oxLDL通过TLR4诱导脂质累积和炎症反应促进动脉粥样硬化的分子机制
|
摘要
目的·探究TLR4诱导脂质累积和炎症反应从而调控动脉粥样硬化的分子机制。方法·采用TLR4特异性siRNA敲除巨噬细胞,通过油红O染色法比较对照组和实验组中细胞内脂质含量;通过Western blotting检测CD36和血凝素样氧化低密度脂蛋白受体1(LOX-1)表达;通过ELISA检测白介素-6(IL-6)、IL-8、单核细胞趋化蛋白-1(MCP-1)和基质金属蛋白酶-9(MMP-9)等炎症因子的表达。结果·动脉粥样硬化斑块中巨噬细胞(CD68~+)大量聚集,且TLR4表达明显上调。ox LDL刺激巨噬细胞,可促进细胞中脂质累积(P<0.01)并导致ox LDL相关受体CD36和LOX-1的表达上调以及炎症因子表达(P<0.01)。特异性si RNA敲除TLR4可抑制ox LDL诱导的巨噬细胞内脂质累积(P<0.01)和炎症因子分泌(P<0.01),并影响ox LDL诱导的CD36表达,但对LOX-1表达没有影响。结论·ox LDL/TLR4可能通过上调CD36介导巨噬细胞中脂质累积和炎症反应,从而促进动脉粥样硬化发生和发展。
Objective·To investigate the possible role of TLR4 signaling pathway in the mediation of atherosclerosis. Methods·TLR4 were knocked down via transfection with TLR4-specific si RNA, and the lipid accumulation was further detected in control and TLR4-knockdown groups by oil red O staining. The expression of CD36 and Lectin-like ox LDL receptor 1(LOX-1) in macrophages were detected by Western blotting to investigate the role of TLR4 in the expression of ox LDL-related receptors. Cytokines such as interleukin-6(IL-6), IL-8, monocyte chemoattractant protein 1(MCP-1), and matrix metalloproteinase-9(MMP-9) were tested by ELISA to confirm the possible role of TLR4 in the secretion of inflammatory factors. Results·Macrophages(namely CD68~+ cells) were found to accumulate within atherosclerosis plaques with TLR4 highly expressed on the surface of macrophages; the stimulation with ox LDL promoted the lipid accumulation(P<0.01), the secretion of inflammatory factors(P<0.01), and the expression of CD36 and LOX-1. The ox LDL-associated expression of CD36 was decreased but the expression of LOX-1 was not affected. The knockdown of TLR4 inhibits ox LDL-induced lipid accumulation(P<0.01)and inflammatory cytokines(IL-6, IL-8, MCP-1 and MMP-9) secretion(P<0.01). Conclusion·TLR4 signaling pathway possibly promotes the lipid accumulation and the secretion of inflammatory factors via up-regulating the expression of CD36 to affect the formation and development of atherosclerosis.
Objective·To investigate the possible role of TLR4 signaling pathway in the mediation of atherosclerosis. Methods·TLR4 were knocked down via transfection with TLR4-specific si RNA, and the lipid accumulation was further detected in control and TLR4-knockdown groups by oil red O staining. The expression of CD36 and Lectin-like ox LDL receptor 1(LOX-1) in macrophages were detected by Western blotting to investigate the role of TLR4 in the expression of ox LDL-related receptors. Cytokines such as interleukin-6(IL-6), IL-8, monocyte chemoattractant protein 1(MCP-1), and matrix metalloproteinase-9(MMP-9) were tested by ELISA to confirm the possible role of TLR4 in the secretion of inflammatory factors. Results·Macrophages(namely CD68~+ cells) were found to accumulate within atherosclerosis plaques with TLR4 highly expressed on the surface of macrophages; the stimulation with ox LDL promoted the lipid accumulation(P<0.01), the secretion of inflammatory factors(P<0.01), and the expression of CD36 and LOX-1. The ox LDL-associated expression of CD36 was decreased but the expression of LOX-1 was not affected. The knockdown of TLR4 inhibits ox LDL-induced lipid accumulation(P<0.01)and inflammatory cytokines(IL-6, IL-8, MCP-1 and MMP-9) secretion(P<0.01). Conclusion·TLR4 signaling pathway possibly promotes the lipid accumulation and the secretion of inflammatory factors via up-regulating the expression of CD36 to affect the formation and development of atherosclerosis.
引文
[1]Hopkins PA,Sriskandan S.Mammalian Toll-like receptors:to immunity and beyond[J].Clin Exp Immunol,2005,140(3):395-407.
[2]Modlin RL.Mammalian toll-like receptors[J].Ann Allergy Asthma Immunol,2002,88(6):543-547;quiz 548-550,583.
[3]Li H,Sun B.Toll-like receptor 4 in atherosclerosis[J].J Cell Mol Med,2007,11(1):88-95.
[4]den Dekker WK,Cheng C,Pasterkamp G,et al.Toll like receptor 4 in atherosclerosis and plaque destabilization[J].Atherosclerosis,2010,209(2):314-320.
[5]Chukkapalli SS,Velsko IM,Rivera-Kweh MF,et al.Global TLR2 and4 deficiency in mice impacts bone resorption,inflammatory markers and atherosclerosis to polymicrobial infection[J].Mol Oral Microbiol,2017,32(3):211-225.
[6]Feng Y,Cai ZR,Tang Y,et al.TLR4/NF-kappa B signaling pathwaymediated and ox LDL-induced up-regulation of LOX-1,MCP-1,and VCAM-1expressions in human umbilical vein endothelial cells[J].Genet Mol Res,2014,13(1):680-695.
[7]Janeesh PA,Sasikala V,Dhanya CR,et al.Robinin modulates TLR/NF-kappa B signaling pathway in oxidized LDL induced human peripheral blood mononuclear cells[J].Int Immunopharmacol,2014,18(1):191-197.
[8]Chen C,Khismatullin DB.Oxidized low-density lipoprotein contributes to atherogenesis via co-activation of macrophages and mast cells[J].PLo S One,2015,10(3):e0123088.
[9]Di Pietro N,Formoso G,Pandolfi A.Physiology and pathophysiology of ox LDL uptake by vascular wall cells in atherosclerosis[J].Vascul Pharmacol,2016,84:1-7.
[10]Singh V,Rana M,Jain M,et al.Curcuma oil attenuates accelerated atherosclerosis and macrophage foam-cell formation by modulating genes involved in plaque stability,lipid homeostasis and inflammation[J].Br J Nutr,2015,113(1):100-113.
[11]Rios FJ,Koga MM,Pecenin M,et al.Oxidized LDL induces alternative macrophage phenotype through activation of CD36 and PAFR[J].Mediators Inflamm,2013,2013(4):198193.
[12]Chistiakov DA,Bobryshev YV,Orekhov AN.Macrophage-mediated cholesterol handling in atherosclerosis[J].J Cell Mol Med,2016,20(1):17-28.
[13]Toledo J,Esteve M,Grasa M,et al.Data related to inflammation and cholesterol deposition triggered by macrophages exposition to modified LDL[J].Data Brief,2016,8:251-257.
[14]Sun N,Wang H,Wang L.Ghrelin inhibits ox LDL-induced inflammation in RAW264.7 mouse macrophages through down-regulation of LOX-1 expression via NF-kappa B signaling pathway[J].Cell Mol Biol(Noisy-le-grand),2016,62(2):57-61.
[15]Sun S,Cheng B,Wu X,et al.Chlamydia pneumoniae disrupts lipid metabolism in human umbilical vein endothelial cells[J].Mol Med Rep,2014,10(2):1150-1156.
[16]Endemann G,Stanton LW,Madden KS,et al.CD36 is a receptor for oxidized low density lipoprotein[J].J Biol Chem,1993,268(16):11811-11816.
[17]Rao LN,Ponnusamy T,Philip S,et al.Hypercholesterolemia induced immune response and inflammation on progression of atherosclerosis in Apob(tm2Sgy)Ldlr(tm1Her)/J mice[J].Lipids,2015,50(8):785-797.
[18]Yu XH,Fu YC,Zhang DW,et al.Foam cells in atherosclerosis[J].Clin Chim Acta,2013,424:245-252.
[19]Yang K,Zhang XJ,Cao LJ,et al.Toll-like receptor 4 mediates inflammatory cytokine secretion in smooth muscle cells induced by oxidized low-density lipoprotein[J].PLo S One,2014,9(4):e95935.
[20]Hirose K,Iwabuchi K,Shimada K,et al.Different responses to oxidized lowdensity lipoproteins in human polarized macrophages[J].Lipids Health Dis,2011,10(1):1.
[21]Dushkin MI.Macrophage/foam cell is an attribute of inflammation:mechanisms of formation and functional role[J].Biochemistry(Mosc),2012,77(4):327-338.
[22]Fenyo IM,Gafencu AV.The involvement of the monocytes/macrophages in chronic inflammation associated with atherosclerosis[J].Immunobiology,2013,218(11):1376-1384.
[23]Yang K,Wang X,Liu Z,et al.Oxidized low-density lipoprotein promotes macrophage lipid accumulation via the toll-like receptor 4-Src pathway[J].Circ J,2015,79(11):2509-2516.
[24]Medzhitov R,Preston-Hurlburt P,Janeway CA,et al.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity[J].Nature,1997,388(6640):394-397.
[2]Modlin RL.Mammalian toll-like receptors[J].Ann Allergy Asthma Immunol,2002,88(6):543-547;quiz 548-550,583.
[3]Li H,Sun B.Toll-like receptor 4 in atherosclerosis[J].J Cell Mol Med,2007,11(1):88-95.
[4]den Dekker WK,Cheng C,Pasterkamp G,et al.Toll like receptor 4 in atherosclerosis and plaque destabilization[J].Atherosclerosis,2010,209(2):314-320.
[5]Chukkapalli SS,Velsko IM,Rivera-Kweh MF,et al.Global TLR2 and4 deficiency in mice impacts bone resorption,inflammatory markers and atherosclerosis to polymicrobial infection[J].Mol Oral Microbiol,2017,32(3):211-225.
[6]Feng Y,Cai ZR,Tang Y,et al.TLR4/NF-kappa B signaling pathwaymediated and ox LDL-induced up-regulation of LOX-1,MCP-1,and VCAM-1expressions in human umbilical vein endothelial cells[J].Genet Mol Res,2014,13(1):680-695.
[7]Janeesh PA,Sasikala V,Dhanya CR,et al.Robinin modulates TLR/NF-kappa B signaling pathway in oxidized LDL induced human peripheral blood mononuclear cells[J].Int Immunopharmacol,2014,18(1):191-197.
[8]Chen C,Khismatullin DB.Oxidized low-density lipoprotein contributes to atherogenesis via co-activation of macrophages and mast cells[J].PLo S One,2015,10(3):e0123088.
[9]Di Pietro N,Formoso G,Pandolfi A.Physiology and pathophysiology of ox LDL uptake by vascular wall cells in atherosclerosis[J].Vascul Pharmacol,2016,84:1-7.
[10]Singh V,Rana M,Jain M,et al.Curcuma oil attenuates accelerated atherosclerosis and macrophage foam-cell formation by modulating genes involved in plaque stability,lipid homeostasis and inflammation[J].Br J Nutr,2015,113(1):100-113.
[11]Rios FJ,Koga MM,Pecenin M,et al.Oxidized LDL induces alternative macrophage phenotype through activation of CD36 and PAFR[J].Mediators Inflamm,2013,2013(4):198193.
[12]Chistiakov DA,Bobryshev YV,Orekhov AN.Macrophage-mediated cholesterol handling in atherosclerosis[J].J Cell Mol Med,2016,20(1):17-28.
[13]Toledo J,Esteve M,Grasa M,et al.Data related to inflammation and cholesterol deposition triggered by macrophages exposition to modified LDL[J].Data Brief,2016,8:251-257.
[14]Sun N,Wang H,Wang L.Ghrelin inhibits ox LDL-induced inflammation in RAW264.7 mouse macrophages through down-regulation of LOX-1 expression via NF-kappa B signaling pathway[J].Cell Mol Biol(Noisy-le-grand),2016,62(2):57-61.
[15]Sun S,Cheng B,Wu X,et al.Chlamydia pneumoniae disrupts lipid metabolism in human umbilical vein endothelial cells[J].Mol Med Rep,2014,10(2):1150-1156.
[16]Endemann G,Stanton LW,Madden KS,et al.CD36 is a receptor for oxidized low density lipoprotein[J].J Biol Chem,1993,268(16):11811-11816.
[17]Rao LN,Ponnusamy T,Philip S,et al.Hypercholesterolemia induced immune response and inflammation on progression of atherosclerosis in Apob(tm2Sgy)Ldlr(tm1Her)/J mice[J].Lipids,2015,50(8):785-797.
[18]Yu XH,Fu YC,Zhang DW,et al.Foam cells in atherosclerosis[J].Clin Chim Acta,2013,424:245-252.
[19]Yang K,Zhang XJ,Cao LJ,et al.Toll-like receptor 4 mediates inflammatory cytokine secretion in smooth muscle cells induced by oxidized low-density lipoprotein[J].PLo S One,2014,9(4):e95935.
[20]Hirose K,Iwabuchi K,Shimada K,et al.Different responses to oxidized lowdensity lipoproteins in human polarized macrophages[J].Lipids Health Dis,2011,10(1):1.
[21]Dushkin MI.Macrophage/foam cell is an attribute of inflammation:mechanisms of formation and functional role[J].Biochemistry(Mosc),2012,77(4):327-338.
[22]Fenyo IM,Gafencu AV.The involvement of the monocytes/macrophages in chronic inflammation associated with atherosclerosis[J].Immunobiology,2013,218(11):1376-1384.
[23]Yang K,Wang X,Liu Z,et al.Oxidized low-density lipoprotein promotes macrophage lipid accumulation via the toll-like receptor 4-Src pathway[J].Circ J,2015,79(11):2509-2516.
[24]Medzhitov R,Preston-Hurlburt P,Janeway CA,et al.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity[J].Nature,1997,388(6640):394-397.