Rutin protects against H2O2-triggered impaired relaxation of placental arterioles and induces Nrf2-mediated adaptation in Human Umbilical Vein Endothelial Cells exposed to oxidative stress.

BACKGROUND: Rutin intake is associated with a reduced risk of cardiovascular disease (CVD). The exact mechanism by which rutin can protect against CVD development is still enigmatic. Since, rutin is a compound with a relatively short half-life, the direct antioxidant effect of rutin cannot explain the long-lasting effect on human health. We hypothesized that rutin next to its direct antioxidant effect that improves endothelial function, may also induce an adaptive response in endogenous antioxidant systems. METHODS AND RESULTS: In Human Umbilical Vein Endothelial Cells (HUVECs), the direct antioxidant effect was confirmed. During scavenging of Reactive Oxygen Species (ROS), rutin is oxidized into a quinone derivative. HUVECs pretreated with rutin quinone became better protected against a second challenge with oxidative stress 3h later. LC-MS/MS analysis indicated that rutin quinone targets cysteine 151 of Keap1. Moreover, we found that the quinone is an inhibitor of the selenoprotein thioredoxin reductase 1. These properties correlated with an activation of Nrf2 and upregulation of Glutamate Cysteine Ligase, the rate-limiting enzyme of glutathione synthesis, while NF-κB and HIF activation became blunted by rutin treatment. Furthermore, rutin was found to prevent hydrogen peroxide from impairing relaxation of human chorionic plate placental vessels, which may help to protect endothelial function. CONCLUSION AND SIGNIFICANCE: Rutin functions as an antioxidant and is oxidized into a quinone that upregulates the Nrf2-mediated endogenous antioxidant response. This mechanism suggests that rutin selectively exerts its protective effects in regions with increased oxidative stress, and explains how rutin reduces the risk of developing CVD. GENERAL SIGNIFICANCE: The newly found mechanism behind the long-term protection of rutin against cardiovascular disease, the selective upregulation of endogenous antioxidant systems, contributes to the further understanding why rutin can reduce the risk on developing cardiovascular disease.

Withaferin A induces heme oxygenase (HO-1) expression in endothelial cells via activation of the Keap1/Nrf2 pathway.

Withaferin A (WA), a natural phytochemical derived from the plant Withania somnifera, is a well-studied bioactive compound exerting a broad spectrum of health promoting effects. To gain better insight in the potential therapeutic capacity of WA, we evaluated the transcriptional effects of WA on primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line (EA.hy926). RNA microarray analysis of WA treated HUVEC cells demonstrated increased expression of the antioxidant gene heme oxygenase (HO-1). Transcriptional regulation of this gene is strongly dependent on the transcription factor NF-E2-related factor 2 (Nrf2), which senses chemical changes in the cell and coordinates transcriptional responses to maintain chemical homeostasis via expression of antioxidant genes and cytoprotective Phase II detoxifying enzymes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein controlling the half-life of Nrf2 via constant proteasomal degradation. In this study we demonstrate that WA time- and concentration-dependently induces HO-1 expression in endothelial cells via upregulation and increased nuclear translocation of Nrf2. According to the crucial negative regulatory role of Keap1 in Nrf2 expression levels, a direct interaction of WA with Keap1 could be demonstrated. In vitro and in silico evaluations suggest that specific cysteine residues in Keap1 might be involved in the interaction with WA.

The contribution of the major metabolite 4'-O-methylmonoHER to the antioxidant activity of the flavonoid monoHER.

The antioxidant flavonoid 7-mono-O-(ß-hydroxyethyl)-rutoside (monoHER) effectively protects against doxorubicin-induced cardiotoxicity in mice. Doxorubicin is a very effective anticancer drug. The clinical use of doxorubicin is limited by severe cardiotoxicity. Free radicals, i.e., hydroxyl and superoxide radicals play a crucial role in this toxicity. In this study the involvement of the major metabolite of monoHER, 4'-O-methylmonoHER (methylmonoHER) in the protective effect of monoHER is studied. MethylmonoHER displayed antioxidant activity i.e., TEAC, hydroxyl and superoxide radical scavenging activity; nevertheless monoHER appeared to be superior compared to methylmonoHER. As a result of scavenging, flavonoids are oxidized and display reactivity towards thiols. Oxidized methylmonoHER, is far less thiol reactive towards creatine kinase than monoHER, which indicates that methylmonoHER is less toxic towards thiol containing enzymes. The thiol-reactivity of oxidized methylmonoHER was also negligible towards KEAP1 compared to monoHER. These results indicate that methylmonoHER hardly protects against radical damage via scavenging or via activating the NRF2 defense system. Also in HUVECs, methylmonoHER provided far less protection against oxidative stress (EC50>100µM) than monoHER which was a very potent protector (EC50=80nM). The results indicate that the contribution of methylmonoHER to the protection against doxorubicin-induced cardiotoxicity by monoHER is relatively low.

The flavonoid monoHER promotes the adaption to oxidative stress during the onset of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease. An evidence-based pharmacological treatment for NAFLD is still lacking, but flavonoids have shown therapeutic potential. The present study was designed to investigate the effect of the flavonoid monoHER on the onset of NAFLD in Ldlr(-/-) mice on a high-fat and high-cholesterol diet. The focus was put on the effect on oxidative stress as well as the adaptive response. Wild type mice served as a control and the effect of monoHER was compared to that of a placebo. In the Ldlr(-/-) group, monoHER provided only a mild protection against oxidative stress. In the placebo Ldlr(-/-) group an adaptive response elicited by the NRF2 antioxidant defense system was observed, evidenced by a higher HO-1 and Gpx3 gene expression, as well as an increased redox status, evidenced by the higher GSH/GSSG ratio. In the monoHER treated Ldlr(-/-) group both the adaptive response as well as the increase in redox status tended to be higher, although this did not reach significance on a group level. Unexpectedly, a strong within animal relationship was found that links a high adaptive response to a low redox status in the monoHER Ldlr(-/-) group. This correlation was absent in the placebo and wild type group. The concept that emerges is that a thiol-reactive oxidation product of monoHER, formed during oxidative stress, selectively induces the NRF2 pathway and enforces the endogenous antioxidant shield, to provide protection against NAFLD.

The minor structural difference between the antioxidants quercetin and 4'O-methylquercetin has a major impact on their selective thiol toxicity.

Antioxidants act as intermediates by picking up the high unselective reactivity of radicals and transferring it to other molecules. In this process the reactivity is reduced and becomes selective. This channeling of the reactivity can cause selective toxicity. The antioxidant quercetin is known to channel the reactivity towards thiol groups. The present study compares the thiol reactivity of quercetin with that of 4'O-methylquercetin (tamarixetin) towards creatine kinase (CK), a vital protein that contains a critical thiol moiety. Our results showed that oxidized quercetin and oxidized tamarixetin both adduct CK, which then loses its enzymatic function. Ascorbate, an important representative of the antioxidant network, is able to prevent adduction to and thus the inhibition of the enzyme by tamarixetin but not by quercetin. Apparently, tamarixetin is less thiol toxic than quercetin, because--rather than adduction to CK--tamarixetin quinone prefers to pass reactivity to the antioxidant network, i.e., to ascorbate. The findings exemplify that radical scavenging flavonoids pick up the reactivity of radicals and act as a pivot in directing the way the reactivity is channeled. A mere minor structural difference of only one methyl moiety between quercetin and tamarixetin appears to have a high impact on the selective, thiol toxicity.

The flavonoid 7-mono-O-(ß-hydroxyethyl)-rutoside is able to protect endothelial cells by a direct antioxidant effect.

The flavonoid 7-mono-O-(ß-hydroxyethyl)-rutoside (monoHER) is an effective protector against doxorubicin induced toxicity which has been related to the antioxidant activity of monoHER. The present study examines the potential relevance of the direct scavenging activity of the flavonoid. The potency of the direct antioxidant effect was confirmed by its instantaneous protection against intracellular oxidative stress in human umbilical vein endothelial cells at therapeutically achievable concentrations (EC50=60 nM) underpinning the involvement of a direct scavenging activity. This direct effect of monoHER is substantiated by (i) its site specific scavenging effect, i.e. on a molecular level monoHER is positioned at the location of radical formation, (ii) its position in the antioxidant network, i.e. on a biochemical level oxidized monoHER quickly reacts with ascorbate or glutathione, (iii) its location in the vascular system, i.e. on a cellular level monoHER is localized in the endothelial and smooth muscle cells in the vascular wall. It is concluded that the flavonoid monoHER can display a physiologically important direct antioxidant effect.