Suppression of oxLDL-Induced MMP-9 and EMMPRIN Expression by Berberine via Inhibition of NF-╬║B Activation in Human THP-1 Macrophages.код для вставкиСкачать
THE ANATOMICAL RECORD 295:78–86 (2012) Suppression of oxLDL-Induced MMP-9 and EMMPRIN Expression by Berberine via Inhibition of NF-jB Activation in Human THP-1 Macrophages ZHOUQING HUANG,1,2 SHU MENG,3 LIANSHENG WANG,2 YUE WANG,2 TING CHEN,2 AND CHANGQIAN WANG2* 1 Department of Cardiology, First Afﬁliated Hospital, Wenzhou Medical College, Wenzhou 325000, China 2 Department of Cardiology, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200011, China 3 Department of Cardiology, Xin Hua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China ABSTRACT Upregulation of matrix metalloproteinases (MMPs) and extracellular matrix metalloproteinase inducer (EMMPRIN) by macrophages leads to atherosclerotic plaque rupture by degradation of the extracellular matrix. NF-jB activation regulates many key inﬂammatory genes linked to atherosclerosis. In the present study, the function of berberine, a natural extract from Rhizoma coptidis, on MMP-9 and EMMPRIN expression, the role of NF-jB activation in oxLDL-stimulated macrophages, and the possible mechanism in which NF-jB activation is involved were investigated. Berberine inhibited the expression of MMP-9 and EMMPRIN at both mRNA and protein levels. The phosphorylation of IjB-a and nuclear translocation of p65 protein were reduced by berberine, suggesting that NF-jB activation was inhibited by berberine in oxLDL-stimulated macrophages. Overall, berberine suppressed the expression of MMP-9 and EMMPRIN by at least reducing partly the activity of NF-jB in oxLDL-induced macroC 2011 Wiley Periodicals, Inc. phages. Anat Rec, 295:78–86, 2012. V Key words: berberine; MMP-9; EMMPRIN; stimulated macrophages 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, C 2011 WILEY PERIODICALS, INC. V NF-B; oxLDL- 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 Grant sponsor: Science and Technology Commission of Shanghai Municipality; Grant number: 08411966900; Grant sponsor: Traditional Chinese Medicine of Zhejiang; Grant number: 2010ZA085. *Correspondence to: Changqian Wang, Department of Cardiology, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, 639 Zhizaoju Road, Shanghai 200011, China. Fax: þ86-21-6313-6856. E-mail: firstname.lastname@example.org Received 14 June 2010; Accepted 20 August 2011 DOI 10.1002/ar.21489 Published online 5 December 2011 in Wiley Online Library (wileyonlinelibrary.com). BERBERINE INHIBIT MMP-9 AND EMMPRIN EXPRESSION mediated NF-jB activation by inducing the production of MMP-9, IL-6, and TNF-a (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-jB plays a key role in atherosclerosis (Majdalawieh and Ro, 2010). A strong signal of active NF-jB can be detected in macrophages and endothelial cells in the aorta with evident atherosclerotic lesions (Brand et al., 1996). NF-jB 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-jB in atherosclerosis. Interestingly, oxLDL is potentially capable of activating NF-jB 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-jB abrogates macrophage recruitment to the atherosclerotic lesions and attenuates the development of atherosclerosis (Majdalawieh and Ro, 2010). Brieﬂy, in unstimulated cells, the NF-jB canonical signaling pathway p65 is localized predominantly in the cytoplasm and remains inactivated through binding with its inhibitory protein known as IjB-a. In the presence of stimuli with oxLDL, IjB-a is phosphorylated and subsequently degraded, which allows p65 to be liberated from IjB-a, 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- jB (p65, IjB-a) in oxLDL-stimulated macrophages was investigated. MATERIAL AND METHODS VIA NF-jB 79 Fig. 1. Effects of berberine on the viability of macrophages. Macrophages were treated with indicated concentrations of berberine (5–100 lM), and cell viability was assessed after 48 hr using MTT assay. Cells incubated in a medium without berberine served as the control variables and were considered 100% viable. 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 b-actin (ab8227) was produced by Abcam (Cambridge, UK). An EMMPRIN antibody was obtained from Zymed (South San Francisco, CA). MMP-9, phospho-IjB-a, 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-jB 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 lM) for 1 hr prior to incubation with oxLDL (50 lg/mL) for 24 hr. To observe berberine’s inhibitory effects on the NF-jB signal pathway, cells (2 106/mL) were pretreated with or without berberine for 1 hr prior to incubation with oxLDL for the indicated times. Reagents 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 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) 80 HUANG ET AL. Fig. 2. Effects of berberine on MMP-9 expression in oxLDL-stimulated macrophages. MMP-9 mRNA and protein levels were measured by real-time PCR and Western blot analysis. (A, B) Representative Western blots of MMP-9 expression affected by berberine in different concentrations and time courses. (C, D) Effects of berberine on MMP9 expression (mRNA and protein). Respective densitometric measure- ment results are given. The band densities were measured by Quantity one. Effects of berberine on MMP-9 mRNA expression. Data represent mean S.D. (*) P < 0.05 and (**) P < 0.01 when compared with the control group; (#) P < 0.05 and (##) P < 0.01 when compared with the (**) marked group. (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. TTCGTTGT-30 (antisense), and 50 -fam-AACAGCGACACC CACTCCTCCACC-tamra-30 (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. 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 lg) 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 RealTime PCR System with the following primer sequences: MMP-9(192 bp), 5 0 -CAGACATCGTCATCCAGTTTG-30 (sense), 50 -CGCCATCTGCGTTTCCAA-30 (antisense), and 50 -fam-CCGAGTTGGAACCACGACGCCCTTG-tamra-30 (probe); EMMPRIN, 50 -CTACACATTGAGAACCTGAAC AT-30 (sense), 50 -TTCTCGTAGATGAAGATGATGGT-30 (antisense), and 50 -fam-CAGCACCAGCACCTCAGCCAC GATG-tamra-30 (probe); and GAPDH, 5 0 -CCAGGTGGT CTCCTCTGACTT-30 (sense), 50 -GTTGCTGTAGCCAAA 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 IjB-a 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-IjB-a, 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 BERBERINE INHIBIT MMP-9 AND EMMPRIN EXPRESSION VIA NF-jB 81 Fig. 3. Effects of berberine on EMMPRIN expression in oxLDLstimulated macrophages. EMMPRIN mRNA and protein expression were determined by real-time PCR and Western blot analysis. (A, B) Representative Western blots analysis of EMMPRIN expression. (C, D) Effects of berberine on EMMPRIN expression (mRNA and protein). Respective densitometric measurement results are given. The band densities were measured by Quantity One. Data represent mean S.D. (*) P < 0.05 and (**) P < 0.01 when compared with the control group; (#) P < 0.05 and (##) P < 0.01 when compared with the (**) marked group. again performed using Quantity the signals. Statistical Analysis One (Bio-Rad) to scan Confocal Laser Scanning Fluorescence Microscopy of NF-jB Cells were seeded in a six-well tissue culture plate and pretreated in the presence or absence of 25 lM 1 hr prior to incubation with oxLDL for the indicated times (1, 2, 3, and 6 hr). The cells were immunoﬂuorescencelabeled using a cellular NF-jB 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-jB 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-jB p65 was visualized with a confocal laser scanning microscope (FluoViewTM 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. RESULTS 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 lM caused no signiﬁcant reduction (about 5–10%) in cell viability. Therefore, a berberine dose at or lower than 75 lM was considered to be noncytotoxic, and doses ranging from 5 to 50 lM 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 82 HUANG ET AL. Fig. 4. Effects of berberine on phosphorylated IjB-a (p-IjB-a) and nuclear protein p65 expression in oxLDL-stimulated macrophages. Cells were pretreated with vehicle or berberine (25 lM) for 1 hr, followed by oxLDL for 1, 2, 3, and 6 hr. The cell lysates were collected for Western blotting. (A) Effect of berberine on p-IjB-a expression. (B) Effect of berberine on p65 expression (p65 translocation). (C, D) Respective densitometric measurement results of p-IjB-a and p65. b-actin and lamin B expression was used for protein level normalization. Data represent mean S.D. (*) P < 0.05 and (**) P < 0.01 when compared with the control group; (#) P < 0.05 and (##) P < 0.01. 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 dosedependent manner (Fig. 2A,C). Macrophages, which were stimulated by oxLDL, were treated with berberine (25 lM) for 3, 6, and 12 hr. As depicted in Fig. 2B,D, MMP-9 expression was reduced by berberine in a timedependent manner. Therefore, berberine reduced MMP-9 expression at both transcription and translation levels in oxLDL-stimulated macrophages. 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. 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 Signiﬁcant Inhibition of NF-jB Activation by Berberine in oxLDL-Stimulated Macrophages One emerging concept is that as a major regulator, NF-jB 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-jB is also involved in the inhibitory effect of berberine on oxLDL-stimulated macrophages is interesting. As shown in Fig. 4, the phosphorylation of IjB-a in cytoplasmic protein, as well as p65 in nuclear protein extraction was increased signiﬁcantly in macrophages after stimulation by oxLDL (50 lg/mL) for 1, 2, 3, and 6 hr. In contrast, berberine (25 lM) blocked partially the degradation of IjB-a 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. DISCUSSION Berberine suppressed effectively the mRNA and protein expression of both MMP-9 and EMMPRIN in BERBERINE INHIBIT MMP-9 AND EMMPRIN EXPRESSION VIA NF-jB 83 Fig. 5. Representative microphotographs showing the effect of berberine on the translocation of NF-jB p65 in oxLDL-treated macrophages. NF-jB localization was visualized by binding with a Cy3conjugated secondary antibody. Cells were then incubated with DAPI to stain the nuclei. Microscopic images were obtained using a confocal laser scanning microscope (three independent experiments), and the red area (representative of the area that contains p65) and blue area (representative of the nucleus part that is DAPI conjugated) images were overlaid to create a purple ﬂuorescence in areas of colocalization. In untreated macrophages, the NF-jB p65 subunit was localized predominantly in the cytoplasm (control group). Cells stimulated with oxLDL only showed a signiﬁcant translocation of p65 to the cell nucleus. In cells pretreated with 25 lM berberine for indicated times and exposed to 50 lg/L oxLDL for 1, 2, 3, and 6 hr, NF-jB p65 was retained signiﬁcantly in the cytoplasm. oxLDL-stimulated macrophages. Berberine can also suppress NF-jB 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-jB 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 84 HUANG ET AL. 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-a, 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-jB at indicated times in macrophages (Rajavashisth et al., 1995; Brand et al., 1997). Recent studies have shown that NF-jB 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-jB binding sequence in the promoters of MMPs and the overexpression of IjB-a protein are accompanied signiﬁcantly by reduced MMP production in macrophages (Monaco et al., 2004; Ogawa et al., 2004). Therefore, the inhibition of NF-jB activation will impede the progress of atherosclerosis. In fact, the suppression of NF-jB activity by blocking IjB 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-jB activity and the incidence of myocardial infarction (Thiemermann, 2004; Majdalawieh and Ro, 2010). These results suggest that NF-jB is a promising therapeutic target for reducing the risks of atherosclerosis. The current study demonstrates that berberine reduces the phosphorylation of IjB-a in the cytoplasm (blocking IjB-a degradation) and suppresses the nuclear translocation of p65 induced by oxLDL in macrophages, indicating that NF-jB activation is inhibited by berberine. The same tendency is observed in the stain of NFjB p65 nucleus translocation. Collectively, berberine suppresses not only MMP-9 and EMMPRIN expression but also NF-jB activation. Further, the activation of NF-jB 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-jB activation. NF-jB activation is involved in EMMPRIN expression (Hagemann et al., 2005). As a novel platelet receptor, EMMPRIN in turn activates NF-jB (Schmidt et al., 2008), suggesting that a positive feedback between EMMPRIN and NF-jB exists. That is, the upregulation of EMMPRIN expression regulates more NF-jB-related inﬂammatory cellular activities (e.g., IL-6 and TNF-a.) than only MMP activity. On the other hand, berberine may play its multiple roles (as a potential inhibitor of EMMPRIN and NF-jB) in regulating associated inﬂammatory genes linked to atherosclerosis and in exerting its protective effect to stabilize atherosclerostic plaque. 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