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北京大学学报（医学版） ›› 2020, Vol. 52 ›› Issue (4): 743-749. doi: 10.19723/j.issn.1671-167X.2020.04.028

• 论著 • 上一篇    下一篇

牙龈卟啉单胞菌感染对载脂蛋白e基因敲除小鼠动脉粥样硬化的影响

轩艳1,蔡宇2,王啸轩2,石巧2,邱立新1,△(),栾庆先2,△()   

  1. 1.北京大学口腔医学院·口腔医院,第四门诊部,北京 100081
    2.北京大学口腔医学院·口腔医院,牙周科 国家口腔疾病临床医学研究中心 口腔数字化医疗技术和材料国家工程实验室 口腔数字医学北京市重点实验室,北京 100081
  • 收稿日期:2018-10-11 出版日期:2020-08-18 发布日期:2020-08-06
  • 通讯作者: 邱立新,栾庆先 E-mail:qiulixin@263.com;kqluanqx@126.com
  • 基金资助:
    国家自然科学基金(81271148);国家自然科学基金(8140030482)

Effect of Porphyromonas gingivalis infection on atherosclerosis in apolipoprotein-E knockout mice

Yan XUAN1,Yu CAI2,Xiao-xuan WANG2,Qiao SHI2,Li-xin QIU1,△(),Qing-xian LUAN2,△()   

  1. 1. Fourth Clinical Division, Peking University School and Hospital of Stomatology, Beijing 100081, China
    2. Department of Periodontology, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
  • Received:2018-10-11 Online:2020-08-18 Published:2020-08-06
  • Contact: Li-xin QIU,Qing-xian LUAN E-mail:qiulixin@263.com;kqluanqx@126.com
  • Supported by:
    National Natural Science Foundation of China(81271148);National Natural Science Foundation of China(8140030482)

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摘要：

目的: 探讨牙龈卟啉单胞菌(Porphyromonas gingivalis,P. gingivalis)引起的炎症和氧化应激反应对动脉粥样硬化的影响及作用机制。方法: 采用8周龄载脂蛋白e基因敲除(ApoE knockout,ApoE-/-)小鼠建立动脉粥样硬化动物模型,将小鼠随机分为两组:(1)磷酸盐缓冲液(phosphate buffered saline,PBS)健康对照组:8只ApoE-/-小鼠,普通饮食+PBS鼠尾静脉注射;(2)P. gingivalis感染组:8只ApoE-/-小鼠,普通饮食+P. gingivalis鼠尾静脉注射。1周3次,隔天1次,共10次。4周后处死,取心脏组织进行油红O染色,血清进行酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA),主动脉进行实时荧光定量PCR以及Western blot检测。结果: P. gingivalis 感染组较PBS健康对照组可以显著加重ApoE-/-小鼠动脉粥样硬化斑块的形成,增加血清中炎症介质,如白细胞介素(interleukin,IL)-1β、IL-6和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)以及氧化应激介质8-羟脱氧鸟苷(8-hydroxy-2-deoxyguanosine,8-OHDG)表达,增加主动脉组织中IL-1β、IL-6、TNF-α、NADPH 氧化酶(NADPH oxidase,NOX)-2和NOX-4基因的mRNA水平。P. gingivalis感染后在主动脉组织中观察到核转录因子-κB(nuclear factor-kappa B,NF-κB)表达有增高趋势。结论: P.gingivalis感染会加速ApoE-/-小鼠动脉粥样硬化进程,诱导氧化应激和炎症反应;NF-κB信号通路可能是P. gingivalis加速动脉粥样硬化形成的重要作用机制。

关键词: 牙龈卟啉单胞菌, 动脉粥样硬化, 炎症, 氧化应激, NF-κB信号通路

Abstract:

Objective: Studies have indicated that periodontal pathogen Porphyromonas gingivalis (P. gingivalis) infection may contributed to accelerate the development of atherosclerosis. The aim of this study was to investigate the effect of inflammation, oxidative stress and the mechanism on atherosclerosis in apolipoprotein-E knockout (ApoE-/-) mice with P. gingivalis infection. Methods: Eight-week-old male ApoE-/- mice (C57BL/6) were maintained under specific pathogen-free conditions and fed regular chow and sterile water after 1 weeks of housing. The animals were randomly divided into two groups: (a) ApoE-/- + PBS (n=8); (b) ApoE-/- + P.gingivalis strain FDC381 (n=8). Both of the groups received intravenous injections 3 times per week for 4 weeks since 8 weeks of age. The sham control group received injections with phosphate buffered saline only, while the P. gingivalis-challenged group with P.gingivalis strain FDC381at the same time. After 4 weeks, oxidative stress mediators and inflammation cytokines were analyzed by oil red O in heart, Enzyme linked immunosorbent assay (ELISA) in serum, quantitative real-time PCR and Western blot in aorta. Results: In our study, we found accelerated development of atherosclerosis and plaque formation in aorta with oil red O staining, increased oxidative stress markers [8-hydroxy-2-deoxyguanosine (8-OHdG), NADPH oxidase (NOX)-2 and NOX-4], as well as increased inflammation cytokines [interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α)] in the serum and aorta of the P. gingivalis-infected ApoE-/- mice. Compared with the control group, there was a significant increase protein level of nuclear factor-kappa B (NF-κB) in aorta after P. gingivalis infection. Conclusion: Our results suggest that chronic intravenous infection of P. gingivalis in ApoE-/- mice could accelerate the development of atherosclerosis by disturbing the lipid profile and inducing oxidative stress and inflammation. The NF-κB signaling pathway might play a potential role in the P. gingivalis-accelerated atherogenesis.

Key words: Porphyromonas gingivalis, Atherosclerosis, Inflammation, Oxidative stress, NF-κB signaling pathway

中图分类号: 

  • R781.42

表1

Real-time PCR引物序列"

Primer Forward(5'-3') Reverse(3'-5')
IL-1β CTATACCTGTCCTGTGTAATGAAAGA TCTGCTTGTGAGGTGCTGATGTA
IL-6 TAGCTACCTGGAGTACATGAAGAACA TGGTCCTTAGCCACTCCTTCTG
TNF-α AGGCGGTGCCTATGTCTCAG GCCATTTGGGAACTTCTCATC
NOX-2 GCCCAAAGGTGTCCAAGC TCCCCAACGATGCGGATAT
NOX-4 ACCCTGTTGGATGACTGGAA ACCAACGGAAAGGACTGGATA
GAPDH TGTGTCCGTCGTGGATCTGA TTGCTGTTGAAGTCGCAGGAG

图1

ApoE-/-小鼠感染P. gingivalis后,主动脉窦腔动脉粥样硬化斑块组织病理学变化"

图2

ELISA检测PBS健康对照组和P. gingivalis感染组血清样本中8-OHdG(A)、IL-1β(B)、IL-6(C)以及TNF-α(D)蛋白表达水平"

图3

实时荧光定量PCR比较PBS对照组和P. gingivalis感染组ApoE-/-小鼠主动脉中各基因mRNA水平的表达"

图4

Western blot检测PBS对照组和P. gingivalis感染组ApoE-/-小鼠主动脉组织NF-κB蛋白水平"

[1] Cutler CW, Kalmar JR, Genco CA. Pathogenic strategies of the oral anaerobe, Porphyromonas gingivalis[J]. Trends Microbiol, 1995,3(2):45-51.
[2] Holt SC, Kesavalu L, Walker S, et al. Virulence factors of Porphyromonas gingivalis[J]. Periodontol 2000,1999(20):168-238.
[3] DeStefano F, Anda RF, Kahn HS, et al. Dental disease and risk of coronary heart disease and mortality[J]. BMJ, 1993,306(6879):688-691.
[4] Southerland JH, Taylor GW, Moss K, et al. Commonality in chronic inflammatory diseases: Periodontitis, diabetes, and coronary artery disease[J]. Periodontol 2000, 2006,40(1):130-143.
[5] Dogan B, Buduneli E, Emingil G, et al. Characteristics of periodontal microflora in acute myocardial infarction[J]. J Periodontol, 2005,76(5):740-748.
pmid: 15898935
[6] Jain A, Batista EL Jr, Serhan C, et al. Role for periodontitis in the progression of lipid deposition in an animal model[J]. Infect Immun, 2003,71(10):6012-6018.
doi: 10.1128/iai.71.10.6012-6018.2003 pmid: 14500522
[7] Ishihara K, Nabuchi A, Ito R, et al. Correlation between detection rates of periodontopathic bacterial DNA in carotid coronary stenotic artery plaque and in dental plaque samples[J]. J Clin Microbiol, 2004,42(3):1313-1315.
[8] Nakano K, Inaba H, Nomura R, et al. Distribution of Porphyromonas gingivalis fimA genotypes in cardiovascular specimens from Japanese patients[J]. Oral Microbiol Immunol, 2008,23(2):170-172.
[9] Kozarov EV, Dorn BR, Shelburne CE, et al. Human atherosclerotic plaque contains viable invasive Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis[J]. Arterioscler Thromb Vasc Biol, 2005,25(3):e17-e18.
[10] Fukasawa A, Kurita-Ochiai T, Hashizume T, et al. Porphyromonas gingivalis accelerates atherosclerosis in C57BL/6 mice fed a high-fat diet[J]. Immunopharmacol Immunotoxicol, 2012,34(3):470-476.
doi: 10.3109/08923973.2011.627866 pmid: 22047042
[11] Yu H, Qi LT, Liu LS, et al. Association of carotid intima-media thickness and atherosclerotic plaque with periodontal status[J]. J Dent Res, 2014,93(8):744-751.
doi: 10.1177/0022034514538973 pmid: 24935064
[12] Lin G, Chen S, Lei L, et al. Effects of intravenous injection of Porphyromonas gingivalis on rabbit inflammatory immune response and atherosclerosis [J/OL]. Mediators Inflamm, 2015: 364391. doi: 10.1155/2015/364391.
[13] Paigen B, Morrow A, Holmes PA, et al. Quantitative assessment of atherosclerotic lesions in mice[J]. Atherosclerosis, 1987,68(3):231-240.
pmid: 3426656
[14] Bélanger M, Rodrigues PH, Dunn WA, et al. Autophagy: A highway for Porphyromonas gingivalis in endothelial cells[J]. Autophagy, 2006,2(3):165-170.
[15] Iwai T. Periodontal bacteremia and various vascular diseases[J]. J Periodontal Res, 2009,44(6):689-694.
[16] Campbell LA, Rosenfeld ME. Infection and atherosclerosis deve-lopment[J]. Arch Med Res, 2015,46(5):339-350.
doi: 10.1016/j.arcmed.2015.05.006 pmid: 26004263
[17] Mattila KJ, Nieminen MS, Valtonen VV, et al. Association between dental health and acute myocardial infarction[J]. BMJ, 1989,298(6676):779-781.
doi: 10.1136/bmj.298.6676.779 pmid: 2496855
[18] Chiu B. Multiple infections in carotid atherosclerotic plaques[J]. Am Heart J, 1999,138(5 Pt 2):S534-S536.
pmid: 10539867
[19] Haraszthy VI, Zambon JJ, Trevisan M, et al. Identification of periodontal pathogens in atheromatous plaques[J]. J Periodontol, 2000,71(10):1554-1560.
[20] Nakano K, Miyamoto E, Tamura K, et al. Distribution of 10 periodontal bacterial species in children and adolescents over a 7-year period[J]. Oral Dis, 2008,14(7):658-664.
doi: 10.1111/j.1601-0825.2008.01452.x pmid: 18565147
[21] Wada K, Kamisaki Y. Roles of oral bacteria in cardiovascular diseases. From molecular mechanisms to clinical cases: Involvement of Porphyromonas gingivalis in the development of human aortic aneurysm[J]. J Pharmacol Sci, 2010,113(2):115-119.
doi: 10.1254/jphs.09r22fm pmid: 20501967
[22] Miyakawa H, Honma K, Qi M, et al. Interaction of Porphyromonas gingivalis with low-density lipoproteins: implications for a role for periodontitis in atherosclerosis[J]. J Periodontal Res, 2004,39(1):1-9.
[23] Li XY, Wang C, Xiang XR, et al. Porphyromonas gingivalis lipopolysaccharide increases lipid accumulation by affecting CD36 and ATP-binding cassette transporter A1 in macrophages[J]. Oncol Rep, 2013,30(3):1329-1336.
pmid: 23835648
[24] Qi M, Miyakawa H, Kuramitsu HK. Porphyromonas gingivalis induces murine macrophage foam cell formation[J]. Microb Pathog, 2003,35(6):259-267.
pmid: 14580389
[25] Ross R. Atherosclerosis is an inflammatory disease[J]. Am Heart J, 1999,138(5 Pt 2):S419-S420.
[26] Hansson GK. Inflammation and immune response in atherosclerosis[J]. Curr Atheroscler Rep, 1999,1(2):150-155.
pmid: 11122704
[27] Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis[J]. Nature, 2011,473(7347):317-325.
pmid: 21593864
[28] Ramji DP, Davies TS. Cytokines in atherosclerosis: Key players in all stages of disease and promising therapeutic targets[J]. Cytokine Growth Factor Rev, 2015,26(6):673-685.
doi: 10.1016/j.cytogfr.2015.04.003 pmid: 26005197
[29] Rosenson RS, Elliott M, Stasiv Y, et al. Randomized trial of an inhibitor of secretory phospholipase A2 on atherogenic lipoprotein subclasses in statin-treated patients with coronary heart disease[J]. Eur Heart J, 2011,32(8):999-1005.
doi: 10.1093/eurheartj/ehq374 pmid: 21081550
[30] Hayashi C, Papadopoulos G, Gudino CV, et al. Protective role for TLR4 signaling in atherosclerosis progression as revealed by infection with a common oral pathogen[J]. J Immunol, 2012,189(7):3681-3688.
doi: 10.4049/jimmunol.1201541 pmid: 22956579
[31] Gibson FC 3rd, Ukai T, Genco CA. Engagement of specific innate immune signaling pathways during Porphyromonas gingivalis induced chronic inflammation and atherosclerosis[J]. Front Biosci, 2008(13):2041-2059.
[32] 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.
doi: 10.1016/j.intimp.2014.10.026 pmid: 25445963
[33] Huang CY, Shih CM, Tsao NW, et al. The GroEL protein of Porphyromonas gingivalis regulates atherogenic phenomena in endothelial cells mediated by up-regulating toll-like receptor 4 expression[J]. Am J Transl Res, 2016,8(2):384-404.
pmid: 27158334
[34] Khlgatian M, Nassar H, Chou HH, et al. Fimbria-dependent activation of cell adhesion molecule expression in Porphyromonas gingivalis-infected endothelial cells[J]. Infect Immun, 2002,70(1):257-267.
doi: 10.1128/iai.70.1.257-267.2002 pmid: 11748191
[35] Gitlin JM, Loftin CD. Cyclooxygenase-2 inhibition increases lipopolysaccharide-induced atherosclerosis in mice[J]. Cardiovasc Res, 2009,81(2):400-407.
doi: 10.1093/cvr/cvn286 pmid: 18948273
[36] Huang KT, Kuo L, Liao JC. Lipopolysaccharide activates endothelial nitric oxide synthase through protein tyrosine kinase[J]. Biochem Biophys Res Commun, 1998,245(1):33-37.
doi: 10.1006/bbrc.1998.8384 pmid: 9535778
[37] Obermeier F, Gross V, Scholmerich J, et al. Interleukin-1 production by mouse macrophages is regulated in a feedback fashion by nitric oxide[J]. J Leukoc Biol, 1999,66(5):829-836.
pmid: 10577516
[38] Sorescu D, Weiss D, Lassègue B. Superoxide production and expression of nox family proteins in human atherosclerosis[J]. Circulation, 2002,105(12):1429-1435.
doi: 10.1161/01.cir.0000012917.74432.66 pmid: 11914250
[39] Szöcs K, Lassègue B, Sorescu D, et al. Upregulation of Nox-based NAD(P)H oxidases in restenosis after carotid injury[J]. Arterioscler Thromb Vasc Biol, 2002,22(1):21-27.
doi: 10.1161/hq0102.102189 pmid: 11788456
[40] Anrather J, Racchumi G, Iadecola C. NF-kappaB regulates pha-gocytic NADPH oxidase by inducing the expression of gp91phox[J]. J Biol Chem, 2006,281(9):5657-5667.
doi: 10.1074/jbc.M506172200 pmid: 16407283
[41] Morris KR, Lutz RD, Choi HS, et al. Role of the NF-kappaB signaling pathway and kappaB cis-regulatory elements on the IRF-1 and iNOS promoter regions in mycobacterial lipoarabinomannan induction of nitric oxide[J]. Infect Immun, 2003,71(3):1442-1452.
doi: 10.1128/iai.71.3.1442-1452.2003 pmid: 12595462
[42] Deng WG, Zhu Y, Wu KK. Up-regulation of p300 binding and p50 acetylation in tumor necrosis factor-alpha-induced cyclooxygenase-2 promoter activation[J]. J Biol Chem, 2003,278(7):4770-4777.
doi: 10.1074/jbc.M209286200 pmid: 12471036
[43] Brown K, Gerstberger S, Carlson L, et al. Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation[J]. Science, 1995,267(5203):1485-1488.
doi: 10.1126/science.7878466 pmid: 7878466
[44] Luoma JS, Stralin P, Marklund SL, et al. Expression of extracellular SOD and iNOS in macrophages and smooth muscle cells in human and rabbit atherosclerotic lesions: colocalization with epitopes characteristic of oxidized LDL and peroxynitrite-modified proteins[J]. Arterioscler Thromb Vasc Biol, 1998,18(2):157-167.
doi: 10.1161/01.atv.18.2.157 pmid: 9484979
[45] Morgan MJ, Liu ZG. Crosstalk of reactive oxygen species and NF-kappa B signaling[J]. Cell Res, 2011,21(1):103-115.
doi: 10.1038/cr.2010.178 pmid: 21187859
[46] Stocker R, Keaney JF Jr. Role of oxidative modifications in atherosclerosis[J]. Physiol Rev, 2004,84(4):1381-1478.
doi: 10.1152/physrev.00047.2003 pmid: 15383655
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