Cardiovascular Mechanisms of Action of Anthocyanins May Be Associated with the Impact of Microbial Metabolites on Heme Oxygenase-1 in Vascular Smooth Muscle Cells

Abstract

Anthocyanins are reported to have cardio-protective effects, although their mechanisms of action remain elusive. We aimed to explore the effects of microbial metabolites common to anthocyanins and other flavonoids on vascular smooth muscle heme oxygenase-1 (HO-1) expression. Thirteen phenolic metabolites identified by previous anthocyanin human feeding studies, as well as 28 unique mixtures of metabolites and their known precursor structures were explored for their activity on HO-1 protein expression in rat aortic smooth muscle cells (RASMCs). No phenolic metabolites were active when treated in isolation; however, five mixtures of phenolic metabolites significantly increased HO-1 protein expression (127.4-116.6%, p ≤ 0.03). The present study demonstrates that phenolic metabolites of anthocyanins differentially affect HO-1 activity, often having additive, synergistic or nullifying effects.

Keywords: anthocyanin; antioxidant; atherosclerosis; metabolism; smooth muscle cells.

Figure 1

Structures of (A) flavonoids and (B) metabolites included in treatments. OH, hydroxyl; Glc, oxygen-linked-glucuronide; OGlu, oxygen-linked glycoside; Sul, sulfate; OCH₃, oxygen-linked methyl group.

Figure 2

Effect of 10 µM flavonoids and phenolic acid metabolites on HO-1 protein expression in RASMCs. (A) precursor flavonoids; (B) benzoic acid metabolites; (C) protocatechuic acid metabolites; and (D) vanillic acid metabolites. Data are presented as a percentage of an untreated control (media only). Filled columns represent vehicle control (0.02% DMSO) and clear columns represent treatments (10 µM). All columns are representative of the mean ± SD, n = 3 independent samples, ** p ≤ 0.01, *** p ≤ 0.001 (ANOVA with post hoc Dunnett relative to vehicle control (0.02% DMSO)). 4HBA, 4-hydroxybenzoic acid; BA4G, benzoic acid-4-O-glucuronide; BA4S, benzoic acid-4-sulfate; C3G, cyanidin-3-O-glucoside; EPI, (−)-epicatechin; HES, hesperetin; IVA, isovanillic acid; IVA3G, isovanillic acid-3-O-glucuronide; IVA3S, isovanillic acid-3-sulfate; NAR, naringenin; P3G, peonidin-3-O-glucoside; PCA, protocatechuic acid; PCA3G, protocatechuic acid-3-O-glucuronide; PCA4G, protocatechuic acid-4-O-glucuronide; PCA3S, protocatechuic acid-3-sulfate; PCA4S, protocatechuic acid-4-sulfate; QUE, quercetin; VA, vanillic acid; VA4G, vanillic acid-4-O-glucuronide; VA4S, vanillic acid-4-sulfate.

Figure 3

Effect of 10 µM mixtures of flavonoids and phenolic acid metabolites on HO-1 protein expression in RASMCs. (A) precursor flavonoids; (B) phenolic acids; (C) benzoic acid metabolites; (D) protocatechuic acid metabolites; (E) vanillic acid metabolites; and (F) all metabolites. Data are presented as a percentage of an untreated control (media only). Filled columns represent vehicle control (0.02% DMSO) and clear columns represent treatments (10 µM). All columns are representative of the mean ± SD, n = 3 independent samples. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (ANOVA with post hoc Dunnett relative to vehicle control (0.02% DMSO)). ‘ALL’ is a mixture composed of 13 conjugated and unconjugated phenolic acids at equimolar concentrations to a cumulative concentration of 10 µM. 4HBA, 4-hydroxybenzoic acid; BA4G, benzoic acid-4-O-glucuronide; BA4S, benzoic acid-4-sulfate; C3G, cyanidin-3-O-glucoside; DMSO, dimethyl sulfoxide; EPI, (−)-epicatechin; HES, hesperetin; IVA, isovanillic acid; IVA3G, isovanillic acid-3-O-glucuronide; IVA3S, isovanillic acid-3-sulfate; NAR, naringenin; P3G, peonidin-3-O-glucoside; PCA, protocatechuic acid; PCA3G, protocatechuic acid-3-O-glucuronide; PCA4G, protocatechuic acid-4-O-glucuronide; PCA3S, protocatechuic acid-3-sulfate; PCA4S, protocatechuic acid-4-sulfate; QUE, quercetin; VA, vanillic acid; VA4G, vanillic acid-4-O-glucuronide; VA4S, vanillic acid-4-sulfate.