Activation of the Keap1-Nrf2 pathway by specioside and the n-butanol extract from the inner bark of Tabebuia rosea (Bertol) DC

Abstract

Background: A large number of chemical compounds exert their antioxidant effects by activation of key transcriptional regulatory mechanisms, such as the transcription factor Nrf2. The aim of this study was to evaluate the activation of the Keap1-Nrf2 pathway by both the n-butanol extract obtained from the inner bark of Tabebuia rosea (Bertol) DC and specioside isolated from this extract. Methods: The antioxidant activity of the extract and specioside isolated from the inner bark of T. rosea were evaluated using the oxygen radical absorbance capacity (ORAC) and the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity (DPPH) techniques, whereas their effects on the viability of HepG2 cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The effects of the compound and the extract on activating the Keap1-Nrf2 pathway were evaluated using a Nrf2 Transcription Factor Assay kit. Induction of the Nrf2-mediated antioxidant response genes HMOX-1 and NQO1 was evaluated by real-time PCR. The protective effects against H ₂O ₂-induced oxidative stress in HepG2 cells was determined as the percent protection using the MTT method. Results: Both the n-butanol extract and specioside exhibited activity at low concentrations without affecting cellular viability, since the cell viability was greater than 80% after 24 hours of exposure at each tested concentration. In addition, Nrf2 dissociated from Keap1 after treatment with the n-butanol extract at a concentration of 0.25 µg/mL after 4 hours of exposure. An increase in the Nrf2 level in the cytoplasm after 4 hours of exposure to 2 μM specioside was observed. Nrf2 levels stabilized in the nucleus 12 hours after stimulation with both specioside and the extract. After 6 hours of stimulation, both the extract and specioside induced the expression of HMOX-1 and NQO1. Conclusion: The n-butanol extract from the inner bark of T. rosea and specioside produced protective effects against H ₂O ₂-induced oxidative stress in HepG2 cells.

Keywords: Bignoniaceae; Nrf2; Tabebuia rosea; antioxidant agents; extracts; specioside.

Figure 1.

Chemical structure of specioside ( A) and catalposide ( B).

Figure 2.

Nrf2 levels in cytosol ( a) and nucleus ( b) after 0, 4, 12 and 24 hours of exposure to 0.5 μM specioside, catalposide, controls and 0.25 µg/mL n-butanol extract. Kruskal-Wallis, Dunn's post hoc. * p<0.05, ** p<0.01, *** p<0.001. ALA, α-lipoic acid; CUR, curcumin; CHAL, 2-trifluoromethyl-2ʹ-methoxychalcone.

Figure 3.

Nrf2 levels in cytosol ( a) and nucleus ( b) after 0, 4, 12 and 24 hours of exposure to 2 μM specioside, catalposide, controls and 1 µg/mL n-butanol extract. Kruskal Wallis, Dunn's post hoc. * p<0.05, ** p<0.01, *** p<0.001. ALA, α-lipoic acid; CUR, curcumin; CHAL, 2-trifluoromethyl-2ʹ-methoxychalcone.

Figure 4.

Relative HMOX-1 ( a) and NQO1 ( b) mRNA levels after 0, 4, 6 and 8 hours post-exposure to 0.5 µM (pure compounds), 0.25 µg/mL ( n-butanol extract) and induction of oxidative stress with 0.98 mM (H ₂O ₂). Kruskal Wallis Dunn's post hoc. * p<0.05, ** p<0.01, *** p<0.001. ALA, α-lipoic acid; CUR, curcumin; CHAL, 2-trifluoromethyl-2ʹ-methoxychalcone.

Figure 5.

Relative HMOX-1 ( a) and NQO1 ( b) mRNA levels after 0, 4, 6 and 8 hours post-exposure to 2 µM (pure compounds), 1 µg/mL ( n-butanol extract) and 0.98 mM (H ₂O ₂). Kruskal Wallis, Dunn's post hoc * p<0.05, ** p<0.01, *** p<0.001. ALA, α-lipoic acid; CUR, curcumin; CHAL, 2-trifluoromethyl-2ʹ-methoxychalcone.

Figure 6.. Effect of the n-butanol extract, pure compounds and controls on the HepG2 cell line against H ₂O ₂-induced oxidative stress.

a. Percentage of cell viability. b. Protective effect. Kruskal-Wallis, Dunn's post hoc. * p<0.05, ** p<0.01, *** p<0.001. ALA, α-lipoic acid; CUR, curcumin; CHAL, 2-trifluoromethyl-2ʹ-methoxychalcone.