EGCG protects endothelial cells against PCB 126-induced inflammation through inhibition of AhR and induction of Nrf2-regulated genes

Abstract

Tea flavonoids such as epigallocatechin gallate (EGCG) protect against vascular diseases such as atherosclerosis via their antioxidant and anti-inflammatory functions. Persistent and widespread environmental pollutants, including polychlorinated biphenyls (PCB), can induce oxidative stress and inflammation in vascular endothelial cells. Even though PCBs are no longer produced, they are still detected in human blood and tissues and thus considered a risk for vascular dysfunction. We hypothesized that EGCG can protect endothelial cells against PCB-induced cell damage via its antioxidant and anti-inflammatory properties. To test this hypothesis, primary vascular endothelial cells were pretreated with EGCG, followed by exposure to the coplanar PCB 126. Exposure to PCB 126 significantly increased cytochrome P450 1A1 (Cyp1A1) mRNA and protein expression and superoxide production, events which were significantly attenuated following pretreatment with EGCG. Similarly, EGCG also reduced DNA binding of NF-κB and downstream expression of inflammatory markers such as monocyte chemotactic protein-1 (MCP-1) and vascular cell adhesion protein-1 (VCAM-1) after PCB exposure. Furthermore, EGCG decreased endogenous or base-line levels of Cyp1A1, MCP-1 and VCAM-1 in endothelial cells. Most of all, treatment of EGCG upregulated expression of NF-E2-related factor 2 (Nrf2)-controlled antioxidant genes, including glutathione S transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), in a dose-dependent manner. In contrast, silencing of Nrf2 increased Cyp1A1, MCP-1 and VCAM-1 and decreased GST and NQO1 expression, respectively. These data suggest that EGCG can inhibit AhR regulated genes and induce Nrf2-regulated antioxidant enzymes, thus providing protection against PCB-induced inflammatory responses in endothelial cells.

Figure 1

EGCG attenuates PCB 126-mediated induction of Cyp1A1 and cellular oxidative stress. (A) Expression of mRNA was analyzed in endothelial cells pretreated with 25–50 µM of EGCG for 3 h, followed by treatment with PCB 126 at 0.25 µM for 4 h. Real-time PCR technique was used to measure expressed mRNA levels. (B) Expression of CYP1A1 protein in endothelial cells pretreated with 0–50 µM of EGCG for 3 h, followed by treatment with PCB 126 for 4 h using Western blot technique. Densitometry results were normalized to β-actin. The Western blot picture shown is a representative of three independent blots. Real-time PCR and Western blot results represent the mean ± SEM, with n=3. (C) Superoxide production in endothelial cells using DHE staining. DHE red fluorescence was assessed using fluorescence microscopy and the strength of signal was quantified. Experiments were repeated a minimum of three times. *Significantly increased compared to DMSO control. ^‡Significantly decreased compared to DMSO control. ^# Significantly different compared to the PCB treatment group.

Figure 2

EGCG attenuates PCB 126-induced activation of NF-κB and AhR. (A) Endothelial cells were pretreated with EGCG (25–50 µM) for 3 h, followed by PCB 126 exposure at 0.25 µM for 3 h. EMSA for NF-κB was performed with nuclear proteins extracted from endothelial cells. EMSA competition assay demonstrated the specificity of NF-κB probe using a competitor (unlabeled oligonucleotide probe), and a mutator (unlabelled probe with a mutation) and antibodies. Figure 2A: Lane 1, DMSO; lane 2, PCB 126; lane 3, EGCG 25 µM; lane 4, EGCG 25 µM/PCB 126; lane 5, EGCG 50 µM; lane 6, EGCG 50 µM/PCB 126; lane 7, PCB 126; lane 8, PCB and competitor; lane 9, PCB and mutator; lane 10, PCB and p50 antibody; lane 11, PCB and p65 antibody; lane 12, PCB and c-Rel antibody; lane 12, PCB and p300 antibody. (B) Endothelial cells were pretreated with EGCG (25–50 µM) for 3 h, followed by PCB 126 exposure at 0.25 µM for 6 h. EMSA for AhR was performed with nuclear proteins extracted from endothelial cells. EMSA competition assay showed the specificity of AhR probe using a competitor and a mutator and antibodies. Figure 2B: Lane 1, DMSO; lane 2, PCB 126; lane 3, EGCG 25 µM; lane 4, EGCG 25 µM/PCB 126; lane 5, EGCG 50 µM; lane 6, EGCG 50 µM/PCB 126; lane 7, PCB 126; lane 8, PCB and competitor; lane 9, PCB and mutator; lane 10, PCB and AhR antibody; lane 11, PCB and Myc antibody. Images are representative of three independent experiments, and image splicing was carried out only for the same experiment, same gel and the same exposure times.

Figure 3

PCB 126-induced expression of MCP-1 and VCAM-1, and adhesion of monocytes to endothelial cells is modulated by EGCG. mRNA expression of (A) MCP-1 and (C) VCAM-1 was analyzed in endothelial cells pretreated with 0–50 µM of EGCG for 3 h, followed by treatment with PCB 126 at 0.25 µM for 16 h. Real-time PCR technique was used to measure mRNA levels. Figure 3 shows protein expression of (B) MCP-1 and (D) VCAM-1 in endothelial cells pretreated with 0–50 µM of EGCG for 3 h, followed by treatment with PCB 126 at 0.25 µM for 16 h. MCP-1 protein levels in cell culture media were assessed using ELISA. Western blot was used to detect VCAM-1 protein in whole cell lysates. The Western blot picture shown is a representative of three independent blots. (E) Endothelial cells were pretreated with EGCG (25–50 µM) for 3 h, followed by treatment with PCB 126 at 0.25 µM for 16 h. Human THP-1 monocytes were activated with TNF-α and loaded with the fluorescent probe calcein. Activated and calcein loaded monocytes were added to endothelial monolayers for activation. After washing, adhered monocytes were counted using a fluorescent microscope. Results represent the mean ± SEM, with n=3. Experiments were repeated a minimum of three times. *Significantly increased compared to DMSO control. ^‡Significantly decreased compared to DMSO control. ^#Significantly different compared to PCB 126 treatment group.

Figure 4

EGCG increases expression of the phase II enzymes GST and NQO1. Expression of (A) GST and (B) NQO1 was analyzed in endothelial cells pretreated with 25–50 µM of EGCG for 3 h, followed by treatment with PCB 126 at 0.25 µM for 16 h. Real-time PCR technique was used to measure expressed mRNA levels. Results represent the mean ± SEM, with n=3. Experiments were repeated a minimum of three times. *Significantly different compared to DMSO control.

Figure 5

Nrf2 silencing increases expression of Cyp1A1, VCAM-1 and MCP-1. Primary endothelial cells were treated with Nrf2 siRNA or control siRNA, and then pretreated with 25–50 µM of EGCG for 3 h, followed by treatment with PCB 126 at 0.25 µM for 16 h. Expression of Cyp1A1 (A), MCP-1 (B), VCAM-1 (C), GST (D) and NQO1 (E) were analyzed in cells. Real-time PCR technique was used to measure expressed mRNA levels. Results represent the mean ± SEM, with n=3. Experiments were repeated a minimum of three times. *Significantly different compared to corresponding DMSO control. ^#Significantly different between control siRNA and Nrf2 siRNA within the same treatment.

Figure 6

Schematic illustration of the inhibition of PCB 126-induced endothelial inflammatory responses by EGCG. EGCG reduces oxidative stress through up-regulation of Nrf2-regulated genes, such as phase II enzymes, and down-regulation of AhR-regulated genes, such as Cyp1A1. These cellular events lead to decreased NF-κB signaling, including reduced expression of adhesion molecules.