Abstract

Isoliquiritigenin (ILTG) is a flavonoid with chalcone structure (4,2′,4′-trihydroxychalcone), an active component present in plants like Glycyrrhiza and Dalbergia which showed various biological activities including anti-inflammatory, anti-carcinogenic and antihistamic. As very little is known in regard to the underlying mechanism involved in explaining the various activities of the compound, we carried out a detailed study on the effect of ILTG on the expression of cell adhesion molecules on human primary endothelial cells. We demonstrate here that ILTG inhibits TNF-α induced adhesion of neutrophils to endothelial monolayer by blocking the expression of ICAM-1, VCAM-1 and E-selectin. Since NF-κB is a major transcription factor involved in the transcriptional regulation of cell adhesion molecules, thus we studied the status of NF-κB activation in ILTG treated endothelial cells. We demonstrate that ILTG inhibits the translocation and activation of nuclear factor-κB (NF-κB) by blocking the phosphorylation and subsequent degradation of IκBα. As oxidative stress is also known to regulate the activation of NF-κB to modulate TNF-α signaling cascade, we tested the effect of ILTG on reactive oxygen species (ROS). We found that it inhibits TNF-α induced ROS production in endothelial cells. These results have important implications for using ILTG or its derivatives towards the development of effective anti-inflammatory molecules.

Keywords: Cell adhesion molecules; Endothelial cells; IκBα; Isoliquiritigenin; NF-κB; ROS

Abbreviations: CAMs, cell adhesion molecules; ICAM-1, intercellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; TNF-α, tumor necrosis factor-α; NF-κB, nuclear factor-κB; EMSA, electrophoretic mobility shift assay; HUVECs, human umbilical cord vein endothelial cells; NEMO, NF-κB essential modulator

Article Outline

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Human endothelial cell culture

2.3. Cell viability assay

2.4. Neutrophil adhesion assay

2.5. Cell-ELISA for measurement of ICAM-1, VCAM-1 and E-selectin

2.6. Flow cytometry analysis

2.7. Total RNA isolation and reverse transcription polymerase chain reaction

2.8. Preparation of nuclear extracts

2.9. Preparation of total cell lysate

2.10. Western blot analysis for p65

2.11. Western blot analysis for IκBα degradation and phosphorylation

2.12. NF-κB activation assay

2.13. NF-κB dependent reporter gene assay

2.14. IκB kinase assay

2.15. Measurement of intracellular ROS generation by flow cytometry

2.16. Statistical analysis

3. Results

3.1. ILTG is non-toxic to cells

3.2. ILTG inhibits adhesion of neutrophils to endothelial monolayer

3.3. ILTG inhibits the TNF-α induced expression of ICAM-1, VCAM-1 and E-selectin on endothelial cells

3.4. ILTG decreases transcript levels of ICAM-1, VCAM-1 and E-selectin

3.5. ILTG inhibits TNF-α induced nuclear translocation of p65

3.6. ILTG inhibits TNF-α induced activation of NF-κB but not AP-1 and Oct-1

3.7. ILTG inhibits TNF-α induced IκBα degradation

3.8. ILTG inhibits TNF-α induced IκBα phosphorylation

3.9. ILTG inhibits TNF-α induced kinase activity of IκB kinase (IKK)

3.10. ILTG inhibits intracellular ROS generation

4. Discussion

Acknowledgements

References