Anti-Inflammatory Effect of the Blueberry Anthocyanins Malvidin-3-Glucoside and Malvidin-3-Galactoside in Endothelial Cells

Blueberry fruits have a wide range of health benefits because of their abundant anthocyanins, which are natural antioxidants. The purpose of this study was to investigate the inhibitory effect of blueberry’s two main anthocyanins (malvidin-3-glucoside and malvidin-3-galactoside) on inflammatory response in endothelial cells. These two malvidin glycosides could inhibit tumor necrosis factor-alpha (TNF-α) induced increases of monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) production both in the protein and mRNA levels in a concentration-dependent manner. Mv-3-glc at the concentration of 1 μM could inhibit 35.9% increased MCP-1, 54.4% ICAM-1, and 44.7% VCAM-1 protein in supernatant, as well as 9.88% MCP-1 and 48.6% ICAM-1 mRNA expression (p < 0.05). In addition, they could decrease IκBα degradation (Mv-3-glc, Mv-3-gal, and their mixture at the concentration of 50 μM had the inhibition rate of 84.8%, 75.3%, and 43.2%, respectively, p < 0.01) and block the nuclear translocation of p65, which suggested their anti-inflammation mechanism was mediated by the nuclear factor-kappa B (NF-κB) pathway. In general malvidin-3-glucoside had better anti-inflammatory effect than malvidin-3-galactoside. These results indicated that blueberry is good resource of anti-inflammatory anthocyanins, which can be promising molecules for the development of nutraceuticals to prevent chronic inflammation in many diseases.

present study, we further investigated the anti-inflammatory properties of Mv-3-glc and Mv-3-gal, as well as their synergistic effect in human vascular umbilical endothelial cells.

Effects of Mv-3-glc and Mv-3-gal on TNF-α-Induced IκB Degradation
In this study, the amount of IκBα was greatly reduced after exposure to 10 μg/L TNF-α (p < 0.01). The inhibitory effect of Mv-3-glc, Mv-3-gal and their mixture at different concentrations on TNF-α-induced expression of IκBα degradation were all significant. They also were in the concentration-dependent manner. Mv-3-glc always exhibited better inhibitory capacity than Mv-3-gal in all the four concentrations. Mv-3-glc and Mv-3-gal mixture at the concentration of 10 μM and 50 μM had the inhibition rate of 78.0% and 84.8%, respectively, which were more than those of Mv-3-glc (63.7% and 75.3%) or Mv-3-gal (34.1% and 43.2%). Mv-3-glc and the mixture at the concentration of 100 μM could completely inhibit the IκBα degradation (p < 0.01), since the IκBα expression was even more than the control (Figure 4).

Effects of Mv-3-glc and Mv-3-gal on TNF-α-Induced NF-κB Translocation
On activation of the NF-κB pathway, the p65 protein is released from the cytosol and migrates into the cell nucleus where it interacts with the promoter regions of various proteins which up-regulated in inflammation [21]. Immunocytochemistry was performed by using NF-κB and fluorescein isothiocyanate (FITC)-conjugated antibody. In un-stimulated cells, the levels of p65 in the nucleus were very low. Upon simulation of TNF-α, the levels of p65 in the nucleus were increased. On the other hand, upon treatment of the cells with Mv-3-glc, Mv-3-gal, and their mixture, the fluorescence intensity levels of p65 were decreased in the nucleus ( Figure 5). The high concentration of Mv-3-glc, Mv-3-gal, and their mixture inhibited the nuclear translocation of p65, with the p65 protein level in the cell nucleus similar to the control.

Discussion
Anthocyanins are one of the largest and most important groups of water-soluble pigments in most fruits. Berries, as colored fruits, are highly chemoprotective because of their bioactive anthocyanins [22,23]. Different anthocyanins offer different antioxidant capacity. A theoretical study evaluated the antioxidant character of three widespread anthocyanidins (cyanidin, delphinidin, and malvidin) [24] according to different parameters (bond dissociation enthalpy, ionization potential, proton affinity, and electron transfer enthalpy) and the atomic charges corresponding to the O atoms of the hydroxyl groups. It is found that antioxidant effect of anthocyanins is based on the free radical scavenging by means of the OH groups. Wild lowbush blueberries (Vaccinium angustifolium Ait) are a rich source of anthocyanins and other flavonoids with anti-inflammatory activities, in which malvidin-3-glucoside was significantly more effective than epicatechin or chlorogenic acid in reducing the expression of pro-inflammatory genes in vitro [17]. In our studied Rabbiteye blueberry (V. ashei), Mv-3-glc and Mv-3-gal which are the two main glycoside forms of malvidin were the most abundant anthocyanins [15,25]. Malvidin possesses great antioxidant activity, and cytotoxicity against human monocytic leukemia cells and HT-29 colon cancer cells [26,27], and anti-hypertensive activity by inhibiting angiotensin I-converting enzyme (ACE) [28]. Our previous study reported the inhibitory effect of malvidin on TNF-α induced inflammatory response [16]. This study showed that Mv-3-glc and Mv-3-gal also possessed potential anti-inflammatory capacity. Mv-3-glc had better inhibitory effect than Mv-3-gal, indicating the differences in the two glycosides' effects. In addition, Mv-3-glc and Mv-3-gal sometimes showed additive effects. An isobologram could be constructed in the future over a range of concentrations to verify whether they have synergy or not. Previous studies confirmed that Mv-3-glc could increase NO bioavailability, as well as inhibit peroxynitrite-induced NF-κB activation, which supported its benefits in cardiovascular health. A treatment of cyanidin 3-rutinoside and cyanidin 3-glucoside from the berry Morus alba L. also resulted in an inhibition on the activation of c-Jun and NF-κB, therefore it could decrease the in vitro invasiveness of cancer cells [29]. The anti-invasive activity on human colon cancer cells of the anthocyanins from fruits of Vitis coignetiae Pulliat was associated with modulation of constitutive NF-κB activation through suppression IκBα phosphorylation [30]. Lipopolysaccharide-induced NF-κB p65 translocation to the nucleus was markedly attenuated by anthocyanins of blueberry, blackberry, and blackcurrant, which mostly included malvidin-3-glucoside, cyanidin-3-glucoside and delphinidin-3-rutinoside. Their anti-inflammatory effects in macrophages were relative to their antioxidant capacity [31], so the antioxidant capacity made anthocyanins promising to develop nutraceuticals to improve endothelial function [32]. These indicate the anthocyanins could be the potential alternatives to prevent and treat the inflammation in many diseases [7]. Recent reports indicated action mechanisms of protecting vascular endothelium including modulation of crucial signaling pathways and gene regulation [33,34]. The present study showed that Mv-3-glc and Mv-3-gal also possessed anti-inflammatory properties by NF-κB pathway in endothelial cells.
Chronic inflammation is a common factor linking various pathologies in many diseases such as atherosclerosis and cancer. Vascular inflammation is a complex process, including the accumulation and activation of immune suppressor cells, pro-inflammatory cytokines, chemokines, growth and angiogenic factors and activation of several inflammatory signaling pathways mediated predominantly by NF-κB transcription factors [7]. The transcriptional activation of NF-κB plays a key role in the development of the inflammatory response [35]. It is well established that NF-κB is normally in an inactive form bound to inhibitory proteins, the IκBs. Exposed to external stimuli such as TNF-α, IKB kinase (IKK) phosphorylates IκBα, which lead to ubiquitination-dependent degradation of IκBα [36]. It is well accepted that the activation of NF-κB in endothelial cells is associated with mononuclear cell infiltration and an increased transcription of adhesion molecules, chemokines, and cytokines [21,37]. TNF-α can activate NF-κB, and then MCP-1, ICAM-1, and VCAM-1 were over expressed in vascular endothelial cells [38,39]. In this study, Mv-3-glc and Mv-3-gal inhibited TNF-α-induced MCP-1, ICAM-1 and VCAM-1 production as well as IκBα degradation. In addition, Mv-3-glc and Mv-3-gal were able to inhibit the nuclear translocation of p65, one subunit of NF-κB, suggesting the mechanism by which Mv-3-glc and Mv-3-gal could block pro-inflammatory signaling downstream of IκB.

Antibodies
Rabbit monoclonal primary antibody against ICAM-1, rabbit polyclonal primary antibodies against VCAM-1, mouse polyclonal primary antibody to the β-Actin antibody, and goat anti-rabbit/mouse HRP-conjugated secondary antibody, were purchased from Boster Biotechnology Inc. Rabbit monoclonal primary antibody to IκBα was bought from Beyotine Technology Inc. Primary antibodies were used at 1:200 dilutions, and secondary antibodies were used at 1:1000 dilutions.

Endothelial Cell Culture and Treatment
Human umbilical vein endothelial cells (HUVECs) are a representative model system for studying inflammation and oxidative stress in the vasculature [40]. HUVECs were saved in National Technical Research Centre of Veterinary Biological Products (Nanjing, China). The second to 6th passage cells were used for all experiments at 80%-90% confluence. HUVECs were quiesced in a reduced serum medium for 4 h prior to experiment. In a separate set of experiments, the cells were treated with 1, 10, 50, and 100 μM Mv-glc, Mv-gal, or their mixture for 18 h, followed by TNF-α (10 μg/L) stimulation for 6 h. DMSO was used as control. The supernatants were collected for ELISA analysis. The cells were prepared for western blotting.

ELISA Analysis and Western Blotting
The levels of MCP-1, ICAM-1, and VCAM-1 in the supernatants were quantified using ELISA kits. The assay procedure was employed according to the kit protocol booklet instructions. The absorbance of the resulting yellow color was measured at 450 nm on a StatFax-2100 Microplate Reader (Awareness Technology Inc., Palm City, FL, USA). The reader was controlled via Hyper Terminal Applet ELISA software. Western blotting was performed on the HUVEC lysates as described before [41]. Beside ICAM-1 and VCAM-1, IκBα was also analyzed by western blotting. Data were normalized by re-probing the membrane with an antibody against β-Actin which was used as a loading control.

Real-Time qRT-PCR
The total RNA was isolated from HUVECs using the Trizol reagent (TaKaRa Bio Inc.). It was reverse-transcribed into cDNA using PrimeScript RT master mix. For reverse-transcription, the SYBR Green 2-step qRT-PCR kit (TaKaRa Bio Inc.) was used. The real-time quantitative PCR analysis was carried out using the LightCycler 480 (Roche Diagnostics Inc., Rotkreuz, Switzerland). The primers for amplification were followed: forward primer 5-GTTGTCCCAAAGAAGCTGTGA-3 and reverse primer 5-AATCCGAACCCACTTCTGC-3 for MCP-1 (83 bp); forward primer 5-CCACAGTCACCTATGGCAAC-3 and reverse primer 5-AGTGTCTCCTGGCTCTGGTT-3 for ICAM-1 (124 bp); forward primer 5-TGGACTTCGAGCAAGAGATG-3 and reverse primer 5-GAAGGAAGGCTGGAAGAGTG-3 for β-actin (137 bp). The reaction was conducted with an initial denaturing at 94 °C for 30 s, then involved 40 cycles of 60 °C for 20 s, and at 65 °C for 15 s in the end. Relative gene expression data was analyzed using the 2 −ΔΔCt method.

Immunofluorescence
Nuclear translocation of p65 is widely used as a measure for NF-κB activation. HUVECs were fixed in 3.75% paraformaldehyde, and permeabilized with 0.1% Triton-X-100 incubated overnight with primary antibody against p65. On the following day, the cells were incubated with secondary antibody (goat anti-rabbit conjugated with FTTC) for 1 h. The cell nuclei were stained with DAPI (4',6-diamidino-2-phenylindole), and visualized under an Axiovision 4 Fluorescent Microscope (Zeiss, Oberkochen, Germany). All images presented are in (×100) magnification.

Statistical Analysis
All data presented are mean value ± standard deviation (SD). The data were analyzed by a one-way ANOVA using the SPSS 19.0 Statistical Software. Differences were considered significant with p value < 0.05.

Conclusions
In the present study, treatment with Mv-3-glc, Mv-3-gal, and their mixture significantly attenuated monocyte adhesion in TNF-α-stimulated HUVECs by inhibiting MCP-1, ICAM-1, and VCAM-1 protein and mRNA expressions both in endothelial cell and supernatants. In addition, they affected IκBα degradation and the nuclear translocation of p65, indicating that they possessed anti-inflammatory effects by blocking the NF-κB pathway mechanism. Mv-3-glc, Mv-3-gal, and their mixture all showed their inhibitory effects on TNF-α-induced inflammatory response in a concentration-dependent manner. Mv-3-glc had better potential anti-inflammatory effect than Mv-3-gal, and they showed synergistic effect sometimes. Blueberries are a good source of anthocyanins such as Mv-3-glc and Mv-3-gal, which can be a promising molecules for the development of nutraceuticals to improve endothelial function and thereby to prevent the progression of chronic inflammation.