Baicalein Inhibits Benzo[a]pyrene-Induced Toxic Response by Downregulating Src Phosphorylation and by Upregulating NRF2-HMOX1 System - PubMed (original) (raw)

Baicalein Inhibits Benzo[a]pyrene-Induced Toxic Response by Downregulating Src Phosphorylation and by Upregulating NRF2-HMOX1 System

Yuka Tanaka et al. Antioxidants (Basel). 2020.

Abstract

Benzo[a]pyrene (BaP), a major environmental pollutant, activates aryl hydrocarbon receptor (AHR), induces its cytoplasmic-to-nuclear translocation and upregulates the production of cytochrome P450 1A1 (CYP1A1), a xenobiotic metabolizing enzyme which metabolize BaP. The BaP-AHR-CYP1A1 axis generates reactive oxygen species (ROS) and induces proinflammatory cytokines. Although the anti-inflammatory phytochemical baicalein (BAI) is known to inhibit the BaP-AHR-mediated CYP1A1 expression, its subcellular signaling remains elusive. In this study, normal human epidermal keratinocytes and HaCaT keratinocytes were treated with BAI, BaP, or BAI + BaP, and assessed for the CYP1A1 expression, antioxidative pathways, ROS generation, and proinflammatory cytokine expressions. BAI and BAI-containing herbal medicine Wogon and Oren-gedoku-to could inhibit the BaP-induced CYP1A1 expression. In addition, BAI activated antioxidative system nuclear factor-erythroid 2-related factor-2 (NRF2) and heme oxygenase 1 (HMOX1), leading the reduction of BaP-induced ROS production. The BaP-induced IL1A and IL1B was also downregulated by BAI. BAI inhibited the phosphorylation of Src, a component of AHR cytoplasmic complex, which eventually interfered with the cytoplasmic-to-nuclear translocation of AHR. These results indicate that BAI and BAI-containing herbal drugs may be useful for inhibiting the toxic effects of BaP via dual AHR-CYP1A1-inhibiting and NRF2-HMOX1-activating activities.

Keywords: Oren-gedoku-to; Src; Wogon; aryl hydrocarbon receptor; baicalein; benzo[a]pyrene; keratinocyte; nuclear factor-erythroid 2-related factor-2; reactive oxygen species.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

Structure of baicalein (BAI). The structure of BAI is shown.

Figure 2

Figure 2

BAI inhibits BaP-induced CYP1A1 expression in keratinocytes. (a) NHEKs or (b) HaCaT cells were treated with DMSO (0.1%), BAI (10 μM for NHEKs and 25 μM for HaCaT cells), BaP (1 μM) or a combination of BAI and BaP and assessed for CYP1A1 mRNA expression (upper panels) at 6 h- or CYP1A1 protein expression (middle and lower panels) at 24 h-post treatment. (c) HaCaT cells were treated with DMSO (0.1%), BAI (25 μM), BaP (1 μM) or a combination of BaP and BAI, BaP and WO, or BaP and OG, and assessed for CYP1A1 mRNA expression (upper panels) at 6 h or CYP1A1 protein expression (middle and lower panels) at 24 h post treatment. Experiments were performed in triplicate wells and each experiment was repeated three times. Representative blot images (middle panels) and mean ± SD of CYP1A1/β-ACTIN ratio (lower panels) are shown. Whole blot images are shown in Supplementary Figure S2. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 3

Figure 3

BAI induces NRF2 nuclear translocation. (a) HaCaT cells were treated with DMOS (0.1%) or BAI (25 μM) for 6 h and nuclear translocation of NRF2 was examined using immunocytochemistry. E-cadherin was stained simultaneously as a marker of plasma membrane. DAPI was used for nuclear staining. Experiments were repeated three times and the representative images (left panels) are shown. Scale bar = 100 μm. (b) HaCaT cells were treated as for (a) and cytoplasmic and nuclear fraction were separately extracted. β-ACTIN and Lamin B1 were used as internal controls of cytoplasmic or nuclear protein. Experiments were performed in triplicate wells and each experiment was repeated three times. Representative blot images (right upper panels) and mean ± SD of relative NRF2/β-ACTIN and NRF2/Lamin B1 ratio (right lower panels) are shown. Whole blot images are shown in Supplementary Figure S3a. * p < 0.05.

Figure 4

Figure 4

BAI inducesHMOX1 expression. (a) NHEKs and (b) HaCaT cells were treated with DMSO (0.1%), BAI (10 μM for NHEKs and 25 μM for HaCaT cells), BaP (1 μM) or a combination of BAI and BaP and assessed for HMOX1 mRNA expression (upper panels) at 6 h or HMOX1 protein expression (middle and lower panels) at 12 h post treatment. Experiments were performed in triplicate wells and each experiment was repeated three times. Representative blot images (middle panels) and mean ± SD of relative HMOX1/β-ACTIN ratio (lower panels) are shown. Whole blot images are shown in Supplementary Figure S3b,c. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 5

Figure 5

BAI exerts antioxidative effects against BaP. (a) NHEKs or (b) HaCaT cells were treated with DMSO (0.1%), BAI (10 μM for NHEKs and 25 μM for HaCaT cells), BaP (1 μM) or a combination of BAI and BaP for 12 h and ROS production was measured by flow cytometry. A representative histogram image of mean fluorescent intensity (MFI) of DCF (excitation: 480 nm, emission: 530 nm) (left) and mean ± SD of MFI (right) are shown. Experiments were performed in triplicate wells and each experiment was repeated three times. *** p < 0.001.

Figure 6

Figure 6

BAI inhibits BaP-induced proinflammatory cytokine expression in keratinocytes. (a,b) NHEKs and HaCaT cells were treated with DMSO (0.1%), BAI (10 μM for NHEKs and 25 μM for HaCaT cells), BaP (1 μM) or a combination of BAI and BaP for 6 h and assessed for (a) IL1A and (b) IL1B expression. (c–f) NHEKs or HaCaT cells were treated as (a,b). After 24 h, cells were harvested and protein expressions of (c–e) IL1A and (c,d,f) IL1B were assessed. Experiments were performed in triplicate wells and each experiment was repeated three times. Representative blot images (left lower panels) and mean ± SD of relative IL1A/β-ACTIN and IL1B/β-ACTIN ratio (right lower panels) are shown. Whole blot images are shown in Supplementary Figure S6. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 7

Figure 7

BaP-induced Src phosphorylation is inhibited by BAI. (a) NHEKs and (b) HaCaT cells were treated with DMSO (0.1%), BAI (10 μM for NHEKs and 25 μM for HaCaT cells), BaP (1 μM) or a combination of BAI and BaP for 24 h and Src phosphorylation was assessed by Western blotting. Experiments were performed in triplicate wells and each experiment was repeated three times. Representative blot images (left) and mean ± SD of relative pSrc/Src ratio (right) are shown. Whole blot images are shown in Supplementary Figure S8a,b. * p < 0.05 and ** p < 0.01.

Figure 8

Figure 8

BaP-induced AHR nuclear translocation is prevented by BAI. (a) HaCaT cells were treated with DMSO (0.1%), BaP (1 μM), the combination of BaP and dasatinib (100 nM) or the combination of BaP and BAI (25 μM) for 24 h and nuclear translocation of AHR was examined using immunocytochemistry. E-cadherin staining indicates plasma membrane and arrows indicate AHR that translocated to the nucleus. DAPI was used for nuclear staining. Scale bar = 100 μm. (b) HaCaT cells were treated as in (a) and cytoplasmic and nuclear fraction were separately extracted. β-ACTIN and Lamin B1 were used as internal controls of cytoplasmic or nuclear protein. Representative blot images (upper panels) and mean ± SD of relative AHR/β-ACTIN and AHR/Lamin B1 ratio (lower panels) are shown. Experiments were performed in triplicate wells and each experiment was repeated three times. Whole blot images are shown in Supplementary Figure S8c. * p < 0.05 and *** p < 0.001.

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