Green Tea Polyphenol EGCG Enhances The Stability Of Atherosclerotic Plaque And Its Related Mechanisms

Part I.green tea polyphenol Epigallocatechin-3-gallate enhances atherosclerotic plaque stability in high-fat diet fed apolipoprotein E-deficient miceObjective:Acute coronary syndrome(ACS)refers to a range of acute myocardial ischaemic states,which includes unstable angina and acute myocardial infarction(AMI)with or without ST elevation.Most of the acute coronary syndromes are considered to result from atherosclerotic plaque rupture and coronary thrombosis.Epidemiological studies have reported that daily consumption of green tea is inversely associated with the prevalence of coronary artery disease(CAD).The antiatherosclerotic effect of green tea has been attributed to its abundant polyphenolic compounds,known as catechins.Green tea catechins contain(-)-epicat-echin(EC),(-)-epicatechin gallate(ECG),(-)-epigallocatechin(EGC)and(-)-epigallocatechin-3-gallate(EGCG).EGCG,the principal constituent in green tea,has a variety of pharmacological and biological properties.The present study was designed to assess whether green tea polyphenol EGCG was able to enhance atherosclerotic plaque stability in apolipoprotein E-deficient mice and investigate the underlying mechanisms.Methods:Thirty male apolipoprotein E-deficient mice(age,6.weeks;.weight,18-20g)were divided into the control group(n=15)and the EGCG group(n=15)randomly.The apolipoprotein E-deficient mice were fed a high-fat diet that contained 21%fat and 0.15%cholesterol.EGCG(10 mg/kg body weight/day)or 0.9%saline was injected intraperitoneally into the apolipoprotein E-deficient mice for 16 weeks.The brachiocephalic arteries were removed and embedded in paraffin.Cross sections of the brachiocephalic arteries were stained with hematoxylin and eosin(HE)for morphometric analysis or stained with Masson's trichrome to evaluate collagen content.Immunohistochemistry was performed to evaluate the expression of macrophages,and smooth muscle cells(SMCs).Protein expression and MMPs(matrix metalloproteinases)activity were assayed by Western blot and Gelatin zymography,respectively.Serum tumor necrosis factor-alpha(TNF-a),interleukin-6(IL-6),monocyte chemotactic protein-1(MCP-1)and interferon-gamma(IFN-y)levels were quantified using ELISA.Results:After 16 weeks feeding of the high-fat diet,there was a clear atherosclerosis formation in the proximal brachiocephalic arteries segment with HE staining.Furthermore,The cross-sectional lesion area was also decreased in the apolipoprotein E-deficient mice treated with EGCG,compared with those in the control group.In the atherosclerotic plaques of EGCG group,the relative contents of macrophages were decreased whereas those of SMCs and collagen were increased.EMMPRIN expression and MMP-2 and 9 activities were substantially suppressed by EGCG treatment.In addition,Administration of EGCG caused down-regulation of circulating TNF-a,IL-6,MCP-1 and IFN-y in apolipoprotein E-deficient mice.Conclusions:EGCG,the most abundant polyphenol in green tea,can promote the stability of atherosclerotic lesions in apolipoprotein E-deficient mice.Furthermore,these effects were mediated through the inhibitory action of EGCG on the expression of inflammatory cytokine,MMPs and EMMPRIN.Part II.Inhibition of EMMPRIN and MMP-9 expression by Epigallocatechin-3-gallate through 67-kDa laminin receptor in PMA-induced macrophagesObjective:It is well documented that overexpression of EMMPRIN(extracellular matrix metalloproteinase inducer)and MMPs(matrix metalloproteinases)by monocytes/macrophages plays an important role in atherosclerotic plaque rupture.Green tea polyphenol epigallocatechin-3-gallate(EGCG)has a variety of pharmacological properties and exerts cardiovascular protective effects.Recently,the 67-kD laminin receptor(67LR)has been identified as a cell surface receptor of EGCG.The aim of the present study was to evaluate the effects of EGCG on the expression of EMMPRIN and MMP-9 in PMA-induced macrophages,and the potential mechanisms underlying its effects.Methods:Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate(PMA).EMMPRIN gene silencing was performed using small-interfering RNA(siRNA).Protein expression and MMP-9 activity were assayed by Western blot and Gelatin zymography,respectively.Real-time PCR was used to examine EMMPRIN and MMP-9 mRNA expression.Results:We showed that EGCG(10-50?mol/L)significantly inhibited the expression of EMMPRIN and MMP-9 and activation of extracellular signal-regulated kinase 1/2(ERK1/2),p38 and c-Jun N-terminal kinase(JNK)in PMA-induced macrophages.Downregulation of EMMPRIN by gene silencing hindered PMA-induced MMP-9 secretion and expression,indicating an important role of EMMPRIN in the inhibition of MMP-9 by EGCG.Moreover,67LR was involved in EGCG-mediated suppression of EMMPRIN and MMP-9 expression.Anti-67LR antibody treatment led to abrogation of the inhibitory action of EGCG on the expression of EMMPRIN and MMP-9 and activation of ERK1/2,p38,and JNK.Conclusion:Our results indicate that EGCG restrains EMMPRIN and MMP-9 expression via 67LR in PMAinduced macrophages,which also suggests that EGCG may be a possible therapeutic agent for stabilizing atherosclerotic plaque.