In this study, we demonstrated that WEP induced early up-regulation of HO-1 and nuclear translocation of Nrf2 following UVA irradiation. At 24 h after UVA irradiation, HO-1 expression markedly decreased in the group treated with WEP and UVA than the HO-1 expression in the group treated with UVA alone (Fig.
1d). This change may be induced by secondary effect of WEP. It is followed by acceleration of HO-1 up-regulation with decreasing ROS expression. This result indicates that total HO-1 expression remained unaltered after pretreatment with WEP, which only accelerates the antioxidative response. Nrf2 acts as an antioxidative transcriptional factor upon accumulation in the nuclei. Therefore, early nuclear translocation of Nrf2 after treatment with both WEP and UVA (Fig.
3) indicated that WEP activates the Nrf2/ARE pathway soon after UVA irradiation. Previous reports have shown that the Nrf2/Keap1 pathway plays an important role in protecting skin fibroblast against UVA-induced apoptosis [
20,
21]. Therefore, it is highly possible that early up-regulation of HO-1 and nuclear translocation of Nrf2 contributed to the protective effect of WEP against UVA irradiation-induced cell death of human skin fibroblast, in our previous report [
19].
Among the four major constituents of WEP, the diphenols significantly induced HO-1. Diphenol is oxidized to quinone under oxidative stress conditions and then reacts with the critical cysteine residues in Keap1 that are essential for its ubiquitin ligase substrate adaptor activity [
22,
23]. Studies have reported various polyphenols with diphenol structures such as epigallocatechin gallate (EGCG) [
24,
25], eckol [
26,
27], and resveratrol [
28,
29] that activate the Nrf2/ARE pathway. Moreover, 1,5-dicaffeoylquinic acid and 3-caffeoyl,4-dihydrocaffeoyl quinic acid were also reported to up-regulate HO-1 expression or induce Nrf2 nuclear translocation under certain oxidative stress conditions induced by oxygen and glucose deprivation/reperfusion or tert-butyl hydroperoxide exposure [
30,
31]. They have a diphenol structure that is similar to that of 3,4-CQA, 3,5-CQA, and CGA. However, the underlying mechanism of HO-1 induction by these compounds remains unclear. For example, a previous report [
30] indicated that 3-caffeoyl,4-dihydrocaffeoyl quinic acid regulates HO-1 expression via the phosphoinositide 3-kinase (PI3K)/Akt-Nrf2 signaling pathways but early up-regulation of HO-1 expression by WEP treatment was not inhibited by LY294002 (the PI3K/Akt pathway inhibitor) pretreatment (data not shown). Moreover, WEP and its main constituents did not induce HO-1 expression and nuclear translocation of Nrf2 in the absence of UVA irradiation. In contrast, the previous reports [
30,
31] showed that the compounds induce the expression of these proteins without pre-induction of oxidative stress. Further investigation is required to elucidate these differences. In a previous study, we showed that
p-CA, which does not possess a diphenol structure, also had protective effects [
19]. Besides, in present study, we could not detect Nrf2 at 3 h after UVA irradiation (Figs.
3a and
4). NQO1, which the other downstream target protein of Nrf2, was not affected WEP pretreatment at 3, 6 h after UVA irradiation (Fig.
5). Based on these results, other mechanisms may also contribute to the protective effects of the main constituents of WEP. Since WEP acts as an HO-1 activator only after oxidative stress occurred in the cells, it can activate HO-1 only tissue under the pathological condition. These results suggest that WEP is useful for treatment of skin diseases as oxidation damaged portion-specific an HO-1 inducer.