The stability of an atherosclerotic plaque determines whether a patient will experience stable angina pectoris or a life-threatening acute coronary syndrome (Shin et al.,2003). Macrophage infiltration and expression of matrix metalloproteinase-9 (MMP-9), degrading the extracellular matrix, contribute to atherosclerotic plaque instability (Galis et al.,1995; Kunte et al.,2008). This finding has also been supported by in vivo studies showing that plasma levels of matrix metalloproteinases (MMPs) can determine future coronary events (Kai et al.,1998; Blankenberg et al.,2003).
Extracellular matrix metalloproteinase inducer (EMMPRIN) triggers the synthesis of MMP-9 in a paracrine or autocrine manner (Zhou et al.,2005; Schmidt et al.,2006). Increasing evidence shows that in response to certain proatherogenic stimuli such as oxLDL, EMMPRIN is upregulated strongly in atherosclerosis-related cells, including macrophages and coronary smooth muscle cells (Major et al.,2002; Haug et al.,2004; Schmidt et al.,2006). It also increases in activated platelets and mediated NF-κB activation by inducing the production of MMP-9, IL-6, and TNF-α (Schmidt et al.,2008). This evidence suggests that EMMPRIN plays a key regulatory role in the development of atherosclerosis.
EMMPRIN and MMP-9 can promote the instability of atherosclerotic plaques. Therefore, hypothetically, the inhibitors of EMMPRIN and MMP-9 are potential candidates for improving the development of atherosclerosis. In this context, we are interested in the isoquinoline alkaloid berberine. Berberine can be isolated from many medicinal herbs such as Hydrastis canadensis (goldenseal), Cortex phellodendri (Huangbai), and Rhizoma coptidis (Huanglian) (Ikram,1975). When present as a major ingredient, berberine can have pharmacological effects on cancer (Anis et al.,2001) and bacterial diseases (Stermitz et al.,2000). Recently, berberine has been found to have cholesterol- (Kong et al.,2004) and glucose-lowering effects (Yin et al.,2008). Further, when THP-1 cells differentiate into macrophages, berberine inhibits MMP-9 and EMMPRIN expression (Huang et al.,2009). However, the role of berberine on the expression of MMP-9 and EMMPRIN and its possible mechanism in oxLDL-stimulated macrophages remain unclear.
NF-κB plays a key role in atherosclerosis (Majdalawieh and Ro,2010). A strong signal of active NF-κB can be detected in macrophages and endothelial cells in the aorta with evident atherosclerotic lesions (Brand et al.,1996). NF-κB activation is believed to promote the expression of some MMPs (Yokoo and Kitamura,1996; Mengshol et al.,2000) and several factors that mediate various processes such as inflammation and thrombosis, which are key events in atherogenesis (Brand et al.,1997). This evidence proves the critical role of active NF-κB in atherosclerosis. Interestingly, oxLDL is potentially capable of activating NF-κB in macrophages in culture systems as well as in atherosclerotic lesions (Rajavashisth et al.,1995; Brand et al.,1997). Some studies have also demonstrated that inhibition of NF-κB abrogates macrophage recruitment to the atherosclerotic lesions and attenuates the development of atherosclerosis (Majdalawieh and Ro,2010). Briefly, in unstimulated cells, the NF-κB canonical signaling pathway p65 is localized predominantly in the cytoplasm and remains inactivated through binding with its inhibitory protein known as IκB-α. In the presence of stimuli with oxLDL, IκB-α is phosphorylated and subsequently degraded, which allows p65 to be liberated from IκB-α, and to be translocated into a nucleus where it binds to a specific sequence in the promoter of target genes, resulting in increased gene expression, including MMP expression (Yamamoto and Gaynor,2004). Hence, to observe the role of berberine in atherosclerosis, the effects of berberine on the expression of MMP-9 and EMMPRIN and on the activity of NF- κB (p65, IκB-α) in oxLDL-stimulated macrophages was investigated.
MATERIAL AND METHODS
RPMI 1640 medium, fetal bovine serum, and penicillin/streptomycin (pen/strep, 10,000 U/mL each) were purchased from GIBCO Company. Phorbol 12-myristate 13-acetate (PMA) was obtained from Calbiochem (San Diego, CA). Dimethyl sulfoxide and berberine were acquired from Sigma-Aldrich (St. Louis, MO). Trizol reagent for RNA isolation was purchased from Invitrogen. Omniscript reverse transcriptase for first-strand cDNA synthesis was obtained from Qiagen. A rabbit monoclonal antibody to β-actin (ab8227) was produced by Abcam (Cambridge, UK). An EMMPRIN antibody was obtained from Zymed (South San Francisco, CA). MMP-9, phospho-IκB-α, and p65 antibodies were obtained from Cell Signaling Technology (Danvers, MA). Lamin B antibodies were obtained from Sigma-Aldrich (St. Louis, MO). NE-PER nuclear and cytoplasmic extraction reagents were purchased from Pierce. All goat antirabbit secondary antibodies (Catalog No. A-21109) used in Western blotting were from Invitrogen (Carlsbad, CA). A cellular NF-κB translocation kit was purchased from Beyotime Biotech. All other chemicals used for Western blotting were of the highest purity commercially available.
Cell Culture and Treatment
Human monocytic cell line THP-1 was obtained from American Type Culture Collection (Rockville, MD) and was maintained at a density of 106/mL in RPMI 1,640 medium containing 10% FBS, 10 mM HEPES (Sigma–Aldrich) and 1% pen/strep solution at 37°C in a 5% CO2 incubator. The cells were cultured in six-well plates for 48 hr in the presence of 100 nM PMA, which allowed them to differentiate into adherent macrophages (Tsuchiya et al.,1982). The cells were pretreated with berberine (0–50 μM) for 1 hr prior to incubation with oxLDL (50 μg/mL) for 24 hr. To observe berberine's inhibitory effects on the NF-κB signal pathway, cells (2 × 106/mL) were pretreated with or without berberine for 1 hr prior to incubation with oxLDL for the indicated times.
Determination of Cell Viability (MTT Assay)
MTT assay was used to assess the cytotoxicity of berberine on macrophages. After the indicated treatments, the cells were incubated with 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Roche Applied Science) in a culture medium for an additional 4 hr. Next, the blue formazan crystals of viable cells were dissolved in dindimethyl sulfoxide and measured spectrophotometrically at 570 nm.
RNA Isolation, cDNA Synthesis, and TaqMan Teal-Time PCR
Total RNA was extracted from oxLDL-stimulated macrophages using Trizol reagent according to the manufacturer's instructions. Total RNA (2 μg) was reverse-transcribed into cDNA using random primers. The resultant cDNA was amplified by TaqMan real-time polymerase chain reaction (PCR). The PCR reaction was monitored directly by the Applied Biosystems 7500 Real-Time PCR System with the following primer sequences: MMP-9(192 bp), 5′-CAGACATCGTCATCCAGTTTG-3′ (sense), 5′-CGCCATCTGCGTTTCCAA-3′ (antisense), and 5′-fam-CCGAGTTGGAACCACGACGCCCTTG-tamra-3′ (probe); EMMPRIN, 5′-CTACACATTGAGAACCTGAACAT-3′ (sense), 5′-TTCTCGTAGATGAAGATGATGGT-3′ (antisense), and 5′-fam-CAGCACCAGCACCTCAGCCACGATG-tamra-3′ (probe); and GAPDH, 5 ′-CCAGGTGGTCTCCTCTGACTT-3′ (sense), 5′-GTTGCTGTAGCCAAATTCGTTGT-3′ (antisense), and 5′-fam-AACAGCGACACCCACTCCTCCACC-tamra-3′ (probe). The whole amplification course was initiated at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, and 60°C for 45 sec. All results were normalized against GAPDH. All real-time PCRs were run in duplicate.
Protein Isolation and Western Blot Analysis
Protein was isolated from the cytoplasm and nuclear fractions using NE-PER nuclear and cytoplasmic extraction reagents according to the manipulation for the analysis of IκB-α phosphorylation and nuclear p65/lamin B levels. Protein isolation and Western blot analysis of the cell lysates were performed as previously described (Huang et al.,2008), except that the membranes were probed with primary antibodies for rabbit antiMMP-9, antiEMMPRIN, antiphospho-IκB-α, antilamin B, and antip65 antibodies (diluted 1:1,000 in TBST) for 2 hr or for rabbit antiactin (diluted 1:5,000 in TBST) for 1 hr, followed by incubation with goat antirabbit secondary antibodies labeled with far-red-fluorescent Alexa Fluor 680 dye. Briefly, all signals were detected by an Odyssey imaging system (Li-cor). Densitometric analysis was again performed using Quantity One (Bio-Rad) to scan the signals.
Confocal Laser Scanning Fluorescence Microscopy of NF-κB
Cells were seeded in a six-well tissue culture plate and pretreated in the presence or absence of 25 μM 1 hr prior to incubation with oxLDL for the indicated times (1, 2, 3, and 6 hr). The cells were immunofluorescence-labeled using a cellular NF-κB translocation kit (Beyotime Biotech) according to the manufacturer's protocol. Briefly, after washing and fixing, the cells were incubated with a blocking solution at 4°C overnight and then with the NF-κB p65 antibody for 2 hr. After washing three times, a rabbit IgG antibody conjugated with Cy3 was added and incubated for 1 hr. To stain the nucleus, the cells were then incubated with DAPI for 5 min. The activation of NF-κB p65 was visualized with a confocal laser scanning microscope (FluoView™ FV1000, Olympus) at excitation wavelengths of 350 nm for DAPI and 540 nm for Cy3. The red and blue images were overlaid to create a two-color image in which purple fluorescence indicated the areas of colocalization.
The results were expressed as mean ± S.D. Differences were compared by one-way ANOVA (LSD, S-N-K, Dunnet) using SPSS 11.0 software. Statistical significance was defined as P < 0.05. All experiments were performed at least three times.
Effects of Berberine on Cell Viability
MTT assay was used to evaluate the effect of berberine on the viability of the macrophages. As shown in Fig. 1, a concentration of berberine ranging from 5 to 75 μM caused no significant reduction (about 5–10%) in cell viability. Therefore, a berberine dose at or lower than 75 μM was considered to be noncytotoxic, and doses ranging from 5 to 50 μM were used in subsequent experiments.
Reduction of MMP-9 Levels by Berberine in oxLDL-Stimulated Macrophages at Both mRNA and Protein Levels
To determine whether berberine has effects on MMP-9 at the mRNA and protein levels, the macrophages were pretreated with the indicated concentration of berberine for 1 hr, followed by a culture with oxLDL for 24 hr. The amounts of MMP-9 mRNA were determined by real-time PCR, and the MMP-9 protein levels were quantified by Western blotting. Berberine inhibited the expression of MMP-9 both at protein and mRNA levels in a dose-dependent manner (Fig. 2A,C). Macrophages, which were stimulated by oxLDL, were treated with berberine (25 μM) for 3, 6, and 12 hr. As depicted in Fig. 2B,D, MMP-9 expression was reduced by berberine in a time-dependent manner. Therefore, berberine reduced MMP-9 expression at both transcription and translation levels in oxLDL-stimulated macrophages.
Reduction of EMMPRIN by Berberine at Both mRNA and Protein Levels in oxLDL-Stimulated Macrophages
As MMP-9 expression is positively regulated by EMMPRIN (Zhou et al.,2005; Yoon et al.,2005) and because berberine can apparently downregulate MMP-9 expression in oxLDL-stimulated macrophages, whether the inhibitory effect of berberine on MMP-9 expression might be a consequence of the inhibition of EMMPRIN expression in oxLDL-stimulated macrophages was examined next. Consistently, the results show that berberine has a similar effect on EMMPRIN at both protein and mRNA levels in oxLDL-stimulated macrophages in a dose-dependent or time-dependent manner (Fig. 3). These results indicate that the downregulation of EMMPRIN by berberine is at least partly responsible for the reduction of MMP-9 expression in oxLDL-stimulated macrophages.
Significant Inhibition of NF-κB Activation by Berberine in oxLDL-Stimulated Macrophages
One emerging concept is that as a major regulator, NF-κB plays a role in MMP-9 (Yoo et al.,2002; Chase et al.,2002; Lu and Wahl,2005) and EMMPRIN (Hagemann et al.,2005) expression. Therefore, examining whether the activation of NF-κB is also involved in the inhibitory effect of berberine on oxLDL-stimulated macrophages is interesting. As shown in Fig. 4, the phosphorylation of IκB-α in cytoplasmic protein, as well as p65 in nuclear protein extraction was increased significantly in macrophages after stimulation by oxLDL (50 μg/mL) for 1, 2, 3, and 6 hr. In contrast, berberine (25 μM) blocked partially the degradation of IκB-α and the nuclear translocation of p65 at indicated times. As shown in Fig. 5, the same tendency was found; the stain levels of p65 diminished in berberine-treated macrophages.
Berberine suppressed effectively the mRNA and protein expression of both MMP-9 and EMMPRIN in oxLDL-stimulated macrophages. Berberine can also suppress NF-κB activation induced by oxLDL in macrophages. These results suggest that berberine exerts its inhibitory effect on both expressions of EMMPRIN and MMP-9 by at least partly suppressing NF-κB activity in oxLDL-induced macrophages.
Plaque vulnerability is a primary determinant in thrombus and rupture-mediated complications, most notably unstable angina and acute myocardial infarction. Accumulating evidence now supports the concept that MMPs contribute to plaque rupture or trigger acute coronary syndrome (Lusis,2000; Libby,2001). Blankenberg et al. (2003) reported a strong association between plasma levels of MMP-9 and a subsequent 4.1-year risk for fatal coronary artery disease events among 1,127 subjects with established coronary disease. Fiotti et al. (2008) showed that the expression of MMP-9, except TIMP-1 or MMP-2, increases in plaques caused by acute coronary syndrome. Therefore, while conversely retarding extracellular matrix destruction and subsequent rupture, the inhibitors of MMP-9 may be a useful therapy to prevent plaque rupture or its clinical sequelae.
In the present study, berberine markedly inhibited MMP-9 expression induced by oxLDL in macrophages. More importantly, it also inhibited the expression of EMMPRIN, which in turn caused the reduction in MMP-9. Given the important roles of MMP-9 and EMMPRIN expression in the development of atherosclerosis and the inhibitory effects of berberine on this expression, we concluded that berberine may exert its beneficial properties by stabilizing atherosclerotic plaque, thus improving the development of atherosclerosis.
Berberine has been used to treat diarrhea and gastrointestinal disorders in Chinese traditional medicine (Choi et al.,2003). Recently, Doggrell et al. (2005) and Kong et al. (2004) described the cholesterol-lowering property of berberine. In a clinical study, Yin et al. (2008) found that berberine lowers blood glucose levels effectively in patients with type 2 diabetes. Berberine prevents oxLDL-induced cellular dysfunction (Hsieh et al.,2007), and it is effective in reducing excess body weight (Lee et al.,2006). Therefore, berberine has known properties for diminishing many of the major risk factors for atherosclerosis, such as hyperlipidemia, diabetes, and overweight. It is also notable for its antiinflammatory effects that inhibit the production of TNF-α, MCP-1, and COX-2 expression (Chen et al.,2008; Guo et al.,2008). A recent study reported that berberine prevents the formation of foam cells from macrophages by enhancing LXRa-ABCA1-dependent cholesterol efflux (Lee et al.,2010). These results provide evidence that berberine has a beneficial effect on the prevention of atherosclerosis.
oxLDL is implicated in many proatherogenic events, such as an increase in the expression of MMP-9 and EMMPRIN, as seen in the present study. It can also activate the nuclear transcription factor NF-κB at indicated times in macrophages (Rajavashisth et al.,1995; Brand et al.,1997). Recent studies have shown that NF-κB activation (nuclear translocation of p65), which has been observed in human and experimental atherosclerosis (Brand et al.,1996,1997; Hajra et al.,2000), is enhanced in unstable coronary atherectomies (Wilson et al.,2002) and is involved in the upregulated expression of proinflammatory factors such as MMPs, except TIMP-1 (Monaco et al.,2004). Both the mutation of the NF-κB binding sequence in the promoters of MMPs and the overexpression of IκB-α protein are accompanied significantly by reduced MMP production in macrophages (Monaco et al.,2004; Ogawa et al.,2004). Therefore, the inhibition of NF-κB activation will impede the progress of atherosclerosis. In fact, the suppression of NF-κB activity by blocking IκB degradation results in significantly smaller atherosclerotic lesions as compared with those in control mice (Wolfrum et al.,2007). Several studies have also demonstrated the positive correlation between NF-κB activity and the incidence of myocardial infarction (Thiemermann,2004; Majdalawieh and Ro,2010). These results suggest that NF-κB is a promising therapeutic target for reducing the risks of atherosclerosis.
The current study demonstrates that berberine reduces the phosphorylation of IκB-α in the cytoplasm (blocking IκB-α degradation) and suppresses the nuclear translocation of p65 induced by oxLDL in macrophages, indicating that NF-κB activation is inhibited by berberine. The same tendency is observed in the stain of NF-κB p65 nucleus translocation. Collectively, berberine suppresses not only MMP-9 and EMMPRIN expression but also NF-κB activation. Further, the activation of NF-κB is responsible for MMP-9 expression. Therefore, in oxLDL-stimulated macrophages, the inhibitory effect of berberine on MMP-9 and EMMPRIN expression is at least partly associated with the downregulation of NF-κB activation.
NF-κB activation is involved in EMMPRIN expression (Hagemann et al.,2005). As a novel platelet receptor, EMMPRIN in turn activates NF-κB (Schmidt et al.,2008), suggesting that a positive feedback between EMMPRIN and NF-κB exists. That is, the upregulation of EMMPRIN expression regulates more NF-κB-related inflammatory cellular activities (e.g., IL-6 and TNF-α.) than only MMP activity. On the other hand, berberine may play its multiple roles (as a potential inhibitor of EMMPRIN and NF-κB) in regulating associated inflammatory genes linked to atherosclerosis and in exerting its protective effect to stabilize atherosclerostic plaque.
In conclusion, berberine inhibits the upregulated expression of MMP-9 and EMMPRIN induced by oxLDL in macrophages through the suppression of NF-κB translocation into the nucleus. The downregulation of MMP-9 and EMMPRIN stabilizes atherosclerotic plaque, so our results provide evidence that berberine reduces inflammatory responses and thereby mitigates plaque development. However, the effects of berberine in vivo require further study using animal models of atherosclerosis.