Salvianolic Acid B Accelerated ABCA1-dependent Cholesterol Efflux By Targeting PPAR-γ And LXRα

Backgrounds and aimsDyslipidemia is one of the components of metabolic syndrome, and is closely related to many diseases such as obesity, type 2 diabetes, hypertension, coronary heart disease and stroke. Long term dyslipidemia can lead to atherosclerosis, increase the incidence and mortality of cardiovascular disease. With the improvement of living standards and lifestyle changes, the prevalence of dyslipidemia in China has been significantly increased. The main function of LDL is to transport cholesterol to the liver tissue, which is the important lipoprotein in atherosclerosis. LDL has a stronger role in atherosclerosis after oxidation or other chemical modification. A number of studies have demonstrated that lowering plasma LDL can reduce the incidence and mortality of coronary heart disease. Cholesterol-loaded macrophages, which subsequently developed into foam cells, are the most important participant of the atherosclerotic lesion, and cholesterol efflux from macrophages can protect against the progression of atherosclerosis. Evidences have shown the critical effect that cholesterol efflux played in preventing atherosclerosis in humans and animals.Reverse cholesterol transport is a pathway which is constituted of the efflux of excess cellular cholesterol from peripheral tissues and the return of the cholesterol to the liver for excretion. RCT, as well as cholesterol efflux, has been confirmed to be the major mechanism by which HDL protects against atherosclerosis. Cholesterol has been thought difficult to be catabolized by most peripheral cells and tissues, and thus its metabolism needs effluxing transporters, such as ATP binding cassette transporter A1 (ABCA1) and extra cellular acceptors, such as ApoA I and HDL. ABCA1 is an integrated membrane protein, which can promote the free cholesterol and phospholipids in the cell efflux to ApoA I. ABCA1 is a key regulator and plays a key role in the reverse cholesterol transport.The expression of ABCA1 is regulated by many factors in vivo, Such as peroxisome proliferator activated receptor (PPARs), liver orphan receptor (LXR) and retinol X receptor (RXR), which can regulate the expression of ABCA1 at the transcriptional level. PPARs includes three subtypes of PPAR-α, PPAR-Pand PPAR-y. PPAR-y is one of the most important members of the nuclear receptor family and is involved in the regulation of macrophage lipid metabolism and cell differentiation. As a key transcriptional regulator, PPAR-y can regulate the expression of many genes that mediate cholesterol transport. LXR is a member of the nuclear receptor superfamily, with subtypes of LXRa and LXRβ.LXRa can regulate many target genes of cholesterol metabolism in the liver, macrophages and intestine. LXRα agonist can up-regulate the expression of ABCA1 and lead to the efflux of cholesterol from cells.Salvianolic acid B (Sal B). also known as lithospermic acid B or tanshinoate B, is isolated from the root of the Chinese herb Danshen (Salvia miltiorrhiza Bunge). In the past several years. Sal B. as well as the other substance extracted from Danshen. have been widely used for the prevention and treatment of atherosclerotic vascular diseases including coronary artery disease and stroke in China even the western countries. As a water-soluble compound of three molecules danshennol and one molecule of caffeic acid, Sal B is the main constituent of Salvia phenolic acid substances. The previous studies have confirmed that Sal B can improve the blood hemorrheology, decrease oxidative injury and repair the function of blood vessel endothelium. Sal B was able to scavenge against free hydroxyl radicals (HO), superoxide anion radicals (O2), and inhibit lipid peroxidation, and thus it has been seen as an antioxidant. Sal B also inhibited macrophage uptake of modified low density lipoprotein in a scavenger receptor CD36-dependent manner and was confirmed to be an effective CD36 antagonist. Sal B was also believed to inhibit platelet aggregation and activation in patients and stabilizes plaque by reducing MMP-2, MMP-9 and improve prognosis. The potential of Sal B in the inhibition LDL oxidation, decrease of plasma cholesterol level, reduction of endothelial damage and the severity of atherosclerosis in diet-induced hypercholesteremic rabbits have been also elucidated. Based on these results, cholesterol efflux has been confirmed to be the major mechanism of lipid metabolism by which HDL protects against atherosclerosis, while Sal B played many kinds of important roles in lipid metabolism and protection against atherosclerosis.Therefore, cholesterol reverse transport was confirmed to be a major mechanism for HDL to prevent atherosclerosis. Sal B plays an important role in lipid metabolism and anti-atherosclerosis. However, there is still no investigation focused on the effects of Sal B on cholesterol efflux. The present study was designed to clarify the relationship of Sal B and cholesterol efflux.Materials and methods1. THP-1 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum(BSA), streptomycin (100 mg/mL), and penicillin (100 U/mL) at 37℃ in a humidified atmosphere 5% CO2. When the concentration of THP-1 cells in the 6-well culture dishes reached 5×10’cells per well, medium containing 160nmol/mL of PMA was added into the cells for 72h to stimulate the cells differentiation into macrophages.2. THP-1 macrophages were exposed to 50mg/L of ox-LDL and [3H] cholesterol (1.0μCi/mL) for 24 h in RPMI 1640 supplemented with 0.2% BSA to induce cholesterol-loaded macrophages.3. Cholesterol-loaded THP-1 macrophages ere exposed to different concentrations of Sal B for 24 h or 10μM Sal B for varying times in the presence of ApoA Ⅰ, HDL2 or HDL3. For analysis of cholesterol efflux to ApoA Ⅰ, HDL2 and HDL3, the medium and the THP-1 macrophages were collected respectively, and the radioactive content in and out of the cells was determined by liquid scintillation.4. THP-1 macrophages were treated with different concentrations (0,0.1, 1 and 10μM) of Sal B for 24 hours and Sal B of 10μM was used to stimulate the cells for different time (0,3,6,12 and 24h). ABCA1 was detected by western blot and real-time PCR respectively at protein and mRNA levels.5. Western blot was performed to examine the expression of PPAR-γ and LXRα in THP-1 macrophages after the stimulation of Sal B.6. Western blot and real-time PCR were performed to examine the expression of ABCA1 after the agonists and antagonists of PPAR-γ and LXRα were respectively applied.7. The agonists and antagonists of PPAR-γ and LXRα were respectively applied again to treat the cholesterol-loaded THP-1 macrophages with Sal B for 24h for analysis of the effect of Sal B on cholesterol efflux.Results1. Sal B increases cholesterol efflux to ApoA I, HDL2 and HDL3 in THP-1 macrophages. The cholesterol-loaded THP-1 macrophages were exposed to different concentrations of Sal B (0.0.1,1 and 10μM) for 24h in the presence of ApoA Ⅰ. HDL2 or HDL3 in the medium. 1μM of Sal B could significantly accelerate cholesterol efflux to ApoA I and HDL2 (P<0.05). and 10μM of Sal B dramatically accelerate cholesterol efflux to ApoA I and HDL2 approximately 2.5 and 3.2 folds respectively compared with control group (P<0.01). Sal B could also increase cholesterol efflux to HDL3 (P<0.05) when its concentration reached 10μM.Subsequently,10 μM of Sal B was used to stimulate the cholesterol-loaded THP-1 macrophages for varying times (0,3,6,12,24h). Sal B could not exert its role on cholesterol efflux to the three mediums for 6h, while cholesterol efflux was significantly enhanced after 12h incubation (P<0.05), and dramatically enhanced after 24h to ApoA I and HDL2 (P<0.01).2. The expression of ABCA1 in THP-1 macrophages at both protein and mRNA levels. The cells were treated with different concentrations (0,0.1,1 and 10μM) of Sal B for 24 hours. ABCA1 was detected by western blot and real-time PCR respectively at protein and mRNA levels. Compared with the control group, the expression of ABCA1 was significantly increased under the stimulation of 1 (P<0.05) and 10μM Sal B (P<0.01), while 0.1μM of Sal B failed to enhance the expression of ABCA1. Simultaneously, real-time PCR was used to examine the mRNA level of ABCA1 after the treatment of Sal B, and the similar results were obtained. Furthermore, Sal B of 10μM was used to stimulate the cells for different time (0,3,6,12 and 24h). The relative expression of ABCA1 was not increased significantly compared with the control group until the stimulation time was prolonged to more than 12h (P<0.01). In the other hand, the relative expression of ABCA1 mRNA was significantly increased from 6h.3. The expression of PPAR-y and LXRa. western blot was performed to examine the expression of PPAR-y and LXRa in THP-1 macrophages after the stimulation of Sal B. The expression of PPAR-y was significantly increased under the stimulation of 1 and 10μM Sal B for 24h (P<0.01), while 0.1μM of Sal B didn’t statistically enhance the expression of PPAR-y. In the following experiments involved in the expression of LXRa, we also found that Sal B played a similar regulating role just as PPAR-y.4. The expression of ABCA1 after the agonists and antagonists of PPAR-y and LXRa were respectively applied. Both the PPAR-y antagonist GW9662 and LXRa antagonist GGPP dramatically inhibited the upregulating effect of Sal B on the expression of ABCA1 at both protein and mRNA levels (P<0.01), while PPAR-y agonist rosiglitazone and LXRa agonist GW3965 could dramatically enhance the expression of ABCA1 (P<0.01).5. Specific inhibition and activation of PPAR-y and LXRa attenuates the effect of Sal B on cholesterol efflux. The agonists and antagonists of PPAR-y and LXRa were respectively applied again to treat the THP-1 macrophages. After 24h incubation, Sal B could exert its role on cholesterol efflux to ApoA-I (P<0.01), HDL2 (P<0.01) and HDL3 (P<0.05), while cholesterol efflux was significantly inhibited after co-incubation with Sal B and PPAR-y antagonist GW9662 or LXRa antagonist GGPP. When the THP-1 macrophages were treated with PPAR-y agonist rosiglitazone and LXRa agonist GW3965, cholesterol efflux to the three mediums were all dramatically increased (P<0.01).Conclusions1. Sal B could concentration-and time-dependently accelerate cholesterol efflux to ApoA I, HDL2 and HDL3 in the cholesterol-loaded THP-1 macrophages.2. Sal B enhances the expression of ABCA1 in THP-1 macrophages at both protein and mRNA levels.3. The expression of ABCA1 induced by Sal B is mediated through PPAR-y and LXRa.4. Sal B promotes cholesterol efflux in THP-1 macrophages through ABCA1/PPAR-γ/LXRa pathway.