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

BMC Complementary and Alternative Medicine

Shu-Tong Yao¹  Shu-Cun Qin⁴  Hao Wang⁴  Peng Jiao⁴  Yan-Yan Li⁴  Shou-Dong Guo⁴  Li Zhao⁴  Xiao-Wei Zhang³  Jia-Jun Zhang³  Hong-Wei Sun²  Hua Tian⁴ 

[1] College of Basic Medical Sciences, Taishan Medical University, Taian 271000, China;Taishan Hospital of Shandong province, Taian 271000, China;Affiliated Hospital of Taishan Medical University, Taishan Medical University, Taian 271000, China;Key Laboratory of Atherosclerosis in Universities of Shandong, Institute of Atherosclerosis, Taishan Medical University, Taian 271000, China

关键词: Apoptosis;    Macrophage;    Oxidized low density lipoprotein;    C/EBP homologous protein;    Endoplasmic reticulum stress;    Ethanol extract of propolis;   

Others  :  1220033
DOI  :  10.1186/s12906-015-0759-4

 received in 2015-02-01, accepted in 2015-07-02,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Tian et al.

【 图 表 】

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【 参考文献 】

• [1]Thorp E, Tabas I. Mechanisms and consequences of efferocytosis in advanced atherosclerosis. J Leukoc Biol. 2009; 86(5):1089-95.
• [2]Tabas I. Macrophage apoptosis in atherosclerosis: consequences on plaque progression and the role of endoplasmic reticulum stress. Antioxid Redox Signal. 2009; 11(9):2333-9.
• [3]Tiwari RL, Singh V, Barthwal MK. Macrophages: an elusive yet emerging therapeutic target of atherosclerosis. Med Res Rev. 2008; 28(4):483-544.
• [4]Farooqui T, Farooqui AA. Beneficial effects of propolis on human health and neurological diseases. Front Biosci (Elite Ed). 2012; 4:779-93.
• [5]Medic’-Saric’ M, Rastija V, Bojic’ M, Males Z. From functional food to medicinal product: systematic approach in analysis of polyphenolics from propolis and wine. Nutr J. 2009; 8:33-50. BioMed Central Full Text
• [6]Teixeira EW, Message D, Negri G, Salatino A, Stringheta PC. Seasonal varia -tion, chemical composition and antioxid an tactivity of Brazilian propolis samples. Evid Based Complement Alternat Med. 2010; 7(3):307-15.
• [7]Bankova V, Popova M, Trusheva B. Propolis volatile compounds: chemical diversity and biological activity: a review. Chem Cent J. 2014; 8:28-35. BioMed Central Full Text
• [8]Blonska M, Bronikowska J, Pietsz G, Czuba ZP, Scheller S, Krol W. Effects of ethanol extract of propolis (EEP) and its flavones on inducible gene expression in J774A.1 macrophages. J Ethnopharmacol. 2004; 91(1):25-30.
• [9]Wang K, Ping S, Huang S, Hu L, Xuan H, Zhang C, Hu F. Molecular mechanisms underlying the in vitro anti-inflammatory effects of a flavonoid-rich ethanol extract from Chinese propolis (poplar type). Evid Based Complement Alternat Med. 2013; 2013:127672-82.
• [10]Fang Y, Sang H, Yuan N, Sun H, Yao S, Wang J, Qin S. Ethanolic extract of propolis inhibits atherosclerosis in ApoE-knockout mice. Lipids Health Dis. 2013; 12:123-8. BioMed Central Full Text
• [11]Yu Y, Si Y, Song G, Luo T, Wang J, Qin S. Ethanolic extract of propolis promotes reverse cholesterol transport and the expression of ATP-binding cassette transporter A1 and G1 in mice. Lipids. 2011; 46(9):805-11.
• [12]Fang Y, Li J, Ding M, Xu X, Zhang J, Jiao P, Han P, Wang J, Yao S. Ethanol extract of propolis protects endothelial cells from oxidized low density lipoprotein-induced injury by inhibiting lectin-like oxidized low density lipoprotein receptor-1-mediated oxidative stress. Exp Biol Med. 2014; 239(12):1678-87.
• [13]Gargalovic PS. Atherogenesis on the chopping block. Cell Metab. 2009; 9(5):399-401.
• [14]Thorp E, Li G, Seimon TA, Kuriakose G, Ron D, Tabas I. Reduced apoptosis and plaque necrosis in advanced atherosclerotic lesions of Apoe-/- and Ldlr-/- mice lacking CHOP. Cell Metab. 2009; 9(5):474-81.
• [15]Tsukano H, Gotoh T, Endo M, Miyata K, Tazume H, Kadomatsu T, Yano M, Iwawaki T, Kohno K, Araki K, Mizuta H, Oike Y. The endoplasmic reticulum stress-C/EBP homologous protein pathway-mediated apoptosis in macrophages contributes to the instability of atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2010; 30(10):1925-32.
• [16]Zhou AX, Tabas I. The UPR in atherosclerosis. Semin Immunopathol. 2013; 35(3):321-32.
• [17]Yao S, Zong C, Zhang Y, Sang H, Yang M, Jiao P, Fang Y, Yang N, Song G, Qin S. Activating transcription factor 6 mediates oxidized LDL-induced cholesterol accumulation and apoptosis in macrophages by up-regulating CHOP expression. J Atheroscler Thromb. 2013; 20(1):94-107.
• [18]Yao S, Yang N, Song G, Sang H, Tian H, Miao C, Zhang Y, Qin S. Minimally modified low-density lipoprotein induces macrophage endoplasmic reticulum stress via toll-like receptor 4. Biochim Biophys Acta. 2012; 1821(7):954-63.
• [19]Yao S, Sang H, Song G, Yang N, Liu Q, Zhang Y, Jiao P, Zong C, Qin S. Quercetin protects macrophages from oxidized low-density lipoprotein- induced apoptosis by inhibiting the endoplasmic reticulum stress-C/EBP homologous protein pathway. Exp Biol Med (Maywood). 2012; 237(7):822-31.
• [20]Yao S, Miao C, Tian H, Sang H, Yang N, Jiao P, Han J, Zong C, Qin S. Endoplasmic Reticulum Stress Promotes Macrophage-derived Foam Cell Formation by Up-regulating Cluster of Differentiation 36 (CD36) Expression. J Biol Chem. 2014; 289(7):4032-42.
• [21]Kunjathoor VV, Febbraio M, Podrez EA, Moore KJ, Andersson L, Koehn S, Rhee JS, Silverstein R, Hoff HF, Freeman MW. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J Biol Chem. 2002; 277(51):49982-8.
• [22]Salvamani S, Gunasekaran B, Shaharuddin NA, Ahmad SA, Shukor MY. Antiartherosclerotic effects of plant flavonoids. Biomed Res Int. 2014; 2014:480258-68.
• [23]Song YH, Cai H, Gu N, Qian CF, Cao SP, Zhao ZM. Icariin attenuates cardiac remodelling through down-regulating myocardial apoptosis and matrix metalloproteinase activity in rats with congestive heart failure. J Pharm Pharmacol. 2011; 63(4):541-9.
• [24]Lee YM, Cheng PY, Chen SY, Chung MT, Sheu JR. Wogonin suppresses arrhythmias, inflammatory responses, and apoptosis induced by myocardial ischemia/reperfusion in rats. J Cardiovasc Pharmacol. 2011; 58(2):133-42.
• [25]Zhang ZJ, Cheang LC, Wang MW, Lee SM. Quercetin exerts a neuro- protective effect through inhibition of the iNOS/NO system and pro-inflammation gene expression in PC12 cells and in zebrafish. Int J Mol Med. 2011; 27(2):195-203.
• [26]Mojzisova G, Sarissky M, Mirossay L, Martinka P, Mojzis J. Effect of flavonoids on daunorubicin-induced toxicity in H9c2 cardiomyoblasts. Phytother Res. 2009; 23(1):136-9.
• [27]Choi JS, Kang SW, Li J, Kim JL, Bae JY, Kim DS, Shin SY, Jun JG, Wang MH, Kang YH. Blockade of oxidized LDL-triggered endothelial apoptosis by quercetin and rutin through differential signaling pathways involving JAK2. J Agric Food Chem. 2009; 57(5):2079-86.
• [28]Ferri KF, Kroemer G. Organelle-specific initiation of cell death pathways. Nat Cell Biol. 2001; 3(11):E255-63.
• [29]Miyazaki Y, Kaikita K, Endo M, Horio E, Miura M, Tsujita K, Hokimoto S, Yamamuro M, Iwawaki T, Gotoh T, Ogawa H, Oike Y. C/EBP homologous protein deficiency attenuates myocardial reperfusion injury by inhibiting myocardial apoptosis and inflammation. Arterioscler Thromb Vasc Biol. 2011; 31(5):1124-32.
• [30]Feng B, Yao PM, Li Y, Devlin CM, Zhang D, Harding HP, Sweeney M, Rong JX, Kuriakose G, Fisher EA, Marks AR, Ron D, Tabas I. The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages. Nat Cell Biol. 2003; 5(9):781-92.
• [31]Scull CM, Tabas I. Mechanisms of ER stress-induced apoptosis in atherosclerosis. Arterioscler Thromb Vasc Biol. 2011; 31(12):2792-7.
• [32]Li F, Guo Y, Sun S, Jiang X, Tang B, Wang Q, Wang L. Free cholesterol-induced macrophage apoptosis is mediated by inositol- requiring enzyme 1 alpha-regulated activation of Jun N-terminal kinase. Acta Biochimica et Biophysica Sinica (Shanghai). 2008; 40(3):226-34.
• [33]Collot-Teixeira S, Martin J, McDermott-Roe C, Poston R, McGregor JL. CD36 and macrophages in atherosclerosis. Cardiovasc Res. 2007; 75(3):468-77.
• [34]Yu XH, Fu YC, Zhang DW, Yin K, Tang CK. Foam cells in atherosclerosis. Clin Chim Acta. 2013; 424:245-52.
• [35]Chávez-Sánchez L, Garza-Reyes MG, Espinosa-Luna JE, Chávez-Rueda K, Legorreta-Haquet MV, Blanco-Favela F. The role of TLR2, TLR4 and CD36 in macrophage activation and foam cell formation in response to oxLDL in humans. Hum Immunol. 2014; 75(4):322-9.
• [36]Park YM. CD36, a scavenger receptor implicated in atherosclerosis. Exp Mol Med. 2014; 46:e99-105.
• [37]Syväranta S, Alanne-Kinnunen M, Oörni K, Oksjoki R, Kupari M, Kovanen PT, Helske-Suihko S. Potential pathological roles for oxidized low-density lipoprotein and scavenger receptors SR-AI, CD36, and LOX-1 in aortic valve stenosis. Atherosclerosis. 2014; 235(2):398-407.