Mengjun Hou

Anthocyanins Induce Cholesterol Efflux from Mouse Peritoneal Macrophages THE ROLE OF THE PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR γ-LIVER X RECEPTOR α-ABCA1 PATHWAY

It is widely accepted that stimulation of reverse cholesterol transport, the efflux of excess cholesterol from peripheral tissues and transferring it to the liver for biliary excretion, is becoming an important component in reducing excess cholesterol deposition in atherosclerotic plaques. The ATP-binding cassette transporter has been identified as a key regulator of macrophage cholesterol efflux and apoAI-mediated reverse cholesterol transport. In vivo studies have documented anthocyanins, a large group of naturally phenolic compounds rich in plants, possess substantial capacities in improving plasma cholesterol levels. In this study, we investigated the potential role of anthocyanins in modulating cholesterol efflux from mouse peritoneal macrophages and macrophage-derived foam cells and the possible molecular mechanism linking ABCA1 to cholesterol efflux. Incubation of the mouse peritoneal macrophages and macrophage-derived foam cells with cyanidin-3-O-β-glucoside and peonidin-3-O-β-glucoside led to dose-dependent (1–100 μm) induction in cholesterol efflux and ABCA1 mRNA expression, and this effect could be blocked by the ABCA1 inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, disodium salt, and a general inhibitor of gene transcription actinomycin D. Treatment of the macrophages with anthocyanins also activated peroxisome proliferator-activated receptor γ, liver X receptor α mRNA expression, and their mediated gene expression. Addition of geranylgeranyl pyrophosphate ammonium salt or GW9662 markedly inhibited the anthocyanin-induced increase of ABCA1 gene expression and apoAI-mediated cholesterol efflux. These data demonstrated that anthocyanin induces cholesterol efflux from mouse peritoneal macrophages and macrophage-derived foam cells and that stimulation of cholesterol efflux by anthocyanin is mediated, at least in part, by peroxisome proliferator-activated receptor γ-liver X receptor α-ABCA1 signaling pathway activation.

Cyanidin-3-O-β-glucoside inhibits iNOS and COX-2 expression by inducing liver X receptor alpha activation in THP-1 macrophages

Anthocyanins belong to a large and widespread group of water-soluble phytochemicals and exhibit potent antioxidative and anti-inflammatory properties; however, the molecular mechanisms of these biochemical actions mediated by anthocyanins remain unclear. In this study, our data show that pretreatment of THP-1 macrophages with Cyanidin-3-O-beta-glucoside (C3G) for 12 h can enhance the expression and transcriptional activities of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha). Furthermore, pretreatment of these cells with C3G for 12 h causes dose-dependent inhibition of lipopolysaccharide (LPS)-induced nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both the mRNA and protein levels together with a decrease in nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production. Consequently, addition of geranylgeranyl pyrophosphate ammonium salt (GGPP), an LXRalpha antagonist, significantly downregulates the inhibitory effect of C3G on LPS-induced iNOS and COX-2 expression in THP-1 macrophages, whereas the PPARgamma antagonist GW9662 has no effect. Further investigation revealed that LXRalpha might interfere with LPS-induced iNOS and COX-2 expression by suppressing the functional activation of nuclear factor-kappaB (NF-kappaB), not - as was previously proposed - by reducing NF-kappaB nuclear translocation. Taken together, these results indicate that LXRalpha activation has an essential role in the anti-inflammatory property of C3G. Moreover, they provide new insight into the molecular basis for the anti-inflammatory property of anthocyanins.

Anthocyanin Prevents CD40-Activated Proinflammatory Signaling in Endothelial Cells by Regulating Cholesterol Distribution

Objective—Intracellular tumor necrosis factor receptor-associated factors (TRAFs) translocation to lipid rafts is a key element in CD40-induced signaling. The purpose of this study was to investigate the influence of anthocyanin on CD40-mediated proinflammatory events in human endothelial cells and the underlying possible molecular mechanism. Methods and Results—Treatment of endothelial cells with anthocyanin prevented from CD40-induced proinflammatory status, measured by production of IL-6, IL-8, and monocyte chemoattractant protein-1 through inhibiting CD40-induced nuclear factor-&kgr;B (NF-&kgr;B) activation. TRAF-2 played pivotal role in CD40–NF-&kgr;B pathway as TRAF-2 small interference RNA (siRNA) diminished CD40-induced NF-&kgr;B activation and inflammation. TRAF-2 overexpression increased CD40-mediated NF-&kgr;B activation. Moreover, TRAF-2 almost totally recruited to lipid rafts after stimulation by CD40 ligand and depletion of cholesterol diminished CD40-mediated NF-&kgr;B activation. Exposure to anthocyanin not only interrupted TRAF-2 recruitment to lipid rafts but also decreased cholesterol content in Triton X-100 insoluble lipid rafts. However, anthocyanin did not influence the interaction between CD40 ligand and CD40 receptor. Conclusions—Our findings suggest that anthocyanin protects from CD40-induced proinflammatory signaling by preventing TRAF-2 translocation to lipid rafts through regulation of cholesterol distribution, which thereby may represent a mechanism that would explain the anti-inflammatory response of anthocyanin.

Anthocyanin attenuates CD40-mediated endothelial cell activation and apoptosis by inhibiting CD40-induced MAPK activation

CD40-mediated inflammatory signaling is a potent activator of endothelial cells (ECs) and effective in triggering the pathogenesis of atherosclerosis, a chronic inflammatory disease. Anthocyanin is considered to exert potent cardiovascular-protective effect partially through its anti-inflammatory property, however, the precise mechanism is still unknown. Here we chose cultured human umbilical vein endothelial cells (HUVECs) to explore the influence of anthocyanin on CD40-mediated endothelial activation and apoptosis and the underlying mechanism. Stimulation of human primary HUVECs by CD40 with its physiological ligand CD40L not only augmented MMP-1, -9 secretion and promoted MMP-1, -9 activities, but also induced endothelial cell apoptosis and death. Treatment of ECs with anthocyanins cyanidin-3-O-beta-glucoside (Cy-3-g) and peonidin-3-O-beta-glucoside (Pn-3-g) prevents CD40-induced endothelial activation by inhibiting production of proinflammatory cytokines and matrix metalloproteinases (MMPs). In addition, exposure to anthocyanins inhibits CD40-induced endothelial apoptosis. Anthocyanins also decreased activation of JNK and p38 induced by CD40. Collectively, our findings suggested that the inhibition of JNK and p38 activation interrupts CD40 induced endothelial cell activation and apoptosis, which thereby may represent a mechanism that would explain the anti-inflammatory response of anthocyanin and its athero-protective function.

Folic Acid Represses Hypoxia-Induced Inflammation in THP-1 Cells through Inhibition of the PI3K/Akt/HIF-1α Pathway

Though hypoxia has been implicated as a cause of inflammation, the underlying mechanism is not well understood. Folic acid has been shown to provide protection against oxidative stress and inflammation in patients with cardiovascular disease and various models approximating insult to tissue via inflammation. It has been reported that hypoxia-induced inflammation is associated with oxidative stress, upregulation of hypoxia-inducible factor 1-alpha (HIF-1α), and production of pro-inflammatory molecules. Whether folic acid protects human monocytic cells (THP-1 cells) against hypoxia-induced damage, however, remains unknown. We used THP-1 cells to establish a hypoxia-induced cellular injury model. Pretreating THP-1 cells with folic acid attenuated hypoxia-induced inflammatory responses, including a decrease in protein and mRNA levels of interleukin (IL)-1β and tumor necrosis factor-alpha (TNF-α), coupled with increased levels of IL-10. Folic acid also reduced hypoxia-induced Akt phosphorylation and decreased nuclear accumulation of HIF-1α protein. Both LY294002 (a selective inhibitor of phosphatidyl inositol-3 kinase, PI3K) and KC7F2 (a HIF-1α inhibitor) reduced levels of hypoxia-induced inflammatory cytokines. We also found that insulin (an Akt activator) and dimethyloxallyl glycine (DMOG, a HIF-1α activator) induced over-expression of inflammatory cytokines, which could be blocked by folic acid. Taken together, these findings demonstrate how folic acid attenuates the hypoxia-induced inflammatory responses of THP-1 cells through inhibition of the PI3K/Akt/HIF-1α pathway.