Inhibition Of Toll-like Receptor / Nuclear Factor-¦Êb Pathway <sup> Of The Apoe - / - </ Sup> Mouse Atherosclerotic Lesions

BACKGROUNDAtherosclerosis is a chronic inflammatory disease with immune responses during initiation and progression of this disease. Toll-like receptors (TLRs) are key pattern-recognition receptors of innate immunity and inflammatory response because they sense the existence of host-derived endogenous or exogenous TLR ligands and initiate a proinflammatory signaling cascade. Evidence is accumulating that TLRs play an important role in the pathogenesis of atherosclerosis. The classical mechanism by which endogenous or exogenous TLR ligands-induced TLRs signaling occurs includes activation of nuclear factor-κB (NF-κB), resulting in secretion of proinflammatory cytokines, cellular adhesion molecules, monocyte chemotactic factor. In 1997, human homologues of Toll, designated Toll-like receptor 4(TLR4), was discovered. To date, 13 TLRs have been described in humans with TLR 1-9 conserved between the human and the mouse. In C3H/HeJ mice with the TLR4 point mutation, plaque formation was decreased significantly compared with wild-type mice fed an atherogenic diet for 14 weeks. TLR-4 is preferentially expressed in lipid-rich and macrophage-infiltrated murine and human atherosclerotic plaques. In vitro studies demonstrated basal expression of TLR-4 by macrophages and endothelial cells, which was upregulated by oxidized LDL (ox-LDL). These findings suggest a potential role for TLR-4 in lipid-mediated proinflammatory signaling in atherosclerosis. The activation of TLR4 by endogeneous ligand such as oxLDL and HSP60 resulted in endothelial cells activation and production of multiple adhesion molecules and chemotatic factor, which attracted monocytes to the intima. The expression of SR-A in macrophages is markedly increased by TLR4 activation, which results in the differentiation of monocytes into macrophage foam cells; furthermore, the activation of TLR4 can induce intimal hyperplasia. (-)-Epigallocatechin-3-gallate (EGCG), a flavonoid found in green tea, is a well-characterized antiatherogenic property. EGCG was shown to inhibit the activity of IKKb which is the key kinase in the pathway for NF-κB activation both in MyD88-dependent or independent pathway of TLR4, and decrease subsequent expressions of inflammatory target gene, adhesion molecules and chemotatic factors. Thus, This study proposed the adoption of EGCG, the TLR4/NF-κB inhibitor, to inhibite the TLR4/NF-κB sigaling pathway to observe the development of aorta atherosclerosis and generation of relative inflammatory cytokines in Apolipoprotein E deficient(apoE^-/-) mice so as to explore the TLR4/NF-κB sigaling pathway in the way of atherosclerotic lesions occurred in the role.METHODSFifty male apoE^-/- mice (5wk old) were divided into four groups, control group: basic diet group, high-fat diet group; therapeutic group: EGCG+ basic diet group, high-fat diet+ EGCG group. EGCG (10 mg/kg) was injected intraperitoneally every day until death(14th W).Areas of aorta atherosclerosis were measured by oil red 0 staining, whole aortas we opened longitudinally from the aortic arch to the iliac bifurcation mounted en face, and stained for lipids with SoudanⅣ. Western blotting and immunohistochemistry were used to detect the change of expression of TLR4 and NF-κB proteins in mouse aorta, TLR4 mRNA detected by RT-PCR, Serum concentrations of MCP-l,TNF-a and ICAM-1 determined with enzyme-linked immunoabstorbent assay(ELISA).RESULTSIn basic diet + EGCG group and high-fat diet + EGCG group, EGCG treatment reduced areas of aorta atherosclerosis by 78% and 63% respectively, compared with high-fat diet group (P<0.01),expressions of TLR4 and NF-κB(p65) were obviously higher in aorta atherosclerosis lesions than that of other groups(P<0.01). Compared with high-diet group, the serum concentrations of MCP-1, TNF-αand sICAM-1 were markedly decreased in treated with EGCG groups. Furthermore, mean plasma total cholesterol (TC), triglyceride (TG) and low density cholesterol (LDLc) levels were significantly elevated in high-fat diet control mice (Table 1, p<0.05), compared with basic diet + EGCG group and high-fat diet + EGCG group.mice. CONCLUSIONSThese results suggested that EGCG, the TLR4/NF-κB inhibitor, can inhibit the development of atherosclerosis in aorta and aortic sinus department of apoE^-/- mice effectively, which possibly correlated with the reduction of TC,TG,LDL, the down-regulated expressions of TLR4, NF-κB, TNF-α,sICAM-1 and MCP-1 proteins and the inhibition of TLR4/NF-κB signaling pathway.