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Objective: To investigate the effects of apolipoprotein E deficiency (Apo E(-/-)) on plasma and lipoprotein distribution of sphingosine-1-phosphate (S1P) in mice. Methods: Five male or female Apo E(-/-) or wild type (WT) mice were fed with chow diet and sacrificed at 32-week-age and plasma was collected. The constituents of lipoprotein(very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL)) were separated by ultracentrifuge. The protein concentration of constituents was detected by BCA protein quantitative kit, and the S1P concentration in plasma and various lipoprotein constituents was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Western blot was used to determine the plasma, liver, and kidney protein expression of apolipoprotein M(Apo M), which is considered as specific ligand of S1P.The S1P concentration in plasma and various constituents of lipoprotein in the Apo E(-/-) mice was compared to respective WT mice. Results: (1)Plasma S1P content was significantly higher in the Apo E(-/-) groups than that of WT groups (male: (535.7±78.5)nmol/L vs. (263.3±22.0)nmol/L; female: (601.1±64.0)nmol/L vs. (279.0±33.9)nmol/L; all P <0.01). (2) Compared with WT mice, S1P content in non-HDL(LDL+ VLDL) was significantly higher in Apo E(-/-) mice (male: (504.9±52.8)nmol/L vs. (28.7±9.0)nmol/L; female: (427.7±27.4) vs. (27.8±4.7)nmol/L; after standardization of protein concentration, male: (385.0±41.2)pmol/mg protein vs. (71.4±6.6)pmol/mg protein; female: (330.2±22.0)pmol/mg protein vs. (67.2±12.1)pmol/mg protein; all P <0.01). (3) The expression of Apo M in plasma, liver and kidney was significantly higher in Apo E(-/-) groups than that of WT groups(all P <0.05). Conclusion: The deficiency of Apo E could lead to upregulated S1P expression in the non-HDL, the underlying mechanism might be the increased transfer of HDL into the non-HDL by Apo M-S1P.

Background: Ethanol extract of propolis (EEP), rich in flavones, has been known for various biological activities including antioxidant, antiinflammatory and antibiotic activities. Our previous studies have shown that EEP protects endothelial cells from oxidized low-density lipoprotein (ox-LDL)-induced apoptosis and inhibits atherosclerotic lesion development. In this present study, we explored the protective effect of EEP on ox-LDL-induced cytotoxicity in macrophages and specifically the endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP) pathway-mediated apoptosis., Methods: EEP was prepared and the total flavonoids content of EEP was determined by the colorimetric method of Chinese Standard (GB/T 20574-2006). The effects of EEP on lipid accumulation, cytotoxicity and apoptosis in RAW264.7 cells induced by ox-LDL or tunicamycin (TM, an ER stress inducer) were assayed using oil red O staining, MTT assay, flow cytometric analysis and so on. Immunofluorescence, Western blot and real time-PCR analysis were then used to further investigate the molecular mechanisms by which EEP protects macrophages from ox-LDL-induced apoptosis. 4-phenylbutyric acid (PBA), an ER stress inhibitor, was used as a positive control., Results: EEP (7.5, 15 and 30 mg/L) not only attenuated ox-LDL-induced lipid accumulation in RAW264.7 macrophages in a dose-dependent manner but also inhibited the decreased cell viability and the increased lactate dehydrogenase (LDH) leakage, caspase-3 activation and apoptosis induced by ox-LDL or tunicamycin (TM, a classical ER stress inducer), which were similar to 4-phenylbutyric acid (PBA, an inhibitor of ER stress) treatment. In addition, like PBA, EEP significantly suppressed the ox-LDL- or TM-induced activation of ER stress signaling pathway including the phosphorylation of double-stranded RNA-activated protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2α (eIF2α) as well as upregulation of glucose regulated protein 78 (GRP78) and the pro-apoptotic protein CHOP. Furthermore, EEP significantly suppressed ox-LDL intake by macrophages and the upregulation of CD36 induced by ox-LDL., Conclusion: These data indicate that EEP may protect macrophages from ox-LDL-induced apoptosis and the mechanism at least partially involves its ability to suppress the CD36-mediated ox-LDL intake and subsequent activation of ER stress-CHOP signalling pathway.

The present study was to investigate whether endoplasmic reticulum stress (ERS) was involved in oxidized low density lipoprotein (ox-LDL)-induced scavenger receptor A1 (SR-A1) upregulation in macrophages. RAW264.7 cells were pretreated with 20 mmol/L of 4-phenylbutyric acid (PBA) for 30 min and then treated with ox-LDL (50 mg/L) for 12 h or stimulated with 2 mg/L tunicamycin (TM) or 2 μmol/L thapsigagin (TG) for 4 h. In addition, RAW264.7 cells were incubated with 0.5, 1 and 2 mg/L TM for 4 h or treated with 2 mg/L TM for 1, 2 and 4 h, respectively. The intracellular total cholesterol (TC) content was measured using a tissue/cell total cholesterol assay kit. The protein and mRNA expressions of SR-A1 and glucose-regulated protein 78 (GRP78) were analyzed by Western blot and real-time PCR, respectively. Dil-ox-LDL uptake was detected using a microplate reader. The results showed that ox-LDL-induced cholesterol accumulation in macrophages was attenuated by PBA, an ERS inhibitor. Ox-LDL caused significant SR-A1 upregulation with concomitant activation of ERS as assessed by upregulation of GRP78, whereas PBA significantly inhibited the ox-LDL-induced SR-A1 upregulation (P < 0.05) and slightly decreased GRP78 expression by 39.3% (P = 0.057). TM, an ERS inducer, upregulated SR-A1 protein expression and ox-LDL uptake in dose- and time-dependent manner, but had no significant effect on SR-A1 mRNA level. However, the TM- or TG-induced SR-A1 upregulation and ox-LDL uptake were significantly mitigated by PBA. These data indicate that ERS plays a critical role in ox-LDL-induced SR-A1 upregulation, which in turn enhances the foam cell formation by uptaking more ox-LDL.