β2GPI/aβ2GPI通过激活TLR4抑制THP-1巨噬细胞对氧化型低密度脂蛋白的摄取
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摘要
目的探讨β2糖蛋白I/抗β2糖蛋白I抗体复合物(β2GPI/aβ2GPI)对巨噬细胞脂质摄取功能和B型清道夫受体CD36表达的影响,以及Toll样受体4(TLR4)在该过程中的作用。方法用100 ng/mL佛波酯(PMA)将THP-1人单核细胞诱导为THP-1巨噬细胞,通过形态学变化和1,1'-二十八烷基-3,3,3',3'-四甲基吲哚碳花菁高氯酸盐标记的氧化低密度脂蛋白(DiI-oxLDL)吞噬试验加以鉴定。用RPMI1640培养液、β2GPI、抗β2糖蛋白I抗体(aβ2GPI)和β2GPI/aβ2GPI处理THP-1巨噬细胞,实时荧光定量PCR和Western blot法检测TLR4 mRNA和蛋白水平。用RPMI1640培养液、氧化低密度脂蛋白(oxLDL)、oxLDL联合β2GPI/aβ2GPI和oxLDL联合LPS处理THP-1巨噬细胞,油红O染色观察胞内脂质积累,免疫荧光细胞化学染色法检测CD36的表达,实时荧光定量PCR检测CD36 mRNA水平,Western blot法检测CD36蛋白水平。利用TLR4阻断剂TAK-242(1μg/mL)预处理THP-1巨噬细胞,观察抑制TLR4对上述刺激下THP-1巨噬细胞脂质积累和CD36表达的影响。结果PMA处理后,THP-1细胞呈现巨噬细胞形态并具备脂质吞噬能力;与RPMI1640相比,β2GPI/aβ2GPI处理可显著增加THP-1巨噬细胞TLR4表达;与单独oxLDL处理相比,oxLDL联合β2GPI/aβ2GPI能够抑制THP-1巨噬细胞的脂质积累和CD36表达,阻断TLR4可以部分逆转这一现象。结论β2GPI/aβ2GPI能激活TLR4抑制巨噬细胞对oxLDL的摄取和CD36表达。
Objective To investigate the effects of β2 glycoprotein I/anti-β2 glycoprotein I antibody complex(β2GPI/aβ2GPI) on lipid phagocytosis and class B scavenger receptor CD36 expression of macrophages,and the role of Toll-like receptor 4(TLR4) during the process. Methods THP-1 macrophages were induced from THP-1 cells treated by 100 ng/mL phorbol ester(PMA),and then identified by morphological observation and DiI-oxLDL uptake assay. THP-1 macrophages were treated with RPMI1640 medium,β2GPI,aβ2GPI or β2GPI/aβ2GPI. The protein and mRNA expression of TLR4 were detected by Western blot analysis and real-time fluorescent quantitative PCR(qRT-PCR),respectively. After that,THP-1 macrophages were treated with RPMI1640 medium,oxidized low-density lipoprotein(oxLDL),oxLDL combined with β2GPI/aβ2GPI or oxLDL combined with LPS. Intracellular lipid deposition was observed by oil red O staining; the mRNA and protein expression of CD36 were detected by qRT-PCR,immunofluorescence and Western blot analysis. To unveil the role of TLR4 in this process,THP-1 macrophages were pre-treated with or without TLR4 inhibitor TAK-242(1 μg/mL). Results After PMA treatment,THP-1 cells showed macrophage-like morphology and were able to engulf Di I-oxLDL. Compared with RPMI1640 medium,β2GPI/aβ2GPI treatment significantly increased TLR4 expression in THP-1 macrophages. Compared with oxLDL alone,oxLDL combined with β2GPI/aβ2GPI inhibited lipid deposition and CD36 expression in THP-1 macrophages,which could be partly reversed by TLR4 blockage. Conclusion The β2GPI/aβ2GPI can inhibit the phagocytosis of oxLDL and CD36 expression in macrophages,which is linked to the function of TLR4.
Objective To investigate the effects of β2 glycoprotein I/anti-β2 glycoprotein I antibody complex(β2GPI/aβ2GPI) on lipid phagocytosis and class B scavenger receptor CD36 expression of macrophages,and the role of Toll-like receptor 4(TLR4) during the process. Methods THP-1 macrophages were induced from THP-1 cells treated by 100 ng/mL phorbol ester(PMA),and then identified by morphological observation and DiI-oxLDL uptake assay. THP-1 macrophages were treated with RPMI1640 medium,β2GPI,aβ2GPI or β2GPI/aβ2GPI. The protein and mRNA expression of TLR4 were detected by Western blot analysis and real-time fluorescent quantitative PCR(qRT-PCR),respectively. After that,THP-1 macrophages were treated with RPMI1640 medium,oxidized low-density lipoprotein(oxLDL),oxLDL combined with β2GPI/aβ2GPI or oxLDL combined with LPS. Intracellular lipid deposition was observed by oil red O staining; the mRNA and protein expression of CD36 were detected by qRT-PCR,immunofluorescence and Western blot analysis. To unveil the role of TLR4 in this process,THP-1 macrophages were pre-treated with or without TLR4 inhibitor TAK-242(1 μg/mL). Results After PMA treatment,THP-1 cells showed macrophage-like morphology and were able to engulf Di I-oxLDL. Compared with RPMI1640 medium,β2GPI/aβ2GPI treatment significantly increased TLR4 expression in THP-1 macrophages. Compared with oxLDL alone,oxLDL combined with β2GPI/aβ2GPI inhibited lipid deposition and CD36 expression in THP-1 macrophages,which could be partly reversed by TLR4 blockage. Conclusion The β2GPI/aβ2GPI can inhibit the phagocytosis of oxLDL and CD36 expression in macrophages,which is linked to the function of TLR4.
引文
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[21]Xie H,Kong X,Zhou H,et al. TLR4 is involved in the pathogeniceffects observed in a murine model of antiphospholipid syndrome[J]. Clin Immunol,2015,160(2):198-210.
[22]Matsuura E,Kobayashi K,Tabuchi M,et al. Oxidative modificationof low-density lipoprotein and immune regulation of atherosclerosis[J]. Prog Lipid Res,2006,45(6):466-486.
[23]张贵婷,周红,何超,等. ox LDL/β2GPI/anti-β2GPI antibody复合物促进人脐静脉内皮细胞表达黏附分子的作用探讨[J]细胞与分子免疫学杂志,2017,33(11):1472-1478.
[24] Wang T,Ouyang H,Zhou H,et al. Proatherogenic activation ofA7r5 cells induced by the ox LDL/beta2GPI/antibeta2GPI complex[J]. Int J Mol Med,2018,42(4):1955-1966.
[25] Zhu X,Zhou H,Wang X,et al. Anti-β2GPI antibody promotesrelease of inflammatory and pro-thrombosis molecules from arteries inapolipoprotein E-deficient mice[J]. Xi Bao Yu Fen Zi Mian Yi XueZa Zhi,2017,33(3):295-299.
[2]Abdolmaleki F,Gheibi Hayat S M,Bianconi V,et al. Atherosclerosisand immunity:A perspective[J]. Trends Cardiovasc Med,2018 Sep28. pii:S1050-1738(18)30225-1. DOI:10. 1016/j. tcm.2018. 09. 017.[Epub ahead of print].
[3]Hartley A,Haskard D,Khamis R. Oxidized LDL and anti-oxidizedLDL antibodies in atherosclerosis-Novel insights and future directionsin diagnosis and therapy[J]. Trends Cardiovasc Med, 2019,29(1):22-26.
[4] Cirillo P,Conte S,Cimmino G,et al. Nobiletin inhibits oxidized-LDL mediated expression of tissue factor in human endothelial cellsthrough inhibition of NF-kappaB[J]. Biochem Pharmacol,2017,128:26-33.
[5]Cheng Y C,Sheen J M,Hu W L,et al. Polyphenols and oxidativestress in atherosclerosis-related ischemic heart disease and stroke[J/OL].Oxid Med Cell Longev,2017,2017:8526438. DOI:10. 1155/2017/8526438. Epub 2017 Nov 26.
[6]Wang H H,Garruti G,Liu M,et al. Cholesterol and lipoproteinmetabolism and atherosclerosis:Recent advances in reverse cholesteroltransport[J]. Ann Hepatol,2017,16(Suppl. 1:s3-105.):s27-s42.
[7] Chistiakov D A,Melnichenko A A,Orekhov A N,et al. How domacrophages sense modified low-density lipoproteins?[J]. Int JCardiol,2017,230:232-240.
[8]Corban M T,Duarte-Garcia A,Mc Bane R D,et al. Antiphospholipidsyndrome:Role of vascular endothelial cells and implications for riskstratification and targeted therapeutics[J]. J Am Coll Cardiol,2017,69(18):2317-2330.
[9]Zhang J Y,Ma J,Yu P,et al. Effects of reducedβ2 glycoprotein Ion high glucose induced cell death in HUVECs[J]. Mol Med Rep,2017,16(4):4208-4214.
[10] Zhang Y,Zhang W,Zha C,et al. Platelets activated by theanti-beta2GPI/beta2GPI complex release microRNAs to inhibitmigration and tube formation of human umbilical vein endothelialcells[J/OL]. Cell Mol Biol Lett,2018,23:24. DOI:10. 1186/s11658-018-0091-3. e Collection 2018.
[11]王晓燕,周红,朱晓洁,等.抗β2糖蛋白I抗体加速高脂饮食Apo E缺陷小鼠的动脉粥样硬化形成[J].中华心血管病杂志,2017,45(1):44-48.
[12]Zhou H,Sheng L,Wang H,et al. Anti-beta2GPI/beta2GPI stimulatesactivation of THP-1 cells through TLR4/MD-2/My D88 and NF-kappa Bsignaling pathways[J]. Thromb Res,2013,132(6):742-749.
[13]Zhang X,Xie Y,Zhou H,et al. Involvement of TLR4 in oxidizedLDL/beta2GPI/anti-beta2GPI-induced transformation of macrophages tofoam cells[J]. J Atheroscler Thromb, 2014, 21(11):1140-1151.
[14]Korakas E,Dimitriadis G,Raptis A,et al. Dietary composition andcardiovascular risk:A mediator or a bystander?[J/OL].Nutrients,2018,10(12):E1912. DOI:10. 3390/nu10121912.
[15] Yunoki K,Nakamura K,Miyoshi T,et al. Ezetimibe improvespostprandial hyperlipemia and its induced endothelial dysfunction[J]. Atherosclerosis,2011,217(2):486-491.
[16] Rosengren A,Hawken S,Ounpuu S,et al. Association of psychosocialrisk factors with risk of acute myocardial infarction in 11119 casesand 13648 controls from 52 countries(the INTERHEART study):case-control study[J]. Lancet,2004,364(9438):953-962.
[17]Gordts P L S M,Esko J D. The heparan sulfate proteoglycan grip onhyperlipidemia and atherosclerosis[J]. Matrix Biol, 2018,71-72:262-282.
[18]Rader D J,Alexander E T,Weibel G L,et al. The role of reverse cholesteroltransport in animals and humans and relationship to atherosclerosis[J].J Lipid Res,2009,50(Suppl):S189-S194.
[19] Ganjali S,Momtazi A A,Banach M,et al. HDL abnormalities infamilial hypercholesterolemia:Focus on biological functions[J].Prog Lipid Res,2017,67:16-26.
[20]Rahman M S,Murphy A J,Woollard K J. Effects of dyslipidaemiaon monocyte production and function in cardiovascular disease[J].Nat Rev Cardiol,2017,14(7):387-400.
[21]Xie H,Kong X,Zhou H,et al. TLR4 is involved in the pathogeniceffects observed in a murine model of antiphospholipid syndrome[J]. Clin Immunol,2015,160(2):198-210.
[22]Matsuura E,Kobayashi K,Tabuchi M,et al. Oxidative modificationof low-density lipoprotein and immune regulation of atherosclerosis[J]. Prog Lipid Res,2006,45(6):466-486.
[23]张贵婷,周红,何超,等. ox LDL/β2GPI/anti-β2GPI antibody复合物促进人脐静脉内皮细胞表达黏附分子的作用探讨[J]细胞与分子免疫学杂志,2017,33(11):1472-1478.
[24] Wang T,Ouyang H,Zhou H,et al. Proatherogenic activation ofA7r5 cells induced by the ox LDL/beta2GPI/antibeta2GPI complex[J]. Int J Mol Med,2018,42(4):1955-1966.
[25] Zhu X,Zhou H,Wang X,et al. Anti-β2GPI antibody promotesrelease of inflammatory and pro-thrombosis molecules from arteries inapolipoprotein E-deficient mice[J]. Xi Bao Yu Fen Zi Mian Yi XueZa Zhi,2017,33(3):295-299.
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