Estudos dos efeitos antioxidantes e anti-inflamatórios da própolis sobre as células RAW 264.7 e na resposta inflamatória pulmonar aguda causada pela exposição à fumaça de cigarro em camundongos

Abstract

Chronic obstructive pulmonary disease (DPOC) causes a respiratory capacity reduction and its development is be associated with cigarette smoke. Cigarette has more than 4800 toxic substances in your composition and it causes death of six million people in the world. Studies had been made to find methods to change cigarette-induced health problems. Propolis (P) has been reported as a natural product with several properties. Here, we used two types of experiments, in vitro and in vivo, to study the potential antioxidant use of P. Phytochemical analyze was made to evaluate which compounds are within P. Murine macrophages, RAW 264.7 cell line, are exposed to different concentrations of propolis and following analyses were made: total polyphenols levels; cell viability (MTT); reduction potential (DPPH); total reactive oxygen species levels (ROS) and malondialdehyde (MDA). Thirty C57BL6 mice were divided into 3 groups: Control+P, CS and CS+P. Both CS groups were exposed to 6 cigarettes per day for 5 days. CS group was treated with vehicle. Lung and bronchoalveolar lavage (BAL) were collected for histological analysis and differential cell counts. Analysis for mieloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), oxidized glutathione (GSSG), MDA, nitrite and western blotting for TNF-alpha were performed. Phytochemical analyses of P showed which possesses four main compounds: hydrocinnamic and coumaric acids; artepillin C and drupanin. P showed an increase in total polyphenols (p<0.001), cell viability (p<0.001) and DPPH levels (p<0.001) in concentration-dependent manner, different from MDA (p<0.001) which was inverse pattern. P decreased ROS levels in 15.6 and 31.2 mg/mL groups (p<0.05). Lung histology of Control+P group was similar to CS and CS+P groups, however significant inflammatory cell influx was observed in CS group. Propolis administration reduced significantly macrophages and neutrophils compared with CS group (p<0.01 e p<0.001, respectively). MPO levels was increased in CS (526.5±34.72 mU/mg ptn, p<0.01), but was shown to be reduced in CS+P (385.1±27.64 mU/mg ptn, p<0.05) compared with Control+P (134±12.99 mU/mg ptn, p<0.001), similar pattern was found in antioxidant enzymes: SOD (Controle+P: 523.5±29.6 U/mg ptn; CS: 523.5±29.6 U/mg ptn, p<0.001; CS+P: 246.8±15.69 U/mg ptn, p<0.001), CAT (Controle+P: 37.38±3.39 U/mg ptn; CS: 92.68±6.24 U/mg ptn, p<0.001; CS+P: 59.84±4.55 U/mg ptn, p<0.05) and GPx (Controle+P: 2.23±0.17 (µM/min/mg ptn) x 10-1; CS: 4.51±0.31 (µM/min/mg ptn) x 10-1, p<0.001; CS+P: 2.64±0.19 (µM/min/mg ptn) x 10-1, p<0.05) and different from GSH/GSSG ratio (Controle+P: 1.088±0.17; CS: 0.736±0.07, p<0.05; CS+P: 1.258±0.10, p<0.05). MDA (Controle+P: 0.266±0.05 nMol/mg ptn; CS: 0.94±0.076 nMol/mg ptn, p<0.001; CS+P: 0.498±0.06 nMol/mg ptn, p<0.01) and nitrite levels (Controle+P: 50.01±4.19 µMol/mg ptn; CS: 108.7±7.73 µMol/mg ptn, p<0.001; CS+P: 58.84±3.42 nMol/mg ptn, p<0.01) increased in CS group when compared with other groups. TNF-α expression was observed in CS only. P administration showed an antioxidant action in murine macrophages and reduced cigarette smoke-induced lung inflammation and oxidative stress, probably, by action of its phytochemical compounds actions