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

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.

Keywords: Baccharis trimera; PKC; nicotinamide adenine dinucleotide phosphate oxidase; quercetin; reactive oxygen species; rutin.

Figure 1

RP-UPLC-DAD profiles of hydroethanolic extract of Baccharis trimera. Identified compounds: 1. Coumaroylquinic acid; 2. Coumaroylquinic acid; 3. 5-O-feruloylquinc acid; 4. 3-O-Isoferuloylquinc acid; 5. Coumaroylquinic acid; 6. 5-O-Isoferuloylquinc acid; 7. 6(8)-C-furanosyl-8(6)-C-hexosyl-flavone; 8. 6(8)-C-hexosyl-8(6)-C-furanosyl-flavone; 9. 3,4-di-O-caffeoylquinc acid; 10. 3,5-di-O-caffeoylquinc acid; 11. 4,5-di-O-caffeoylquinc acid; 12. Quercetin; 13. 3′,5-dihydroxy-4′,7-dimethoxyflavone; 14. 3′,5-dihydroxy-4′,6,7-trimethoxyflavone. Analysis conditions: see in experimental section

Figure 2

Identified compounds in hydroethanolic extract of Baccharis trimera by LC-DAD-ESI-MS Caf: caffeoyl; Fer: feruloyl; Isofer: isoferuolyl; Coum: coumaroyl

Figure 3

Viability of SK Hep-1 cells exposed to Baccharis trimera, quercetin and rutin for 12 h. The MTT assay was performed to analyze cell viability. The viability of the control group (cells not exposed to extract, quercetin, or rutin) was considered as 100%, and the other values were compared with the control group. The results are expressed as the means ± SEM. ***P < 0.001 for 100 µg mL⁻¹ of Baccharis trimera hydroethanolic extract, which decreased cell viability. One-way ANOVA, followed by Bonferroni’s post-test was used for statistical analysis. This test was performed in sextuplicate from three independent experiments

Figure 4

ROS production in unstimulated SK Hep-1 cells. The results are expressed as the means ± SEM. (a–c) The cells were exposed to treatments (Baccharis trimera, quercetin and rutin) for 30 min. (d–f) The cells were treated with Baccharis trimera, quercetin or rutin for 6 h. This test was performed in sextuplicate from three independent experiments. *P < 0.05, **P < 0.001, ***P < 0.0001 for values significantly different from C (control cells)

Figure 5

Influence of the PKC/NADPH oxidase pathway in ROS production in SK Hep-1 cells. The results are expressed as the means ± SEM. (a–c) The cells were exposed to treatments for 30 min and stimulated with PMA + Iono for 30 min. (d–f) The cells were treated with Baccharis trimera, quercetin or rutin for 6 h and incubated for 30 min with PMA + iono. This test was performed in sextuplicate from three independent experiments. *P < 0.05 for values significantly different from C (control cells, without stimulus). ^#P < 0.05, ^##P < 0.001, ^###P < 0.0001 for values significantly different from C (PMA + Iono). (g) Cells stained with Carboxy- H₂DCFDA and visualized through fluorescence microscopy. Cells – SK Hep-1 without fluorescence. ROS – cells analyzed with fluorescence labeled with Carboxy-H₂DCFDA. C: Control cells; PMA/iono: positive control cells (C + PMA/Iono). DPI: Negative control cells (C + DPI + PMA/Iono). Bt50: Baccharis trimera-treated cells stimulated with PMA + Iono. Que50: Quercetin-treated cells stimulated with PMA + iono. (A color version of this figure is available in the online journal.)

Figure 6

Reduction of PKC protein expression and inhibition of PKC activity through Baccharis trimera extract and quercetin. (a) Densitometric scanning of PKC after normalization with β-actin. Results of the Western blotting assay showing PKC activation through PMA + Ionomycin in SK Hep-1 cells and the effect of Baccharis trimera hydroethanolic extract and quercetin on this protein. *P < 0.05 for values significantly different from C (PMA + iono). The analyses were performed using Quantity One image analysis software (Bio Rad). (b) PKC activity in cells exposed to Baccharis trimera extract and quercetin, followed by stimulation with PMA + ionomycin was commercially assessed using a kit purchased from Enzo Life Sciences. As a positive control, PKC was supplied in the kit. ^#P < 0.0001 for values significantly different from C (control cells); **P < 0.001 and ***P < 0.0001 for values significantly different from C (PMA + Iono). The results are expressed as the means ± SEM. Statistically significant differences were determined using One-way ANOVA and Dunnett’s post-test. These analyses were performed in triplicate

Figure 7

p47^phox and p47^phox phosphorylated expression in SK Hep-1 cells exposed to Baccharis trimera and quercetin. (a) Densitometric scanning of p47^phox and phospho p47^phox after normalization with β-actin. Results of Western blotting assay showing that p47^phox expression are not altered by PMA + iono exposure nor with Baccharis trimera and quercetin treatments for 6 h. (b) p47^phox phosphorylated expression was reduced by Baccharis trimera and quercetin treatments. *P < 0.05 for values significantly different from C (PMA + iono). The analyses were performed using Quantity One image analysis software (Bio Rad). The results are expressed as the means ± SEM. Statistically significant differences were determined using One-way ANOVA and Dunnett’s post-test. These analyses were performed in triplicate

Figure 8

Proposed mechanisms for the antioxidant effect of Baccharis trimera. (a) Represents the production of ROS through the PKC/NADPH oxidase signaling pathway. (b) Represents the mechanism of the Baccharis trimera-mediated modulation of ROS production. (1) The inhibition of PKC protein expression; (2) the inhibition of enzyme activity; and (3) down-regulation of p47^phox phosphorylation. PLC: phospholipase C; IP3: inositol-1,4,5-trisphosphate; DAG: diacylglycerol; PKC: protein kinase C; Bt: Baccharis trimera; Que: quercetin; iono: ionomycin; PMA: phorbol ester