Isobavachalcone attenuates TNF-α-induced ICAM-1 and VCAM-1 expression in human umbilical vein endothelial cells by regulating the NF-κB signaling pathway

Abstract

Vascular inflammation activated by pro-inflammatory cytokines is an inflammatory response that occurs in the early stages of atherosclerosis. Endothelial dysfunction in vascular inflammation begins with the expression of cell surface adhesion molecules by pro-inflammatory cytokines. The purpose of this study was to evaluate and verify the vascular inflammatory effects of isobavachalcone. In this study, we investigated the effects of isobavachalcone on inflammatory responses in vascular inflammation induced by the tumor necrosis factor-α (TNF-α) in human umbilical vein endothelial cells (HUVECs). TNF-α stimulation significantly increased the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) proteins, and concentration-dependently decreased by isobavachalcone in HUVECs. Isobavachalcone suppressed TNF-α-induced ICAM-1 and VCAM-1 expression in HUVECs, thereby inhibiting TNF-α-induced increase in monocyte adhesion. In addition, isobavachalcone decreased the phosphorylation of the NF-κB (necrosis factor κB) p65 subunit. The findings of this study demonstrate that isobavachalcone prevents TNF-α-induced vascular inflammation and has the potential to protect against the early progression of atherosclerosis.

Introduction

Inflammation is a process in which the system that protects the human body from infection and external stimuli is activated. In acute inflammation, the inflammatory reaction disappears within hours or several days and causes symptoms such as redness, swelling, pain, and fatigue, whereas chronic inflammation is a prolonged inflammatory state that can last several months or years. In the inflammatory process of blood vessels, acute inflammation correlates with vasodilation, increased vascular permeability, and the release of tumor necrosis factor-α (TNF-α), interleukins (ILs), and nitric oxide (NO), commonly known as inflammatory cytokines [1]. TNF-α is critical for increasing the expression of adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells, and activates macrophages and neutrophils [2,3,4]. In addition, TNF-α promotes edema by regulating vascular permeability [5, 6].

Isobavachalcone (IBC, Fig. 1) is an active molecule present in the medicinal plant Psoralea corylifolia and has been reported to have antioxidant, anti-inflammatory, antibacterial, and anticancer activities [7,8,9,10]. However, to our knowledge, studies on the protective effects of isobavachalcone on vascular inflammation have not been reported. In this study, we examined whether isobavachalcone decreased the expression of TNF-α-induced ICAM-1 and VCAM-1 in human umbilical vein endothelial cells (HUVECs), and the adhesion of monocytes to the vascular endothelium. We also investigated the signaling mechanism that mediates the suppressive effect of isobavachalcone on adhesion molecule expression in endothelial cells. Our findings suggest that isobavachalcone inhibits TNF-α-stimulated ICAM-1 and VCAM-1 protein expression in HUVECs by regulating the NF-κB signaling pathway.

Fig. 1

Chemical structure of isobavachalcone

Materials and methods

Materials and cell culture

Isobavachalcone was purchased from Sigma-Aldrich (St. Louis, MO, USA). The human umbilical vein endothelial cells (HUVECs) used in this study were purchased from Lonza (Walkersville, MD, USA). HUVECs were cultured in endothelial cell growth basal medium (EBM-2) supplemented with various growth factors at 37 °C and 5% CO₂. The antibodies anti-ICAM-1, anti-VCAM-1 (Santa Cruz Biotechnology, Santa, CA, USA), anti-phospho-p65 (Ser536), anti-IκB-α, anti-phospho-ERK and anti-ERK (Cell Signaling Technology, Beverly, MA, USA), anti-p65 (Upstate Biotechnology, Lake Placid, NY, USA), and anti-β-actin (Sigma-Aldrich, St Louis, MO, USA) were used in this study.

Western blot analysis

Western blot analysis was performed as described previously [11]. Briefly, the protein samples were mixed with 5 × sample buffer, heated for 9 min, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membrane. Chemiluminescent detection was performed using enhanced chemiluminescence detection reagents and analyzed using a ChemiDoc XRS Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). After stripping the membrane, it was confirmed that each group was loaded with the same amount of protein by blotting with an anti-β-actin antibody.

Monocyte adhesion assay

THP-1 cells (American Type Culture Collection, Manassas, VA, USA) were incubated with the CellTracker dye (Invitrogen) for 15 min at 37 °C. Monocyte-endothelial adhesion was determined by fluorescence labeling of monocytes, as described previously [12]. HUVECs were pretreated with isobavachalcone (50 µM) for 30 min and stimulated with TNF-α (10 ng/mL) and isobavachalcone (50 µM) for 4 h. Fluorescence-labeled monocytes were added to the HUVEC monolayer and allowed to adhere for 2 h. The number of monocytes attached to the HUVECs was expressed as the perceived fluorescence intensity.

Statistical analysis

Data are expressed as mean ± SD. Mean comparisons between two groups were examined for significant differences using analysis of variance, followed by individual comparisons using Tukey’s post hoc test, with a P-value < 0.05 indicating a statistically significant difference.

Results

Cell viability of isobavachalcone on HUVECs

Cell viability was measured after treatment with varying concentrations of isobavachalcone, before determining its active concentration, to ensure the cells were non-toxic. The goal of this experiment was to determine the concentration of isobavachalcone that resulted in over 90% cell viability. HUVEC viability showed a slightly increasing trend with increasing concentrations of isobavachalcone from 0.1 to 50 µM (Fig. 2).

Fig. 2

Cell viability assay of HUVECs. The HUVECs were exposed to concentrations of isobavachalcone from 0.1 to 50 µM for 24 h and viability was detected using the CCK-8 assay. Control cells (CB) received vehicle alone. All data are expressed as the mean ± SD of the three independent experiments. *, p < 0.05 vs. CB

Isobavachalcone attenuates the expression of TNF-α-induced ICAM-1 and VCAM-1 protein in HUVECs

We determined whether isobavachalcone affects the protein expression of ICAM-1 and VCAM-1. The HUVECs were pre-incubated with isobavachalcone in a dose-dependent manner (10, 20, and 50 µM) for 30 min, prior to incubation with or without TNF-α for 6 h. Protein expression of ICAM-1 and VCAM-1 increased with TNF-α alone treatment and was significantly downregulated by isobavachalcone (20 and 50 µM) (Fig. 3A and B).

Fig. 3

Isobavachalcone reduces the expression of TNF-α-induced ICAM-1 and VCAM-1 proteins in HUVECs. Immunoblot analysis results are shown for ICAM-1 (A) and VCAM-1 (B) protein expression in TNF-α-stimulated HUVECs co-treated with isobavachalcone. β-Actin protein was used as an internal control. The bar graphs (bottom panel) represent the mean ± SD of three different experiments. ***, p < 0.001 vs. CB; ###, p < 0.001 vs. TNF-α

Isobavachalcone extract inhibits TNF-α-induced NF-κB p65 phosphorylation

TNF-α is a transcription factor in NF-κB signaling responsible for the expression of cell adhesion molecules that increase inflammatory responses [13, 14]. We measured the phosphorylation of p65 of NF-κB to investigate the effect of isobavachalcone on NF-κB activity. TNF-α-induced phosphorylation of the p65 subunit of NF-κB increased approximately 1.7-fold compared to CB alone, but phosphorylation of the p65 subunit of NF-κB was decreased by isobavachalcone (50 μM) (Fig. 4A). The results are expressed as relative ratios (Fig. 4B). IκB-α is known to inhibit NF-κB by masking its nuclear localization signal and sequestering it in an inactive state in the cytoplasm. IκB-α was reduced by TNF-α but recovered by co-treatment with isobavachalcone (50 μM) (Fig. 3C and D). These data demonstrate that isobavachalcone reduces the increase in ICAM-1 and VCAM-1 levels by inhibiting NF-κB phosphorylation and IκB-α degradation activated by TNF-α. Isobavachalcone also showed the effect of inhibiting phosphorylation of ERK, which is known as a representative MAPK pathway (Fig. 4E and F).

Fig. 4

Isobavachalcone decreases phosphorylation of NF-κB p65 in TNF-α-stimulated HUVECs. Immunoblot analysis of phosphorylated NF-κB p65 (A) and degradation of IκB-α (C) in total cell extract were conducted. HUVECs were pretreated with 50 µM isobavachalcone for 30 min and exposed to TNF-α (10 ng/mL) for 4 h (p-p65) and 1 h (IκB-α). Total cell extracts were prepared and analyzed for phosphorylation of p65, total p65, IκB-α and β-Actin. Densitometric analyses results are presented as the relative ratios of phospho-p65 to p65, and IκB-α to β-Actin (B and D). E Expression of phospho-ERK and ERK in TNF-α-stimulated HUVECs co-treated with isobavachalcone for 15 min. (F) Densitometric analyses results are presented as the relative ratios of phospho-ERK to ERK. The bars represent the mean ± SD of three different experiments. ***, p < 0.001 vs. CB; ###, p < 0.001 vs. TNF-α

Isobavachalcone disturbs the adhesion of monocytes to HUVEC monolayers

We investigated whether isobavachalcone reduced monocyte adhesion to HUVECs through TNF-α-stimulated ICAM-1 and VCAM-1 expression. Monocytes attached to HUVECs were identified through fluorescent labeling of the monocytes using a cell tracker dye. HUVECs stimulated with TNF-α (10 ng/mL) showed increased monocyte adhesion (approximately 4.8-fold) compared to those treated with CB, and the group co-treated with isobavachalcone (50 μM) showed a 54% decrease in monocyte adhesion (Fig. 5A and B).

Fig. 5

Isobavachalcone disturbs adhesion of monocytes to HUVEC monolayers. A Monocyte adherence to HUVEC monolayers is represented in the images. HUVECs were incubated with the indicated isobavachalcone (50 µM) and TNF-α (10 ng/mL) for 4 h. Monocytes labeled with a cell tracker were added to the HUVEC monolayer and the degree of adhesion was measured. B Monocyte adhesion to HUVECs was quantified. Bars represent the mean ± SD of three experiments. ***, p < 0.001 vs. CB; ##, p < 0.01 vs. TNF-α

Discussion

Despite recent advances in the understanding of the pathogenesis of vascular diseases, there is limited information on the preventive measures against the development of these diseases. Isobavachalcone is a chalcone isolated from the multipurpose medicinal plant Psoralea corylifolia, which is known to possess antioxidant, antiplatelet, antimicrobial, anti-inflammatory, anti-tumor, and neuroprotective properties. It is a report that isobavachalcone exhibits an anti-inflammatory effect by reducing ICAM-1 in the inflammatory response induced by LPS [15]. In this study, human venous endothelial cells were directly stimulated with TNF-α, which has a single mechanism, rather than LPS, to induce an inflammatory response. We also demonstrated the effect of regulating VCAM-1 involved in adhesion of lymphocytes, monocytes, eosinophils, and basophils to the endothelium [16] as well as ICAM-1 limited to leukocyte-endothelium adhesion. Taken together, we demonstrated that isobavachalcone suppresses TNF-α-induced ICAM-1 and VCAM-1 expression by regulating the NF-κB signaling pathway in cultured HUVECs. These data suggest that isobavachalcone has an anti-vascular inflammatory effect that is mediated by the downregulation of adhesion molecule expression.

Leukocyte adhesion to activated endothelial cells is a key process in the inflammatory response and the development of atherosclerosis. NF-κB is functionally important for the expression of pro-inflammatory genes, including vascular cell adhesion molecules [17, 18]. Isobavachalcone inhibits the adherence of leukocytes to adjacent endothelial cells by reducing the expression of ICAM-1 and VCAM-1. These data suggest that isobavachalcone regulates the rate of leukocyte recruitment at inflammatory sites.

In conclusion, our findings suggest that isobavachalcone suppresses TNF-α-induced ICAM-1 and VCAM-1 protein expression in HUVECs by regulating the NF-κB signaling pathway. These results demonstrated that isobavachalcone prevents TNF-α-induced vascular inflammation and has the potential to protect against inflammatory diseases. Isobavachalcone, is believed to impart beneficial effects and play a pivotal role in vascular diseases, such as hypertension, systemic inflammation, and oxidative stress. In addition, isobavachalcone is expected to prevent the early progression of atherosclerosis.