== Taqman probe and primer sequences for real-time PCR -SMA, -easy muscle mass actin; EMMPRIN, extracellular matrix metalloproteinases inducer; ICAM-1, intercellular adhesion molecule-1; MMP-9, metalloproteinase-9; NFB, nuclear factor-B; VCAM-1, vascular cell adhesion molecule-1

== Taqman probe and primer sequences for real-time PCR -SMA, -easy muscle mass actin; EMMPRIN, extracellular matrix metalloproteinases inducer; ICAM-1, intercellular adhesion molecule-1; MMP-9, metalloproteinase-9; NFB, nuclear factor-B; VCAM-1, vascular cell adhesion molecule-1. == Statistical analysis == Adapalene SPSS v17.0 statistical software package was utilized for data analysis. were measured by Western blotting. ApoE/ mice fed with the Western diet for 16 weeks were treated with BBR, dhBBR and Di-MeBBR Adapalene 16 weeks. Aortic atherosclerotic lesion size, plaque matrix proteins, and EMMPRIN and other inflammatory factors were measured using Oil Red O Staining, Massons trichromestaining and immunohistochemical staining and real-time PCR. == Results == Compared with BBR, dhBBR and Di-MeBBR significantly reduced EMMPRIN expression, which was associated with a greater inhibition of p-p38, p-JNK, nuclear NFB p65 and phospho-p65 induced by oxLDL in macrophages. dhBBR and Di-MeBBR, but not BBR, reduced atherosclerotic plaque size and improved plaque stability Adapalene indicated by increased -easy muscle mass actin and collagen content, and thicker Ocln fibrous caps. dhBBR and Di-MeBBR reduced expression of EMMPRIN, CD68, and NFB p65, and Di-MeBBR also reduced expression of matrix metalloproteinase-9, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in aortic plaques. == Conclusions == These results have exhibited that BBR derivatives, dhBBR and Di-MeBBR, are superior to BBR in inhibiting inflammation and reducing plaque size and vulnerability. Keywords:Berberine derivatives, EMMPRIN, Inflammation, Atherosclerosis, Plaque stability == Background == Atherosclerosis is usually a chronic inflammatory disease affecting large and medium-sized arteries throughout the body [1,2]. In the early course of the disease, monocytes adhere to dysfunctional vascular endothelium and migrate into the subendothelial layer of the intima where they differentiate into macrophages. These macrophages transform into foam cells when the subendothelial space is usually enriched with atherogenic lipoproteins [3]. Foam cells aggregate to form the atheromatous core of the atherosclerotic plaques. Macrophages and foam cells key a variety of inflammatory factors, which attract more monocytes to infiltrate into the subendothelial space, propagate inflammatory response and subsequently advance atherosclerotic plaques [4]. Macrophage and foam cells also express several matrix metalloproteinases (MMPs), such as MMP-9 and extracellular matrix metalloproteinase inducer (EMMPRIN) [5,6], which, in turn, contribute to vulnerability of atherosclerotic plaques [7,8]. A plaque with a large lipid core and covered by a thin fibrous cap is at a higher risk for rupture [9]. The ruptured fibrous cap is known to be rich in macrophages that produce MMPs, thus digesting extracellular matrix and weakening the fibrous cap. The rupture of an atherosclerotic plaque followed by thrombus formation prospects to myocardial infarction, stroke, and death [10]. Therefore, reducing atherosclerotic plaque size and preventing the rupture of Adapalene vulnerable plaques are essential to preventing emergency medical conditions. In view of the important functions of MMP-9 and EMMPRIN in vulnerability of atherosclerotic plaques, MMP-9 and EMMPRIN are potential targets for therapeutic inventions to inhibit plaque rupture and acute medical conditions. Berberine (BBR) can be isolated from many different medicinal herbs, such as Berberis, Phellodendron amurense (Huang Po), Coptis chinensis (Huang Lian) [11]. Our previous studies exhibited that BBR markedly inhibited both mRNA and protein levels of EMMPRIN and MMP-9 in phorbol myristate acetate (PMA)- induced macrophages [12,13]. We further exhibited that BBR treatment for 30 days, as an adjunct therapy, reduced serum levels of MMP-9, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 in patients with acute coronary syndrome (ACS) following percutaneous coronary intervention when compared with standard therapy alone [14]. However, whether BBR reduces plaque size and enhances plaque stability in animal models is usually yet to be explored. In addition, several studies have shown that BBR derivatives, such as dihydroberberine (dhBBR) and 8, 8-dimethyldihydroberberine (Di-MeBBR), are more biologically available compared with BBR [15,16], suggesting that BBR derivatives may have greater anti-inflammatory and anti-atherosclerotic effects than BBR. Therefore, the aims of this study are: 1) to evaluate the inhibitory effects of BBR derivatives on EMMPRIN expression in macrophages and foam cells, in comparison with BBR; 2) to explore whether BBR and its derivatives havein vivoanti-atherosclerotic efficacy in apolipoprotein E knock-out (apoE/) mice; 3) to study signaling pathways contributing to their anti-atherosclerotic effects. == Methods == == Cell experiments == Cells from a human monocytic cell collection (THP-1 cells, obtained from the American Type Culture Collection) were cultured at a density of 106/mL in RPMI-1640 medium (Hycolone, USA) supplemented with 10% FBS, and 100 U/mL penicillin/streptomycin answer. To induce differentiation of monocytes into macrophages, THP-1 cells were cultured in 100 nM PMA (Calbiochem) for 48 h as previously Adapalene explained [17]. PMA-induced macrophages (2 106/mL) were pretreated with BBR (25 M), or Di-MeBBR (25 M), dhBBR (25 M) or thBBR (tetrahydroberberine, 25 M) for 1 h prior to incubation with oxLDL (50 g/mL) for the indicated occasions for different assays. Then, flow cytometry analysis and Western blotting were performed. == Circulation.