牙龈卟啉单胞菌感染对人主动脉内皮细胞炎症反应的影响研究
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摘要
目的研究牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)感染对人主动脉内皮细胞(human aortic endothelial cells,HAECs)表达炎症因子的影响并探讨其可能的分子机制。方法在体外以感染复数(multiplicity of infection,MOI)分别为0、25∶1、100∶1和200:1的P.gingivalis刺激HAECs 24 h后,分别采用实时荧光定量PCR、酶联免疫吸附试验和免疫印迹试验检测单核细胞趋化因子-1(monocyte chemotactic protein 1,MCP-1)、白细胞介素-1β(interleukin 1β,IL-1β)、Toll样受体(Toll like receptor,TLR)-2和TLR-4的表达水平;在体外以MOI分别为0和200∶1的P.gingivalis刺激HAECs 30 min后,采用凝胶迁移实验检测核因子κB(nuclear factorκB,NF-κB)的活化水平。结果 P.gingivalis刺激组(MOI分别为25∶1、100∶1和200∶1)的HAECs中MCP-1、IL-1β、TLR-2和TLR-4的表达均显著高于未刺激组(MOI为0)(均P <0.05),且提示这种上调作用呈P.gingivalis刺激剂量依赖性;P.gingivalis刺激组(MOI为200∶1)的HAECs中NF-κB活化水平显著高于未刺激组(MOI为0)(P <0.05)。结论 P.gingivalis可能通过TLRs-NF-κB信号通路上调HAECs中炎症因子的表达,导致血管内皮功能紊乱,提示P. gingivalis可能促进动脉粥样硬化的发生和发展。
Objective To investigate the effects of Porphyromonas gingivalis(P.gingivalis)infection on the expression of inflammatory factors in human aortic endothelial cells(HAECs)and explore its possible molecular mechanism. Methods HAECs were challenged in vitro by P.gingivalis with MOI of 0,25:1,100∶1 and 200∶1 respectively,and after 24 hours the expression of monocyte chemotactic protein 1(MCP-1),interleukin 1β(IL-1β),Toll like receptors(TLR)-2 and TLR-4 was detected by real time quantitative PCR,enzyme linked immunosorbent assay and Western Blot. HAECs were challenged by P.gingivalis with MOI of 0 and 200∶1 respectively in vitro,and after 30 minutes the activation of nuclear factor κB(NF-κB)was detected by electrophoretic mobility shift assay. Results The expressions of MCP-1,IL-1β,TLR-2 and TLR-4 in HAECs were significantly increased in P.gingivalis-challenged groups(MOI = 25∶1,100∶1 and200∶1 respectively)than those in non-challenged group(MOI = 0)(P < 0.05),and the up-regulation was dose-dependent;the activation of NF-κB in HAECs of P.gingivalis-challenged group(MOI = 200∶1)was significantly increased than that of non-challenged group(MOI = 0)(P < 0.05). Conclusion P.gingivalis up-regulates the expression of inflammatory cytokines in HAECs,which leads to its dysfunction,suggesting that P.gingivalis could play a significant role in promoting the initiation and development of atherosclerosis.
Objective To investigate the effects of Porphyromonas gingivalis(P.gingivalis)infection on the expression of inflammatory factors in human aortic endothelial cells(HAECs)and explore its possible molecular mechanism. Methods HAECs were challenged in vitro by P.gingivalis with MOI of 0,25:1,100∶1 and 200∶1 respectively,and after 24 hours the expression of monocyte chemotactic protein 1(MCP-1),interleukin 1β(IL-1β),Toll like receptors(TLR)-2 and TLR-4 was detected by real time quantitative PCR,enzyme linked immunosorbent assay and Western Blot. HAECs were challenged by P.gingivalis with MOI of 0 and 200∶1 respectively in vitro,and after 30 minutes the activation of nuclear factor κB(NF-κB)was detected by electrophoretic mobility shift assay. Results The expressions of MCP-1,IL-1β,TLR-2 and TLR-4 in HAECs were significantly increased in P.gingivalis-challenged groups(MOI = 25∶1,100∶1 and200∶1 respectively)than those in non-challenged group(MOI = 0)(P < 0.05),and the up-regulation was dose-dependent;the activation of NF-κB in HAECs of P.gingivalis-challenged group(MOI = 200∶1)was significantly increased than that of non-challenged group(MOI = 0)(P < 0.05). Conclusion P.gingivalis up-regulates the expression of inflammatory cytokines in HAECs,which leads to its dysfunction,suggesting that P.gingivalis could play a significant role in promoting the initiation and development of atherosclerosis.
引文
[1]闫福华.牙周炎对全身疾病和健康影响的研究进展[J].口腔医学,2018,38(7):577-581.
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[8]Pan S,Lei L,Chen S,et al.Rosiglitazone impedes Porphyromonas gingivalis-accelerated atherosclerosis by downregulating the TLR/NF-κB signaling pathway in atherosclerotic mice[J].Int Immunopharmacol,2014,23(2):701-708.
[9]Onat D,Brillon D,Colombo PC,et al.Human vascular endothelial cells:a model system for studying vascular inflammation in diabetes and atherosclerosis[J].Curr Diab Rep,2011,11(3):193-202.
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[11]Mudau M,Genis A,Lochner A,et al.Endothelial dysfunction:the early predictor of atherosclerosis[J].Cardiovasc J Afr,2012,23(4):222-231.
[12]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].PLoS One,2014,9(4):e95935.
[13]Olofsson PS,Sheikine Y,Jatta K,et al.A functional interleukin-1 receptor antagonist polymorphism influences atherosclerosis development.The interleukin-1beta:interleukin-1 receptor antagonist balance in atherosclerosis[J].Circ J,2009,73(8):1531-1536.
[14]Goulopoulou S,McCarthy CG,Webb RC.Toll-like receptors in the vascular system:sensing the dangers within[J].Pharmacol Rev,2016,68(1):142-167.
[15]Yu XH,Zheng XL,Tang CK.Nuclear factor-κB activation as a pathological mechanism of lipid metabolism and atherosclerosis[J].Adv Clin Chem,2015,70:1-30.
[16]刘斌,程岚,刘大力,等.牙龈卟啉单胞菌脂多糖对人主动脉内皮细胞表达ICAM-1影响的研究[J].牙体牙髓牙周病学杂志,2011,21(10):568-572.
[17]Gibson FC,Yumoto H,Takahashi Y,et al.Innate immune signaling and Porphyromonas gingivalis-accelerated atherosclerosis[J].J Dent Res,2006,85(2):106-121.
[18]Yang J,Wu J,Liu Y,et al.Porphyromonas gingivalis infection reduces regulatory T cells in infected atherosclerosis patients[J].PLoS One,2014,9(1):e86599.
[19]Lin G,Shi X,Chen S,et al.Effects of micro-amounts of Porphyromonas gingivalis lipopolysaccharide on rabbit inflammatory immune response and development of atherosclerosis[J].JPeriodontal Res,2015,50(3):356-362.
[20]Chukkapalli SS,Velsko IM,Rivera-Kweh MF,et al.Polymicrobial oral infection with four periodontal bacteria orchestrates a distinct inflammatory response and atherosclerosis in ApoE null mice[J].PLoS One,2015,10(11):e0143291.
[21]Ho MH,Guo ZM,Chunga J,et al.Characterization of innate immune responses of human endothelial cells induced by Porphyromonas gingivalis and their derived outer membrane vesicles[J].Front Cell Infect Microbiol,2016,6:139.
[22]Olsen I,Taubman MA,Singhrao SK.Porphyromonas gingivalis suppresses adaptive immunity in periodontitis,atherosclerosis,and Alzheimer′s disease[J].J Oral Microbiol,2016,8:33029.
[23]Xu W,Pan Y,Xu Q,et al.Porphyromonas gingivalis ATCC33277 promotes intercellular adhesion molecule-1 expression in endothelial cells and monocyte-endothelial cell adhesion through macrophage migration inhibitory factor[J].BMC Microbiol,2018,18(1):16.
[24]Offenbacher S.Periodontal diseases:pathogenesis[J].Ann Periodontol,1996,1(1):821-878.
[25]徐婉悦,潘亚萍,张冬梅.牙周致病菌与动脉粥样硬化相关关系研究进展[J].中国实用口腔科杂志,2017,10(7):441-444.
[26]Saffi MA,Furtado MV,Polanczyk CA,et al.Relationship between vascular endothelium and periodontal disease in atherosclerotic lesions:Review article[J].World J Cardiol,2015,7(1):26-30.
[27]Satoh S,Yada R,Inoue H,et al.Toll-like receptor-4 is upregulated in plaque debris of patients with acute coronary syndrome more than Toll-like receptor-2[J].Heart Vessels,2016,31(1):1-5.
[28]Yumoto H,Chou HH,Takahashi Y,et al.Sensitization of human aortic endothelial cells to lipopolysaccharide via regulation of Toll-like receptor 4 by bacterial fimbria-dependent invasion[J].Infect Immun,2005,73(12):8050-8059.
[29]Hajra L,Evans AI,Chen M,et al.The NF-kappa B signal transduction pathway in aortic endothelial cells is primed for activation in regions predisposed to atherosclerotic lesion formation[J].Proc Natl Acad Sci U S A,2000,97(16):9052-9057.
[30]Lin G,Chen S,Lei L,et al.Effects of intravenous injection of Porphyromonas gingivalis on Rabbit inflammatory immune response and atherosclerosis[J].Mediators Inflamm,2015,2015:364391.
[31]Tang YL,Jiang JH,Wang S,et al.TLR4/NF-κB signaling contributes to chronic unpredictable mild stress-induced atherosclerosis in ApoE-/-mice[J].PLoS One,2015,10(4):e0123685.
[2]张宇,艾丽思,林莉.牙龈卟啉单胞菌菌体表面毒力因子及其致病性研究进展[J].中国实用口腔科杂志,2015,8(1):43-47.
[3]赵戬,潘亚萍.牙龈卟啉单胞菌对慢性牙周炎致病作用研究进展[J].中国实用口腔科杂志,2015,8(9):567-571.
[4]Amar S,Engelke M.Periodontal innate immune mechanisms relevant to atherosclerosis[J].Mol Oral Microbiol,2015,30(3):171-185.
[5]Li L,Messas E,Batista EL,et al.Porphyromonas gingivalis infection accelerates the progression of atherosclerosis in a heterozygous apolipoprotein E-deficient murine model[J].Circulation,2002,105(7):861-867.
[6]Lalla E,Lamster IB,Hofmann MA,et al.Oral infection with a periodontal pathogen accelerates early atherosclerosis in apolipoprotein E-null mice[J].Arterioscler Thromb Vasc Biol,2003,23(8):1405-1411.
[7]潘盛波,雷浪,李厚轩,等.牙龈卟啉单胞菌促进动脉粥样硬化的机制研究[J].福建医科大学学报,2014,48(6):367-371.
[8]Pan S,Lei L,Chen S,et al.Rosiglitazone impedes Porphyromonas gingivalis-accelerated atherosclerosis by downregulating the TLR/NF-κB signaling pathway in atherosclerotic mice[J].Int Immunopharmacol,2014,23(2):701-708.
[9]Onat D,Brillon D,Colombo PC,et al.Human vascular endothelial cells:a model system for studying vascular inflammation in diabetes and atherosclerosis[J].Curr Diab Rep,2011,11(3):193-202.
[10]Sena CM,Pereira AM,Sei?a R.Endothelial dysfunction-a major mediator of diabetic vascular disease[J].Biochim Biophys Acta,2013,1832(12):2216-2231.
[11]Mudau M,Genis A,Lochner A,et al.Endothelial dysfunction:the early predictor of atherosclerosis[J].Cardiovasc J Afr,2012,23(4):222-231.
[12]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].PLoS One,2014,9(4):e95935.
[13]Olofsson PS,Sheikine Y,Jatta K,et al.A functional interleukin-1 receptor antagonist polymorphism influences atherosclerosis development.The interleukin-1beta:interleukin-1 receptor antagonist balance in atherosclerosis[J].Circ J,2009,73(8):1531-1536.
[14]Goulopoulou S,McCarthy CG,Webb RC.Toll-like receptors in the vascular system:sensing the dangers within[J].Pharmacol Rev,2016,68(1):142-167.
[15]Yu XH,Zheng XL,Tang CK.Nuclear factor-κB activation as a pathological mechanism of lipid metabolism and atherosclerosis[J].Adv Clin Chem,2015,70:1-30.
[16]刘斌,程岚,刘大力,等.牙龈卟啉单胞菌脂多糖对人主动脉内皮细胞表达ICAM-1影响的研究[J].牙体牙髓牙周病学杂志,2011,21(10):568-572.
[17]Gibson FC,Yumoto H,Takahashi Y,et al.Innate immune signaling and Porphyromonas gingivalis-accelerated atherosclerosis[J].J Dent Res,2006,85(2):106-121.
[18]Yang J,Wu J,Liu Y,et al.Porphyromonas gingivalis infection reduces regulatory T cells in infected atherosclerosis patients[J].PLoS One,2014,9(1):e86599.
[19]Lin G,Shi X,Chen S,et al.Effects of micro-amounts of Porphyromonas gingivalis lipopolysaccharide on rabbit inflammatory immune response and development of atherosclerosis[J].JPeriodontal Res,2015,50(3):356-362.
[20]Chukkapalli SS,Velsko IM,Rivera-Kweh MF,et al.Polymicrobial oral infection with four periodontal bacteria orchestrates a distinct inflammatory response and atherosclerosis in ApoE null mice[J].PLoS One,2015,10(11):e0143291.
[21]Ho MH,Guo ZM,Chunga J,et al.Characterization of innate immune responses of human endothelial cells induced by Porphyromonas gingivalis and their derived outer membrane vesicles[J].Front Cell Infect Microbiol,2016,6:139.
[22]Olsen I,Taubman MA,Singhrao SK.Porphyromonas gingivalis suppresses adaptive immunity in periodontitis,atherosclerosis,and Alzheimer′s disease[J].J Oral Microbiol,2016,8:33029.
[23]Xu W,Pan Y,Xu Q,et al.Porphyromonas gingivalis ATCC33277 promotes intercellular adhesion molecule-1 expression in endothelial cells and monocyte-endothelial cell adhesion through macrophage migration inhibitory factor[J].BMC Microbiol,2018,18(1):16.
[24]Offenbacher S.Periodontal diseases:pathogenesis[J].Ann Periodontol,1996,1(1):821-878.
[25]徐婉悦,潘亚萍,张冬梅.牙周致病菌与动脉粥样硬化相关关系研究进展[J].中国实用口腔科杂志,2017,10(7):441-444.
[26]Saffi MA,Furtado MV,Polanczyk CA,et al.Relationship between vascular endothelium and periodontal disease in atherosclerotic lesions:Review article[J].World J Cardiol,2015,7(1):26-30.
[27]Satoh S,Yada R,Inoue H,et al.Toll-like receptor-4 is upregulated in plaque debris of patients with acute coronary syndrome more than Toll-like receptor-2[J].Heart Vessels,2016,31(1):1-5.
[28]Yumoto H,Chou HH,Takahashi Y,et al.Sensitization of human aortic endothelial cells to lipopolysaccharide via regulation of Toll-like receptor 4 by bacterial fimbria-dependent invasion[J].Infect Immun,2005,73(12):8050-8059.
[29]Hajra L,Evans AI,Chen M,et al.The NF-kappa B signal transduction pathway in aortic endothelial cells is primed for activation in regions predisposed to atherosclerotic lesion formation[J].Proc Natl Acad Sci U S A,2000,97(16):9052-9057.
[30]Lin G,Chen S,Lei L,et al.Effects of intravenous injection of Porphyromonas gingivalis on Rabbit inflammatory immune response and atherosclerosis[J].Mediators Inflamm,2015,2015:364391.
[31]Tang YL,Jiang JH,Wang S,et al.TLR4/NF-κB signaling contributes to chronic unpredictable mild stress-induced atherosclerosis in ApoE-/-mice[J].PLoS One,2015,10(4):e0123685.