Effect of Anthocyanin-Rich Extract of Sour Cherry for Hyperglycemia-Induced Inflammatory Response and Impaired Endothelium-Dependent Vasodilation

Abstract

Diabetes mellitus (DM)-related morbidity and mortality are steadily rising worldwide, affecting about half a billion people worldwide. A significant proportion of diabetic cases are in the elderly, which is concerning given the increasing aging population. Proper nutrition is an important component in the effective management of diabetes in the elderly. A plethora of active substances of plant origin exhibit potency to target the pathogenesis of diabetes mellitus. The nutraceutical and pharmaceutical effects of anthocyanins have been extensively studied. In this study, the effect of Hungarian sour cherry, which is rich in anthocyanins, on hyperglycemia-induced endothelial dysfunction was tested using human umbilical cord vein endothelial cells (HUVECs). HUVECs were maintained under both normoglycemic (5 mM) and hyperglycemic (30 mM) conditions with or without two concentrations (1.50 ng/µL) of anthocyanin-rich sour cherry extract. Hyperglycemia-induced oxidative stress and inflammatory response and damaged vasorelaxation processes were investigated by evaluating the level of reactive oxygen species (ROS) and gene expression of four proinflammatory cytokines, namely, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1α (IL-1α), as well as the gene expression of nitric oxide synthase (NOS) endothelin-1 (ET-1) and endothelin-converting enzyme-1 (ECE-1). It was found that hyperglycemia-induced oxidative stress was significantly suppressed by anthocyanin-rich sour cherry extract in a concentration-dependent manner. The gene expression of the tested proinflammatory cytokines increased under hyperglycemic conditions but was significantly reduced by both 1 and 50 ng/µL anthocyanin-rich sour cherry extract. Further, although increased ET-1 and ECE-1 expression due to hyperglycemia was reduced by anthocyanin-rich sour cherry extract, NOS expression was increased by the extract. Collectively, these data suggest that anthocyanin-rich sour cherry extract could alleviate hyperglycemia-induced endothelial dysfunction due to its antioxidant, anti-inflammatory, and vasorelaxant effects.

Keywords: anthocyanins; endothelial dysfunction; hyperglycemia; vasodilation.

Figure 1

Combined use of anthocyanin-rich sour cherry extract and high glucose does not influence viability of human umbilical cord vein endothelial cells (HUVECs). Viability of HUVECs was monitored following 48 h treatment by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) (A) and Nile Red (B) assays. Early necrotic and apoptotic processes of HUVECs were monitored following 48 h treatment by DilC1(5) (C) and SYTOX Green (D) assays. Results are expressed as percentage of untreated control (100%, solid line), with normoglycemia (5 mM) as mean ± SEM of four independent determinations. * and ** mark significant (p < 0.05 and p < 0.01, respectively) differences compared to the vehicle control group.

Figure 2

Anthocyanin-rich sour cherry extract decreases production of reactive oxygen species (ROS) under hyperglycemic condition. Production of ROS by HUVECs was monitored following 48 h treatment by 2′,7′-dichlorofluorescin diacetate (DCFDA) staining. Results are expressed as the percentage of untreated control (100%, solid line) with normoglycemia (5 mM) as mean ± SEM of four independent determinations. * marks a statistically significant difference (p < 0.05) compared to the vehicle control group. ^#, ^## marks significant (p < 0.05, p < 0.001) differences as indicated compared to the untreated control with hyperglycemia (30 mM).

Figure 3

The anti-inflammatory action of anthocyanin-rich sour cherry extract. qPCR analyses of gene expression of tumor necrosis factor-alpha (TNF-α) (A), interleukin-6 (IL-6) (B), interleukin-8 (IL-8) (C), and interleukin-1α (IL-1α) (D) on HUVECs following the indicated seven days of simultaneous treatment. Data are presented using the ΔΔCT method regarding glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-normalized mRNA expressions of the untreated control (100%, solid line) with normoglycemia (5 mM) mean ± SD of two independent determinations. * and *** mark significant (p < 0.05 and p < 0.001, respectively) differences as indicated compared to the untreated control with normoglycemia (5 mM). ^### marks significant (p < 0.001) differences as indicated compared to the untreated control with hyperglycemia (30 mM).

Figure 4

Vasorelaxant effect of anthocyanin-rich sour cherry extract. qPCR analyses of gene expression of endothelin-converting enzyme-1 (ECE-1) (A), endothelin-1 (ET-1) (B), and nitric oxide synthase (NOS) (C) on HUVECs following the indicated seven days of simultaneous treatment. Data are presented using the ΔΔCT method regarding GAPDH-normalized mRNA expressions of the untreated control (100%, solid line) with normoglycemia (5 mm) mean ± SD of two independent determinations. ** and *** mark significant (p < 0.01 and p < 0.001, respectively) differences as indicated compared to the untreated control with normoglycemia (5 mM). ^## and ^### mark significant (p < 0.01 and p < 0.001, respectively) differences as indicated compared to the untreated control with hyperglycemia (30 mM).