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  • Baccharis trimera inhibits reactive oxygen species production through PKC and down-regulation p47 phox phosphorylation of NADPH oxidase in SK Hep-1 cells.

Baccharis trimera inhibits reactive oxygen species production through PKC and down-regulation p47 phox phosphorylation of NADPH oxidase in SK Hep-1 cells.

Experimental biology and medicine (Maywood, N.J.) (2017-01-21)
Glaucy Rodrigues de Araújo, Ana Carolina Silveira Rabelo, Janaína Serenato Meira, Joamyr Victor Rossoni-Júnior, William de Castro-Borges, Renata Guerra-Sá, Maurício Azevedo Batista, Denise da Silveira-Lemos, Gustavo Henrique Bianco de Souza, Geraldo Célio Brandão, Míriam Martins Chaves, Daniela Caldeira Costa
ABSTRACT

Baccharis trimera, popularly known as "carqueja", is a native South-American plant possessing a high concentration of polyphenolic compounds and therefore high antioxidant potential. Despite the antioxidant potential described for B. trimera, there are no reports concerning the signaling pathways involved in this process. So, the aim of the present study was to assess the influence of B. trimera on the modulation of PKC signaling pathway and to characterize the effect of the nicotinamide adenine dinucleotide phosphate oxidase enzyme (NOX) on the generation of reactive oxygen species in SK Hep-1 cells. SK-Hep 1 cells were treated with B. trimera, quercetin, or rutin and then stimulated or not with PMA/ionomycin and labeled with carboxy H2DCFDA for detection of reactive oxygen species by flow cytometer. The PKC expression by Western blot and enzyme activity was performed to evaluate the influence of B. trimera and quercetin on PKC signaling pathway. p47 phox and p47 phox phosphorylated expression was performed by Western blot to evaluate the influence of B. trimera on p47 phox phosphorylation. The results showed that cells stimulated with PMA/ionomycin (activators of PKC) showed significantly increased reactive oxygen species production, and this production returned to baseline levels after treatment with DPI (NOX inhibitor). Both B. trimera and quercetin modulated reactive oxygen species production through the inhibition of PKC protein expression and enzymatic activity, also with inhibition of p47 phox phosphorylation. Taken together, these results suggest that B. trimera has a potential mechanism for inhibiting reactive oxygen species production through the PKC signaling pathway and inhibition subunit p47 phox phosphorylation of nicotinamide adenine dinucleotide phosphate oxidase.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-p47 PHOX antibody produced in rabbit, affinity isolated antibody
Sigma-Aldrich
Anti-Rabbit IgG (whole molecule)–Peroxidase antibody produced in goat, affinity isolated antibody
Sigma-Aldrich
Anti-Mouse IgG (whole molecule)–Peroxidase antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
Sigma-Aldrich
Dulbecco′s Modified Eagle′s Medium - high glucose, HEPES Modification, With 4500 mg/L glucose, L-glutamine, and 25 mM HEPES, without sodium bicarbonate and pyruvate, powder, suitable for cell culture
Sigma-Aldrich
Diphenyleneiodonium chloride, ≥98%
Sigma-Aldrich
Anti-phospho-p47 phox (pSer359) antibody produced in rabbit, affinity isolated antibody
Sigma-Aldrich
Monoclonal Anti-Protein Kinase C (PKC) antibody produced in mouse, clone MC5, ascites fluid
Sigma-Aldrich
Anti-β-Actin antibody, Mouse monoclonal, clone AC-15, purified from hybridoma cell culture
Sigma-Aldrich
Ionomycin calcium salt, Ready Made Solution, from Streptomyces conglobatus, 1 mM in DMSO