Kuntz Sabine, Asseburg Heike, Dold Sebastian, Römpp Andreas, Fröhling Bettina, Kunz Clemens, Rudloff Silvia
Department of Pediatrics, Feulgenstrasse 12, University of Giessen, Germany.
Food Funct. 2015 Apr;6(4):1136-49. doi: 10.1039/c4fo00755g.
Anthocyanins (ACNs) are the most prevalent flavonoids in berries and their health promoting effects on vascular functions are still discussed. The aim of the present study was to identify the anti-inflammatory effect of ACNs on activated human umbilical vein endothelial cells (HUVECs) after their transport across an epithelial monolayer.
We established a transwell epithelial-endothelial co-culture system with Caco-2/HT29-B6 cells mimicking the intestinal layer and HUVECs as endothelial cells mimicking the vascular layer. Caco-2 were seeded alone (100%) or together with HT29-B6 cells (10 and 20%) on transwell inserts in order to simulate different metabolization sides of the gut. ACNs as well as malvidin-3-glucoside (M3G) were applied to the luminal compartment of the transwell-system. Transport and degradation rates were determined by high performance liquid chromatography with ultraviolet detection (HPLC-UV) or by ultra-PLC coupled to mass spectrometry (UPLC-MS). After 4 hours incubation time, co-cultured HUVECs were used immediately (short-term incubation) or after 20 hours (long-term incubation). Thereafter, HUVECs were stimulated for 3 hours with 1 ng mL(-1) TNF-α to mimic a low-grade or 10 ng mL(-1) to mimic a high-grade inflammation. Afterwards, (1.) leukocyte adhesion, (2.) expression of cell adhesion molecules (ICAM-1, VCAM-1 and E-selectin) and (3.) cytokine expression and secretion (IL-6 and IL-8) were determined using flow cytometry and real-time PCR.
Degradation and incubation studies revealed that ACNs were differently degraded depending on the ACN structure and the seeding densities. Incubation of ACNs and M3G to Caco-2 cells (100%) led to a fast decrease, which was not observed when HT29-B6 cells were co-cultured (10 and 20%). Concomitantly, anti-inflammatory effects were only observed using 100% Caco-2 cells, whereas mixtures of Caco-2 and HT29-B6 cells failed to induce an effect. ACN extract and M3G significantly attenuated TNF-α-stimulated low-grade leukocyte adhesion, expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 and cytokine expression and secretion (IL-8 and IL-6) as well as NF-κB mRNA expression. No effects were observed with high TNF-α (10 ng mL(-1)) or after short-term incubation (4 hours).
ACNs in physiological concentrations reached the serosal compartment and reduced inflammation-related parameters, which were related to the initial steps during the pathogenesis of atherosclerosis.
花青素（ACNs）是浆果中最常见的黄酮类化合物，其对血管功能的健康促进作用仍在讨论中。本研究的目的是确定ACNs在穿过上皮单层后对活化的人脐静脉内皮细胞（HUVECs）的抗炎作用。
我们建立了一个Transwell上皮-内皮共培养系统，用Caco-2/HT29-B6细胞模拟肠道层，用HUVECs作为内皮细胞模拟血管层。将Caco-2单独接种（100%）或与HT29-B6细胞一起接种（10%和20%）到Transwell小室中，以模拟肠道的不同代谢面。将ACNs以及矢车菊素-3-葡萄糖苷（M3G）应用于Transwell系统的腔室。通过高效液相色谱-紫外检测（HPLC-UV）或超高效液相色谱-质谱联用（UPLC-MS）测定转运和降解率。孵育4小时后，共培养的HUVECs立即使用（短期孵育）或20小时后使用（长期孵育）。此后，用1 ng/mL TNF-α刺激HUVECs 3小时以模拟低度炎症，或用10 ng/mL刺激以模拟高度炎症。之后，使用流式细胞术和实时PCR测定（1）白细胞黏附、（2）细胞黏附分子（ICAM-1、VCAM-1和E-选择素）的表达以及（3）细胞因子的表达和分泌（IL-6和IL-8）。
降解和孵育研究表明，ACNs根据其结构和接种密度的不同而有不同程度的降解。将ACNs和M3G孵育到Caco-2细胞（100%）中导致快速下降，而当与HT29-B6细胞共培养（10%和20%）时未观察到这种情况。同时，仅在使用100% Caco-2细胞时观察到抗炎作用，而Caco-2和HT29-B6细胞的混合物未能诱导出效果。ACN提取物和M3G显著减弱了TNF-α刺激的低度白细胞黏附、黏附分子E-选择素、VCAM-1和ICAM-1的表达以及细胞因子的表达和分泌（IL-8和IL-6）以及NF-κB mRNA的表达。在高浓度TNF-α（10 ng/mL）或短期孵育（4小时）后未观察到效果。
生理浓度的ACNs到达浆膜腔室并降低了与炎症相关的参数，这些参数与动脉粥样硬化发病机制的初始步骤有关。
