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.