Background
Atherosclerosis is 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 enriched with atherogenic lipoproteins [
3]. Foam cells aggregate to form the atheromatous core of the atherosclerotic plaques. Macrophages and foam cells secret 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 leads to myocardial infarction, stroke, and death [
10]. Therefore, reducing atherosclerotic plaque size and preventing the rupture of vulnerable plaques are essential to preventing emergency medical conditions. In view of the important roles 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 demonstrated 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 demonstrated 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 improves plaque stability in animal models is 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 have in vivo anti-atherosclerotic efficacy in apolipoprotein E knock-out (apoE−/−) mice; 3) to study signaling pathways contributing to their anti-atherosclerotic effects.
Discussion
Our previous studies have found that BBR inhibits MMP-9 and EMMPRIN expression in macrophages [
12] and also decreases plasma MMP-9, ICAM-1 and VCAM-1 in patients with ACS [
14]. However, the clinical use of BBR is limited by its poor bioavailability. In the present study, we evaluate anti-atherosclerotic effects of BBR derivatives (known to have enhanced bioavailability)
in vitro and
in vivo, in comparison with BBR. Our
in vitro study showed that BBR and its derivatives, dhBBR and Di-MeBBR, decreased EMMPRIN expression and inhibited phosphorylation of p38 and JNK and nuclear translocation of NFκB p65 induced by oxLDL in macrophages, with dhBBR and Di-MeBBR showing greater effects in comparison with BBR. Our
in vivo study demonstrated that dhBBR and Di-MeBBR reduced aortic atherosclerotic lesion size, improved plaque stability (increased α-SMA and collagen content, and increased fibrous cap thickness), and decreased EMMPRIN, MMP-9, CD68, ICAM-1, VCAM-1 and NFκB p65 expression when compared with vehicle control in apoE−/− mice fed with the Western diet. However, BBR at the same dosage did not show beneficial effect on atherosclerosis in apoE−/− mice. Take together, our results have shown that dhBBR and Di-MeBBR have better anti-inflammatory effects than BBR, and dhBBR and Di-MeBBR, but not BBR, reduce atherosclerotic plaque size and improve plaque stability in apoE−/− mice. Therefore, dhBBR and Di-MeBBR have therapeutic advantages over BBR as an attractive adjunct therapy in patients with atherosclerosis.
Although our previous
in vitro and clinical studies have shown that BBR has anti-inflammatory effects, we have not explored whether BBR reduces atherosclerosis in animal models. In the present study, we have demonstrated that BBR derivatives at 10 mg/kg/d not only reduce plaque size, but also improve plaque stability in apoE−/− mice, however BBR at the same dosage had no significant anti-atherosclerotic effect. One of the earliest events in atherogenesis is the adhesion of monocytes to the vascular endothelium, which is mainly mediated by ICAM-1 and VCAM-1 [
25]. Following initial adhesion, monocytes subsequently infiltrate into the subendothelial space and differentiate into macrophages. Macrophages and foam cells contribute to the initiation and development of atherosclerosis by forming fatty streak and secreting a variety of inflammatory mediators [
4]. In this study, we found that both dhBBR and Di-MeBBR reduced CD68 expression in atherosclerotic plaques in apoE−/− mice, indicating reduced infiltration of macrophages. Di-MeBBR also decreased ICAM-1 and VCAM-1 expression in atherosclerotic plaques, thus inhibiting the initial step of monocyte recruitment. Although our previous clinical research showed BBR reduced plasma ICAM-1 and VCAM-1 in patients with ACS [
14], BBR had no effect on CD68, ICAM-1 and VCAM-1 in apoE−/− mice. Macrophages also contribute to plaque rupture by secreting MMPs that degrade extracellular matrix proteins and weaken the fibrous cap [
26],[
27]. Among various MMP species, MMP-9, mainly deriving from macrophages and foam cells, plays an important role in plaque rupture and ACS [
7],[
27],[
28]. EMMPRIN is an upstream inducer of MMPs [
6],[
22],[
23], and it induces MMP-9 synthesis in a paracrine or autocrine manner [
29]. We previously found that BBR inhibited MMP-9 and EMMRIN expression in macrophages. In this study, we further showed that dhBBR and Di-MeBBR, compared with BBR, had greater inhibitory effects on EMMPRIN expression in both macrophages stimulated with oxLDL and atherosclerotic plaques in apoE−/− mice. Di-MeBBR also inhibited MMP-9 expression in atherosclerotic plaques. Take together, BBR derivatives reduce plaque size and improve plaque stability through suppressing both the accumulation of macrophages and the production of EMMPRIN and MMP-9 by macrophages.
The protective roles of BBR derivatives against atherosclerosis are likely mediated by NFκB and MAPK signaling pathways. NFκB pathway is a major transcription factor that regulates gene expression of a wide variety of inflammatory mediators including cell adhesion molecules, chemokines, cytokines, and MMPs [
30], which are key players in the pathogenesis and development of atherosclerosis and its complications. There are a variety of stimuli which can activate NFκB including vascular injury, oxLDL, and cytokines etc. [
30]. One of the key steps in activating NFκB pathway is the stimulation of the IκB kinases [
31]. In unstimulated cells, NFκB canonical signaling pathway p65 is localized predominantly in the cytoplasm and remains inactivated through binding with its inhibitory protein, IkB-α. Upon oxLDL stimulation, IκB-α is phosphorylated and subsequently degraded, which allows p65 to be liberated from IkB-α. NFκB is then translocated into the nucleus where it binds to a specific sequence in the promoter of target genes, resulting in increased expression of target genes [
30]. NFκB may also be activated by MAPK signaling pathways [
32]. MAPK signaling pathways are known to play an important role in the pathogenesis of cardiac and vascular disease including atherosclerosis [
33]. Studies have shown that activation of JNK and p38, but not ERK1/2, is required for foam cell formation [
24],[
34],[
35]. Activation of JNK and p38 also induces gene expression of inflammatory mediators such as adhesion molecules and MMPs [
36]-[
38]. Our previous study found that BBR inhibited activation of NFκB and p38 induced by oxLDL in macrophages [
12],[
13]. The current study further demonstrated that dhBBR and Di-MeBBR exhibited greater inhibition on activation of NFκB and MAPK (p38 and JNK) in oxLDL-stimulated macrophages and on expression of NFκB p65 in atherosclerotic plaques in apoE−/− mice. BBR derivatives thus exert anti-inflammatory and anti-atherosclerotic effects by targeting NFκB and MAPK (p38 and JNK) signaling pathways.
Superiority of BBR derivatives over BBR is likely due to their enhanced bioavailability. The structure of BBR is extremely flat, which limits its absorption across the intestinal epithelia. In BBR derivatives, the D ring side chain is filled with various substituents, alternating the double bond of ring C and attaching different substituents at the 13-position and other positions [
16]. Changing to dhBBR opens up the structure, making it more amenable to uptake, which is substantiated by pharmokinetic data [
15]. Enhanced bioavailability of dhBBR leads to improved
in vivo efficacy on metabolism [
15]. Once absorbed, dhBBR is rapidly converted to BBR, so dhBBR is likely the active moiety. However, dhBBR can also be converted to BBR in the stomach, which would hinder
in vivo absorption and reduce its bioavailability [
15]. Blocking aromatization of dhBBR 8,8-disubstitution with an alkyl group optimizes dhBBR into more aqueous soluble and acid stable 8,8-dialkyldihydroberberine hydrochlorides. 8,8-Dimethyl-13,13a- dihydroberberine (Di-MeBBR) has been identified as a promising derivative. With improved aqueous solubility and acid stability, Di-MeBBR has significantly higher bioavailability without conversion back to BBR
in vivo. Di-MeBBR improves glucose and lipid metabolism in diet-induced obese mice at a relatively low dosage and has been shown to be more effective than dhBBR in db/db mice [
16]. In the present study, overall, BBR derivatives have better anti-inflammatory effects than BBR, and reduce atherosclerotic plaque size and instability in apoE−/− mice. There are some differences between Di-MeBBR and dhBBR in terms of improving plaque stability. Compared to vehicle control group, Di-MeBBR group has higher collagen content, whereas dhBBR group has higher α-SMA content and thicker fibrous cap. We have shown that Di-MeBBR, but not dhBBR or BBR, significantly reduces MMP-9 gene and protein expression in apoE−/− mice, which could lead to reduced collagen degradation and higher collagen content in Di-MeBBR group. α-SMA is primarily produced by smooth muscle cells. Whether dhBBR has the effect on smooth muscle cells needs further investigation. In addition, in previous studies [
12],[
13] and this study, we pretreated cells with BBR and its derivatives before oxLDL stimulation. Our next study will investigate whether treatment of BBR and its derivatives at the same time of or after oxLDL stimulation will exert similar anti-inflammatory effects.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JC and JC carried out immunohistochemical staining, real-time PCR and Western blotting, analyzed data, and wrote the manuscript. LF contributed to study design and data analysis and interpretation, and did critical revision of the manuscript. BL, QZ and YS participated in study design and coordination and helped to draft the manuscript. YW carried out flow cytometry and participated in mouse study. YL designed the study, analyzed data and helped to draft the manuscript. SM designed the study, performed experiments (immunohistochemical staining, real-time PCR and Western blotting), analyzed data and wrote the manuscript. All authors read and approved the final manuscript.