Salvianolic acid B inhibits macrophage uptake of modified low density lipoprotein (mLDL) in a scavenger receptor CD36-dependent manner

Abstract

CD36, a class B scavenger receptor, has been implicated in the pathogenesis of a host of vascular inflammatory diseases. Through a high-throughput screening (HTS) assay for CD36 antagonist, we previously identified salvianolic acid B (SAB), a hydrophilic component derived from the herb Danshen, as a potential candidate. Danshen, the dried roots of Salvia miltiorrhiza, has been widely used in China for the prevention and treatment of atherosclerosis-related disorders. Previous studies showed that SAB acted as an anti-oxidant by preventing lipid peroxidation and oxidized LDL (oxLDL) formation. The present study was to investigate the specificity and efficacy of SAB in the inhibition of CD36-mediated lipid uptake. SAB reduced modified LDL (mLDL) uptake in a dose-dependent manner in phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 and RAW 264.7 cells. In the CD36 silenced THP-1 cells, SAB had no effect in reducing mLDL uptake, whereas its overexpression in CHO cells reinstates the effect, indicating a specific involvement of SAB in antagonizing the CD36's function. Surface plasmon resonance (SPR) analysis revealed a direct binding of SAB to CD36 with a high affinity (K_D = 3.74 μM), confirming physical interactions of SAB with the receptor. Additionally, SAB reduced oxLDL-induced CD36 gene expression in the cultured cell lines and primary macrophages. In ApoE KO mice fed a high fat diet, SAB reduced CD36 gene expression and lipid uptake in macrophages, showing its ability to antagonize CD36 pathways in vivo. These results demonstrate that SAB is an effective CD36 antagonist and suggest SAB as a potential anti-atherosclerotic agent.

Introduction

CD36, a member of the scavenger receptor class B family, has been thought to play a critical role in foam cell formation and inflammatory vascular disease [1], [2], [3], [4], [5], [6]. Although a role of CD36 in atherosclerosis has been controversial [4], [5], [7], [8], studies using CD36 knock-out (KO) mice and pharmacological inhibitors suggest that CD36 is a potential therapeutic target to treat inflammatory diseases such as atherosclerosis and stroke [9], [10], [11], [12]. CD36 is expressed on many types of cells and tissues, including monocytes/macrophages [13], [14], adipocytes [15], platelets [16], endothelial cells [17], cardiac and skeletal muscle [18], and retinal pigment epithelial cells [19]. The receptor shows high affinity for lipid-based ligands including oxidized/modified low density lipoprotein (oxLDL/mLDL), long chain fatty acids (LCFA) and structurally distinct non-lipid-based ligands such as thrombospondins, fibrillar β-amyloid, and apoptotic cells [20], [21]. It is involved in regulating an array of functions in different physiological and pathological processes such as atherosclerosis, innate immunity, inflammation, angiogenesis, and lipid metabolism [21], [22].

During the last decade, several pharmacological agents have been identified in antagonizing CD36 activity. Hexarelin, a hexapeptide of the growth hormone releasing peptide (GHRP) family, was reported to be able to act as a CD36 ligand [23]. Treatment of mice with hexarelin or EP 80317, a structurally related analog devoid of any growth hormone releasing activity, resulted in a marked decrease in atherosclerotic lesions which was CD36-dependent [11], [24]. Further studies by Demers et al. [23] showed that the binding domain of hexarelin on CD36 overlaps that of oxLDL, suggesting that hexarelin exerts its protective effect by blocking CD36-mediated uptake of oxLDL. Besides targeting oxLDL binding sites, other approaches have been used to modulate CD36 expression or its downstream effect. For instance, SS31, one of a new class of anti-oxidant SS peptides [25], [26], has been shown to attenuate oxLDL-induced CD36 expression and foam cell formation in mouse peritoneal macrophages and CD36-mediated ischemic injury in vivo [27]. Some other anti-oxidants, such as α-tocopherol or polyphenolic compounds from plant, were reported not only to protect LDL from oxidation but also to reduce the expression of CD36 and uptake of oxLDL into macrophages [28], [29], [30], [31], [32], [33].

Salvianolic acid B (SAB) is a water-soluble polyphenolic anti-oxidant isolated from the root of red-rooted salvia (Salvia miltiorrhiza Bunge), the Chinese herb Danshen. Danshen is widely used for the prevention and treatment of vascular diseases including atherosclerosis and stroke in China and other Asian countries. Early research focused mainly on its lipophilic components such as tanshinone IIA and cryptotanshinone, while recent studies have given more attention to its hydrophilic components, especially SAB [34]. SAB, also known as lithospermic acid B or tanshinoate B, can prevent LDL from oxidation and inhibit lipid peroxidation [35], [36], [37]. However, this study revealed that SAB can also antagonize the binding of oxLDL to CD36 and prevent the following foam cell formation when the LDL has already turned to oxLDL. In addition, SAB attenuates multiple inflammatory factors or inhibits the associated pathways including the expression of cyclooxygenase-2 [38], MMP-2 and MMP-9 [39] and TGF-β/Smad [40] or NF-κB signaling pathways [41], which may be involved in CD36-related pathway and trigger the atherosclerotic and other vascular disease.

Through a high-throughput screening (HTS) for CD36 antagonists based on the competition of soluble CD36-oxLDL binding assay [42], SAB was identified as a potential candidate molecule that antagonized the CD36-oxLDL binding. The present study investigates the specificity and efficacy of SAB in blocking CD36 pathways. Here we report that SAB inhibits CD36 function and expression in culture and in hyperlipidemic ApoE KO mice. The study provides evidence that SAB is a specific CD36 antagonist both in vitro and in vivo.