P69: Analysis of acute and chronic effects of (?)-epicatechin on NO bioavailability and organ redox state in the cardiovascular system in vivo

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• 作者：Miriam M. Cortese-Krott^a ; Thomas Krenz^a ; Ana Rodriguez-Matheos^a ; ^b ; Friederike Oberle^a ; Kim Weber^a ; David Pullmann^a ; Magalie Haeberlein^a ; Sivatharsini Thasian-Sivarajah^a ; Jeremy Spencer^b ; Malte Kelm^a ; Christian Heiss^a
• 刊名：Nitric Oxide
• 出版年：2013
• 出版时间：15 April, 2013
• 年：2013
• 卷：31
• 期：supp_S1
• 页码：S43
• 全文大小：36 K

文摘

Background and Hypothesis

Consumption of cocoa flavanols may have beneficial effects on cardiovascular health. (?)-Epicatechin, the major flavanol monomer present in cocoa, has been shown to positively affect endothelial function and a (?)-epicatechin-rich cocoa drink increased the circulating nitric oxide (NO) metabolites in humans. There is also evidence that flavanols and flavonoids may activate the expression and the activity of Nrf2-dependent phase II genes in cultured non-endothelial cells. We hypothesized that (?)-epicatechin may induce a systemic Nrf2-dependent antioxidant response, thereby contributing to maintain the redox state, NO bioavailability, and the functionality of the vasculature.

Methods

In vivo effects of acute and chronic oral administration of (?)-epicatechin were analyzed in C57/Bl6 mice. (?)-Epicatechin was administrated at 1-10 mg/kg body weight (BW) intragastrically and the effects analyzed after 1 h. Chronic effects were assessed after 5 days (?)-epicatechin administration of 2 and 10 mg/kg BW in drinking water. Vascular function was assessed by measuring reactive hyperemic response of the hindlimb by laser Doppler perfusion imaging (LDPI). Chronic inhibition of nitric oxide synthase (NOS) was achieved by administration of the specific inhibitor l-NNA in drinking water for 6 days. Circulating levels of (?)-epicatechin metabolites were determined with HPLC. In vitro effects of (?)-epicatechin and its main plasma metabolites present in humans were analyzed in human umbilical vein endothelial cells (HUVECs). The H₂O₂ concentration in media was measured by a peroxide assay. The activation of Nrf2 by epicatechin was measured by immunohistochemistry and confocal microscopy of cells cultured on cover slips, Western blot analysis of nuclear and cytoplasmic extracts, as well as by ARE (antioxidant responsive element)-binding assay (TransAM). The expression of eNOS and of phase II genes was assessed in cells and organs by real time RT-PCR and Western blot analysis. Phosphorylation of eNOS was assessed by Western blot analysis. The concentration of reduced glutathione in mouse organs were assessed by the glutathione reductase recycling assay.

Results

Acute administration of (?)-epicatechin dose-dependently increased the circulating levels of epicatechin metabolites along with improved vascular response to reactive hyperemia of the hindlimb. This was paralleled by an increase in eNOS phosphorylation in the aorta of the mice, indicating that (?)-epicatechin application induces NOS activation. Chronic treatment with (?)-epicatechin improved vascular response in a NOS-dependent fashion, and increased cGMP levels in aorta and plasma of the mice, demonstrating enhanced NO bioavailability. Interestingly, we observed a parallel increase in the expression of Nrf2-dependent phase II genes, including glutamate cysteine ligase (Gclc), and GSH levels in the heart and lung of the mice. In vitro we found that (?)-epicatechin induced the translocation of Nrf2 into the nucleus of the cells and the binding to the ARE. Moreover,(?)-epicatechin and its main plasma metabolites increased expression of GCL (mRNA and protein), as well as eNOS expression and phosphorylation.

Conclusions

Taken together, our findings demonstrate that administration of (?)-epicatechin leads to an increase of systemic NO bioavailability and vascular response in vivo and activates eNOS and Nrf2-dependent pathways.

Disclosure

Nothing to disclose.