Epigallocatechin-3-gallate attenuates neointimal hyperplasia in a rat model of carotid artery injury by inhibition of high mobility group box 1 expression

Abstract

Epigallocatechin-3-gallate (EGCG) is a kind of polyphenol compound, called catechin, and is extracted from green tea. EGCG has a wide range of biological activities. The present study aimed to evaluate the effect of EGCG on neointimal hyperplasia in a rat model of carotid artery balloon injury and to explore the molecular mechanisms involved. Various experiments were performed to assess the effects of EGCG on thickening of neointima, expression levels of high mobility group box 1 protein (HMGB1) and receptor of advanced glycation end products (RAGE), the inflammatory response, oxidative stress and activation of nuclear factor (NF)-κB. Results demonstrated that EGCG decreased the intimal area and the ratio of intimal area/medial area compared with the balloon injury group. The expression levels of HMGB1 and RAGE induced by balloon injury were markedly inhibited by EGCG treatment. Furthermore, the inflammatory response and oxidative stress damage, which have close correlations with HMGB1, were restrained by EGCG. Finally, EGCG treatment markedly inhibited NF-κB activation. The present data provided evidence that EGCG attenuates neointimal hyperplasia in a model of carotid artery balloon injury, which indicated that EGCG may serve as a potential drug for restenosis in clinics.

Keywords: epigallocatechin-3-gallate; high mobility group box 1; inflammation; neointimal hyperplasia; oxidative stress.

Figure 1.

Chemical structure of EGCG. The molecular formula of EGCG is C₂₂ H₁₈ O₁₁ and its molecular weight is 458.38. EGCG, epigallocatechin-3-gallate.

Figure 2.

EGCG attenuates neointimal hyperplasia induced by carotid artery balloon injury. (A) Sections of carotid artery tissues 14 days after balloon injury were stained with hematoxylin and eosin (magnification, ×400; scale bar, 50 µm). The areas of (B) intima, (C) lumen and (D) media were shown and the (E) I/M ratios were calculated. Data are presented as the mean + standard deviation (n=6). *P<0.05, **P<0.01 and ***P<0.001 vs. the sham group; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. the injury group. EGCG, epigallocatechin-3-gallate; I/M, neointima/media area.

Figure 3.

EGCG inhibits balloon injury-induced HMGB1 and RAGE expression levels. mRNA expression levels of (A) HMGB1 and (B) RAGE in artery tissues were determined by reverse transcription-quantitative polymerase chain reaction. Protein expression levels of (C) HMGB1 and (D) RAGE in artery tissues were detected by western blotting. β-actin was used as a loading control. The protein bands were quantified by gray scanning. Data are presented as the mean + standard deviation (n=6). *P<0.05, **P<0.01 and ***P<0.001 vs. the sham group; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. the injury group. EGCG, epigallocatechin-3-gallate; HMGB1, high mobility group box 1; RAGE, receptor of advanced glycation end products.

Figure 4.

EGCG inhibits the balloon injury-induced inflammatory response. (A) TNF-α, (B) IL-1β, (C) IL-6, (D) ICAM-1 and (E) VCAM-1 levels in peripheral blood were detected by ELISA. Three independent experiments were performed. Data are presented as the mean + standard deviation (n=6). **P<0.01 and ***P<0.001 vs. the sham group; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. the injury group. EGCG, epigallocatechin-3-gallate; TNF-α, tumor necrosis factor α; IL, interleukin; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; prot, protein.

Figure 5.

EGCG inhibits balloon injury-induced oxidative stress damage. (A) MDA and (B) ROS levels were assessed. Three independent experiments were performed. Data are presented as the mean + standard deviation (n=6). **P<0.01 and ***P<0.001 vs. the sham group; ^#P<0.05 and ^###P<0.001 vs. the injury group. EGCG, epigallocatechin-3-gallate; MDA, malondialdehyde; ROS, reactive oxygen species; prot, protein.

Figure 6.

EGCG inhibits NF-κB activation induced by balloon injury. (A) The expression levels of NF-κB in the nucleus and cytoplasm were assessed by western blot assay. Histone H3 and β-actin were used as loading controls. The protein bands were quantified by gray scanning (n=6). (B) Electrophoretic mobility shift assay was performed to assess the DNA binding activity of NF-κB. Three independent experiments were performed. Data are presented as the mean + standard deviation (n=6). **P<0.01 and ***P<0.001 vs. the sham group; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. the injury group. EGCG, epigallocatechin-3-gallate; NF, nuclear factor.