1. EGCG protects endothelial cells against PCB 126-induced inflammation
EGCG prevents PCB-induced endothelial cell inflammation via epigenetic modifications of NF-κB target genes
Dandan Liu1 , Michael C. Petriello2, Jordan T. Perkins2 , and Bernhard Hennig2
1Department of Biochemistry, University of Kentucky, Lexington KY
2University of Kentucky Superfund Research Center, Lexington, KY
Abstract
Highlights of Results
EGCG attenuates PCB 126-mediated induction of NF-κB-regulated inflammatory genes
Discussion
Figure 5. The mRNA level of inflammatory genes was analyzed using qRT-PCR. Endothelial cells were pretreated with 15 or 30 μM of EGCG for 8 h, and followed by treatment in fresh medium with 0.03 nM of PCB 126 for 16 h.
EGCG reduced PCB 126-induced expression of p65 and suppressed NF-κB signaling by preventing PCB 126-induced p65 nuclear translocation.
EGCG down-regulated the expression of proatherogenic cytokines and adhesion molecules to protect endothelial cells from PCB 126-mediated vascular inflammation.
EGCG prevented vascular inflammation through exchange of HAT and HDAC chromatin recruitment and histone hypo-acetylation, which decreases expression of NF-κB-regulated genes.
EGCG significantly attenuated PCB 126-mediated induction of proatherogenic cytokines and adhesion molecules such as IL-6, CRP, ICAM-1, VCAM-1, and IL-1α/β in human endothelial cells.
EGCG pretreatment prevented PCB 126-induced NF-κB activation by greatly suppressing the increased p65 nuclear translocation.
EGCG modulated HAT and HDAC chromatin recruitment leading to histone hypo-acetylation in downstream target genes. Histone hypo-acetylation creates transcriptionally repressed chromatin structure and causes deactivation of downstream genes.
Consuming diets high in bioactive polyphenols may be a sensible means of preventing pollutant-induced vascular inflammation and atherosclerosis.
Acknowledgments
Research reported was supported by NIEHS/NIH grant P42ES The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. Graduate stipend was provided by AHA.
IL-6 mRNA (arbitrary units)
CRP mRNA (arbitrary units)
ICAM-1 mRNA (arbitrary units)
IL-1α mRNA (arbitrary units)
VCAM-1 mRNA (arbitrary units)
IL-1β mRNA (arbitrary units) #
0.0825 ^ *
EGCG decreases PCB 126-induced p65 expression and its nuclear translocation
p65 mRNA (arbitrary units)
0.0784
DAPI
p65
PCB 126
/EGCG
TNF-α A C B
p-IκB-α
p-IκB-β
DMSO
Actin
IKK-α/β
E15
E30
PCB
PCB/E15
PCB/E30
Figure 4. (A) EGCG decreased PCB 126-induced p65 expression. Cellular p65 mRNA level was measured in endothelial cells which were pre-treated with 15 or 30 μM of EGCG for 8 h, and then treated with 0.03 nM of PCB 126 for 16 h. (B) EGCG decreased PCB 126-mediated p65 nuclear translocation. The intracellular localization of endogenous p65 was analyzed by immunocytochemical staining using anti-p65 (green) and the nucleus was visualized by DAPI staining (blue).
EGCG may be protective by modulating inflammatory pathways regulated through NF-κB signaling
EGCG protects endothelial cells against
PCB 126-induced inflammation
Figure 2. A: PCB 126-induced oxidative stress is modulated by green tea extract (GTE) diet supplementation. Plasma F2-isoprostane and metabolite levels were measured by HPLC/MS MS
B: Relative mRNA levels of representative antioxidant enzyme markers. Overall GTE supplementation did not significantly increase antioxidant mRNA levels in control diets, but, in the presence of environmental perturbation (i.e. PCB 126 gavage), significantly higher antioxidant levels were seen in mouse liver above non-supplemented diet A B
Figure 1. A-C: Endothelial cells pretreated with EGCG were protected from PCB-126 induced upregulation of inflammatory markers MCP1 and VCAM-1 as well as Cyp1A1 (Marker of Aryl hydrocarbon receptor activation).
D: EGCG treatment also increased the expression of GST and NQO1 (Antioxidant genes controlled by Nrf2 signaling pathway).. C D
EGCG has been reported to inhibit NF-κB activation. We hypothesize that EGCG can protect against PCB-induced endothelial inflammation in part through epigenetic regulation of NF-kB-regulated inflammatory genes.
Figure 3. Epigenetics is a broad term referring to cellular control mechanisms that alter expression of genes without changing our DNA sequences. These changes, in principle, are heritable to the next generation.
Anti-inflammatory polyphenols, such as epigallocatechin-3-gallate (EGCG), have been shown to protect against the toxicity of environmental pollutants. We recently have demonstrated that diets enriched with green tea extracts protect against PCB toxicity in mice, which might involve epigenetic regulation. It is well known that bioactive nutrients such as polyphenols may exert their protection by modulating inflammatory pathways regulated through nuclear factor-kappa B (NF-κB) signaling. EGCG has been reported to have anti-NF-κB transactivation activity. We hypothesize that EGCG can protect against PCB-induced endothelial inflammation in part through epigenetic regulation of NF-kB-regulated inflammatory genes. In order to test this hypothesis, vascular endothelial cells were exposed to physiologically relevant levels of coplanar PCB 126 and/or EGCG, followed by quantification of NF-kB subunit p65, histone acetyltransferase (HAT) p300 and histone deacetylases (HDACs) 1-3 accumulation through ChIP assay in the promotor region of inflammatory genes. In addition, the enrichment of the acetylated H3 (ac-H3) was also quantified. Exposure to PCB 126 increased the expression of vascular inflammatory mediators, including interleukin (IL)-6, C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and IL-1α/β, which was prevented by pre-treatment with EGCG. This inhibitory effect by EGCG correlated with abolished nuclear import of p65, decreased chromatin binding of p65 and p300, as well as increased chromatin binding of HDAC1/2. Furthermore, EGCG induced hypoacetylation of H3, which accounts for deactivation of downstream genes. These data suggest that EGCG-induced epigenetic modifications can decrease vascular endothelial toxicity of PCB 126. (NIH/NIEHS P42ES007380)
Mice fed a diet high in green tea polyphenols exhibit increased antioxidant capabilities
Methods
Immunocytochemistry: Endothelial cells were seeded in chamber slides and pre-treated with EGCG for 8 h, followed by treatment with PCB 126 in fresh medium for 8 h. Cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.25% Triton. Cells were first incubated with mouse monoclonal anti-p65 overnight at 4°C and then with Alexa Fluor-labeled secondary antibody for 1 h at room temperature in the dark. The nucleus were stained with 4, 6-diamidino-2-phenylindole (DAPI), and then images were viewed under the fluorescence microscope.
Chromatin immunoprecipitation: Sheared chromatin was immuneprecipitated with specific antibodies to p65, p300, HDAC1, HDAC2, HDAC3, rabbit IgG, ac-H3, and H3. Non-specific IgG was used as a negative control and produced signals much less than those produced by target-specific antibodies. H3 was used to normalize for ac-H3 occupancy. The promoter regulating the expression of GAPDH localizes in euchromatin and was used as a control locus.
Real Time PCR: Cells were grown in 6-well plates, and total RNA was extracted from the cells using TRIzol reagent (Life Technologies). Reverse transcription was performed using the AMV reverse transcription system (Promega). The levels of mRNAs expression were then assessed by real-time PCR using a Biorad CFX96 instrument and SYBR Green master mix. GAPDH was used for normalization.
Animal Study: Forty C57BL/6 mice were purchased from Jackson Laboratory and evenly assigned to the following experimental groups: control diet (10% kcal as fat) +
vehicle, control+1% green tea extract (GTE) + vehicle, control + PCB 126, control+1% GTE +
PCB Sunphenon 30S-O organic green tea extract
containing was obtained from Taiyo International Inc. and incorporated into the control diet formulationGreen tea extract treatment amounts per body weight coincide with approximately 4 cups of tea (~200 ml/cup) per day in humans. Mice were fed the control and GTE-supplemented diets for 12 weeks and were gavaged with PCB 126 (5
μmol/kg mouse) or vehicle (stripped corn oil) on
weeks 10, 11, and 12 (Newsome et. al. 2014).
What is this buzzword “Epigenetics?”