020 Nov;8(21):1418.
doi: 10.21037/atm-20-2426.

Protective effect of selegiline on cigarette smoke-induced oxidative stress and inflammation in rat lungs in vivo

Yuting Cui  1 , Kenneth W K Liu  1   2 , Mary S M Ip  1   2 , Yingmin Liang  1 , Judith C W Mak  1   2   3
Affiliations

Protective effect of selegiline on cigarette smoke-induced oxidative stress and inflammation in rat lungs in vivo

Yuting Cui et al. Ann Transl Med. 2020 Nov.

Abstract

Background: Cigarette smoke (CS)-induced build-up of oxidative stress is the leading cause of chronic obstructive pulmonary disease (COPD). Monoamine oxidases (MAOs) are novel sources of reactive oxygen species (ROS) due to the production of hydrogen peroxide (H2O2). However, it remains unclear whether MAO signaling is involved in CS-induced oxidative stress in vivo. This study aimed at investigating the impact of selegiline, a selective MAO-B inhibitor, on CS-induced lung oxidative stress and inflammation in vivo and its underlying mechanism.

Methods: Sprague Dawley rats were randomly divided into four groups: saline plus sham air (Saline/air), saline plus cigarette smoke (Saline/CS), selegiline plus sham air (Slg/air) and selegiline plus cigarette smoke (Slg/CS). Rats from Saline/air and Saline/CS groups were intraperitoneally injected with saline (2 mL/kg body weight) while rats from Slg/air and Slg/CS groups were injected with selegiline (2 mg/kg body weight) about 30 min prior to exposure daily. The Saline/air and Slg/air groups were exposed to atmospheric air while the Saline/CS and Slg/CS groups were exposed to mainstream CS generated from the whole body inExpose smoking system (SCIREQ, Canada) for twice daily (each for 1 hour with 20 cigarettes). After 7 days, rats were sacrificed to collect bronchoalveolar lavage (BAL) and lung tissues for the measurement of oxidative/anti-oxidative and inflammatory/anti-inflammatory makers respectively.

Results: CS caused significant elevation of MAO-B activity, reduction of total antioxidant capacity (T-AOC) and rGSH/GSSG ratio, and enhancement of superoxide dismutase (SOD) activity in rat lung. Selegiline significantly only reversed CS-induced elevation of MAO-B activity and reduction of rGSH/GSSG ratio. The CS-induced elevation of heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO1) expression via nuclear factor erythroid 2-related factor 2 (Nrf2) was also reversed by selegiline. Despite of CS-induced increase in total cell counts, especially the number of macrophages, selegiline had no effect. Selegiline attenuated CS-induced elevation of pro-inflammatory mediators (CINC-1, MCP-1 and IL-6) and restored CS-induced reduction of anti-inflammatory mediator IL-10 in BAL, which was driven through MAPK and NF-κB.

Conclusions: Inhibition of MAO-B may provide a promising therapeutic strategy for CS-mediated oxidative stress and inflammation in acute CS-exposed rat lungs.

Keywords: Cigarette smoke (CS); inflammation; monoamine oxidase-B; oxidative stress; selegiline.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-2426). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Effect of selegiline on MAO-B activity and ROS levels in rat lungs. Protein from rat lungs was extracted appropriately according to the protocols for the measurement of (A) MAO-B activity, (B) cytoplasmic ROS and (C) mitochondrial ROS levels. Results were corrected for protein concentration. Values are expressed as mean ± SEM (n=6-8). *, P<0.05, compared with Saline/air group; #, P<0.05, compared with Saline/CS group (one-way ANOVA followed by Tukey’s post hoc test).
Figure 2
Figure 2
Effect of selegiline on CS-induced oxidant/antioxidant balance in rat lungs. Oxidant and anti-oxidant markers were measured using commercially available kits. Levels of (A) T-AOC, (B) GSH/GSSG ratio, (C) SOD activity and (D) CAT activity were measured in rat lungs. Results were corrected with protein concentration and expressed as mean ± SEM (n=7-8). *, P<0.05; **, P<0.01, compared with Saline/air group; ###, P<0.001, compared with Saline/CS group (one-way ANOVA followed by Tukey’s post hoc test).
Figure 3
Figure 3
Effect of selegiline on CS-induced HO-1 and NQO1 protein expression and Nrf2 nuclear translocation in rat lungs. Total protein and cytoplasmic and nuclear protein from rat lungs were extracted for Westerm blots. (A) Representative Western blots and quantification of the effect of selegiline on CS-induced HO-1 and NQO1 protein expression. Total protein was normalized to β-actin. (B) Representative Western blots and quantification of the effect of selegiline on CS-induced Nrf2 nuclear translocation. Cytoplasmic protein was normalized to β-actin while nuclear protein was normalized to Lamin A/C. Values are expressed as mean ± SEM (n=8). **, P<0.01; ***, P<0.001, compared with Saline/air group; #, P<0.05, ##, P<0.01, compared with Saline/CS group (one-way ANOVA followed by Tukey’s post hoc test), ++, P<0.01 compared with Saline/air group (unpaired Student’s t test).
Figure 4
Figure 4
Effect of selegiline on total and differential cell counts in BAL after CS exposure. Total cells counts were measured by dual AO/EB staining. BAL was subjected to cytospin and stained by Diff-Quick staining Kit. Macrophages, neutrophils and lymphocytes were identified based on their color and morphology, and counted. (A) Total cell counts, (B) macrophages, (C) neutrophils and (D) lymphocytes in BAL were shown. Values are expressed as mean ± SEM (n=6). **, P<0.01, compared with Saline/air group (one-way ANOVA followed by Tukey’s post hoc test).
Figure 5
Figure 5
Effect of selegiline on CS-induced inflammatory responses in BAL. Levels of (A) CINC-1, (B) MCP-1, (C) IL-6, (D) IL-10 in BAL were measured using ELISA kits. Values are expressed as mean ± SEM (n=8). *, P<0.05; **, P<0.01, compared with SA control; #, P<0.05, compared with CS group (one-way ANOVA followed by Tukey’s post hoc test).
Figure 6
Figure 6
Effect of selegiline on CS-induced activation of MAPK and NF-κB signaling pathways in rat lungs. Total protein from rat lungs was extracted for the measurement of ERK and p38. (A) Representative Western blots and quantification of the effect of selegiline on CS-induced phosphorylation of ERK and p38. Phosphorylation of ERK was normalized to total ERK; phosphorylation of p38 was normalized to total p38. Nuclear and cytoplasmic fractions from rat lungs were extracted for the measurement of NF-κB p65. (B) Representative Western blots and quantification of the effect of selegiline on CS-induced NF-κB p65 protein expression in cytoplasmic and nuclear fractions. Cytoplasmic protein was normalized to β-actin; nuclear protein expression was normalized to Lamin A/C. Values are expressed as mean ± SEM (n=7–8). *, P<0.05; **, P<0.01, compared with Saline/air group; #, P<0.05, compared with Saline/CS group (one-way ANOVA followed by Tukey’s post hoc test).
Figure 7
Figure 7
A schematic diagram to demonstrate the effect of selegiline on CS-induced oxidative stress and inflammation. MAO-B is an important ROS source located at the outer membrane of mitochondria. After CS exposure, MAO-B activity was increased thus contributing to CS-induced oxidative stress and inflammation via activation of Nrf2 and MAPK/NF-κB signaling pathways. The selective MAO-B inhibitor selegiline partially blocked CS-induced oxidative stress and inflammation through inhibition of MAO-B-derived ROS.
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