title: Ethanol extract of propolis protects macrophages from oxidized low density lipoprotein-induced apoptosis by inhibiting CD36 expression and endoplasmic reticulum stress-C/EBP homologous protein pathway.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Key Laboratory of Atherosclerosis in Universities of Shandong, Institute of Atherosclerosis, Taishan Medical University, Taian, 271000, China. liutianfangyu@163.com.
authors.forename: Hua
authors.initials: H
authors.lastname: Tian
authors.affiliation: Taishan Hospital of Shandong province, Taian, 271000, China. hongweisunyst@126.com.
authors.forename: Hong-Wei
authors.initials: HW
authors.lastname: Sun
authors.affiliation: Affiliated Hospital of Taishan Medical University, Taishan Medical University, Taian, 271000, China. gegewv_008@163.com.
authors.forename: Jia-Jun
authors.initials: JJ
authors.lastname: Zhang
authors.affiliation: Affiliated Hospital of Taishan Medical University, Taishan Medical University, Taian, 271000, China. drqingfeng@163.com.
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Atherosclerosis in Universities of Shandong, Institute of Atherosclerosis, Taishan Medical University, Taian, 271000, China. zhaoli20131234@126.com.
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Atherosclerosis in Universities of Shandong, Institute of Atherosclerosis, Taishan Medical University, Taian, 271000, China. gsd20060501@163.com.
authors.forename: Shou-Dong
authors.initials: SD
authors.lastname: Guo
authors.affiliation: Key Laboratory of Atherosclerosis in Universities of Shandong, Institute of Atherosclerosis, Taishan Medical University, Taian, 271000, China. 1228441486@qq.com.
authors.forename: Yan-Yan
authors.initials: YY
authors.lastname: Li
authors.affiliation: Key Laboratory of Atherosclerosis in Universities of Shandong, Institute of Atherosclerosis, Taishan Medical University, Taian, 271000, China. jiaopeng196@163.com.
authors.forename: Peng
authors.initials: P
authors.lastname: Jiao
authors.affiliation: Key Laboratory of Atherosclerosis in Universities of Shandong, Institute of Atherosclerosis, Taishan Medical University, Taian, 271000, China. tywanghao_2005@163.com.
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.affiliation: Key Laboratory of Atherosclerosis in Universities of Shandong, Institute of Atherosclerosis, Taishan Medical University, Taian, 271000, China. shucunqin@hotmail.com.
authors.forename: Shu-Cun
authors.initials: SC
authors.lastname: Qin
authors.affiliation: Key Laboratory of Atherosclerosis in Universities of Shandong, Institute of Atherosclerosis, Taishan Medical University, Taian, 271000, China. yst228@126.com.|College of Basic Medical Sciences, Taishan Medical University, Taian, 271000, China. yst228@126.com.
authors.forename: Shu-Tong
authors.initials: ST
authors.lastname: Yao
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D018955:CD36 Antigens / Q000378:metabolism*; D002460:Cell Line; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D011429:Propolis / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D051742:Transcription Factor CHOP / Q000378:metabolism*
chemical_list: D018955:CD36 Antigens; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717248:Hspa5 protein, mouse; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D051742:Transcription Factor CHOP; D011429:Propolis