Materials
Winfield red cigarettes (Phillip Morris, Australia); apocynin (Sigma Aldrich, Australia); Ketamine/Xylazine (Virbac, Australia); acridine orange/ethidium bromide (Invitrogen, USA); Kwik-Diff®reagent 1, fixative (Thermo Fisher Scientific, USA); RNeasy Mini Kit (Qiagen, Germany); High Capacity RNA-to-cDNA kit (Thermo Fisher Scientific, USA); pre-developed TaqMan primers (Thermo Fisher Scientific, USA), C2C12 murine myoblasts (American Type Culture Collection, USA; CRL-1772); cell culture reagents (Thermo Fisher Scientific, USA); H2O2 (Chem-Supply, Australia); antibody for immunofluorescence (Santa Cruz Biotechnology, USA); Fluoromount-GTM, with DAPI (Thermo Fisher Scientific, USA); MTS Cell Proliferation Assay (Promega, Australia); murine IL-6 ELISA Kit (Thermo Fisher, USA); murine IGF-1 DuoSet ELISA Kit (R&D Systems, USA); Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA), phosphorylation-specific, actin antibodies and p62 for western blots (Cell Signaling Technology, USA); all other antibodies for western blots (Abcam, USA); SuperSignal™ West Femto Maximum Sensitivity Substrate for chemiluminescence detection (Thermo Fisher Scientific, USA).

Results

Apocynin treatmentattenuates the pro-inflammatory lung response induced by CS exposure
Mice displayed no significant difference in starting body weight and food intake. However, CS exposure concomitantly reduced body weight gain (~7% loss) and food intake (~17% loss) which were unaffected by apocynin (5 mg kg-1) administration, suggesting apocynin did not impact on growth or appetiteof these mice at the administered dosage (Figure 1A-B). In line with the reduced body weight gain, tissue mass of testicular (30%) and retroperitoneal (38%) white adipose tissue (WAT), heart (9%) and spleen (21%) were also reduced by CS exposure; however this was prevented by apocynin treatment, except for the heart (Table 2). To examine whether apocynin treatment was effective in attenuating the direct impact of CS on immune cell recruitment to the lung, we performed differential cell count analyses on the bronchoalveolar lavage fluid (BALF). CS exposure caused a 3.7-fold increase in total cell infiltration which was attributed to a marked increase in the number of macrophages, neutrophils and lymphocytes (Figure 1C-F). In line with this, CS exposure caused a marked increase in gross lung weight (Table 2) and the expression of key pro-inflammatory cytokines/chemokines in the lungs, including granulocyte–macrophage colony-stimulating factor (Gmcsf ), CC-chemokine 2 (Ccl2 ), C-X-C motif ligand 1 (Cxcl2 ) and Tnfα(Figure 1G-J). Apocynin treatment significantly attenuated the CS-induced BALF cellularity evidenced by a 28% reduction in total cell counts (Figure 1C), 50% reduction in neutrophil counts (Figure 1E) and 86% reduction in lymphocyte counts in the CS-exposed mice (Figure 1F), without significant alterations in macrophage counts (Figure 1D). Accordingly, the CS-induced expression of Ccl2 , Cxcl2 ,Tnfα in the lungs were significantly attenuated by 84% (Figure 1H), 27% (Figure 1I) and 51% (Figure 1J), respectively; while the expression of Gmcsf remained elevated despite apocynin treatment (Figure 1G). This lung inflammation-attenuating effect of apocynin appeared to be specific to the CS exposure, as no significant effects in BALF cellularity (Figure 1C-F) and gene expression (Figure 1G-J) were observed in the sham-exposed mice.