摘要
在炎症性牙周病中，细菌对牙周组织的直接破坏作用是有限的，而细菌产物激发的宿主反应是造成牙周组织破坏的主要原因。牙周优势菌LPS是公认的炎症启动因子，在牙周炎发病中的作用越来越受到重视。牙周优势菌LPS作用于单核巨噬细胞，诱导产生各种细胞因子和炎性介质，参与牙周病的炎症激活和组织创伤。LPS致病作用的强弱，在一定意义上体现在其刺激机体产生内源性细胞因子的能力。本研究旨在探讨五倍子水提取物对牙周病炎症过程中牙周优势菌LPS及LPS所介导产生的细胞因子和炎性介质的影响，为五倍子水提取物用于预防和治疗牙周病提供实验依据。
1、五倍子水提取物对Pg-LPS活性的影响
采用EDS-99细菌LPS检测系统，用动态浊度法，研究五倍子水提取物对Pg-LPS活性的影响，观察五倍子水提取物体外降解Pg-LPS的时效、量效关系。研究表明，5种浓度的五倍子水提取物均能够显著抑制Pg-LPS的活性，其降解作用在一定范围内呈浓度依赖性，同时具有时间依赖性。
2、五倍子水提取物对Pg-LPS结构的影响
通过透射电镜技术观察五倍子水提取物对Pg-LPS结构的影响，电镜观察发现，经五倍子水提取物作用后的Pg-LPS已失去其正常结构，呈散乱分布的残破网状或短带状。说明五倍子水提取物抑制Pg-LPS活性不是暂时性抑制，而是通过破坏Pg-LPS结构起到拮抗的作用。
3、五倍子水提取物对Pg-LPS介导IL—1β活性的影响
采用放射免疫分析法(RIA)，探讨五倍子水提取物对Pg-LPS介导人外周血单核细胞分泌IL-1β活性的影响。结果显示，五种浓度五倍子水提取物均可显著抑制Pg-LPS介导单核细胞分泌的IL-1β的活性，其抑制作用呈浓度依赖性。提示五倍子水提取物抗炎作用的机理是通过抑制Pg-LPS介导的IL-1β的活性而实现的。
第四军医大学硕士学位论文
4、五倍子水提取物对人PDLC中的PGEz与LTB。合成的影响
采用ELISA法和高效液相色谱法，观察五倍子水提取物对人PDLC中PGE。和
LTB．的影响，探讨五倍子水提取物抗炎作用的机理。结果显示，五倍子水提取物
具有抗炎作用，能显著降低PGEz和LTB．的合成和释放，并具有浓度依赖性。表
明五倍子水提取物抗炎作用可能与抑制环氧化酶、脂氧化酶有关。但五种浓度的
五倍子水提取物抑制效果均低于 100ng加l的消炎痛。
5、五倍子水提取物对牙周炎症中胶原酶的影响
应用羟脯氨酸检测法，探讨五倍子水提取物对牙周炎症中胶原酶的影响。实
验显示五倍子水提取物能显著抑制胶原酶的活性，其作用呈浓度依赖性。说明五
倍子水提取物能够阻断胶原酶对结缔组织的破坏作用，抑制或减缓牙周炎的进
展。但浓度低于 100ug加 时其抑制效果低于 sug加 的强力霉素。
The host response to bacterium products is responsible for the direct destruction of the periodontal tissues, while the effect of intact bacterium is limited during the process of inflammatory periodontal diseases, Lipopolysaccharide (LPS) produced by periodontal dominant bacteria is one of generally-recognized inflammatory factors, and its role in the onset of periodontitis is attracting more and more attention. LPS involves in the inflammatory process and tissue destruction during periodontal diseases through inducing mononuclear macrophages to produce various cell factors and inflammatory mediators. The pathogenicity of LPS, in a sense, can be represented by its ability to stimulate the host to produce endogenous cell factors. Thus, the aim of the present study was to investigate the effect of the gallnut water extract on LPS produced by periodontal dominant bacteria and the LPS-induced cell factors and inflammatory mediators during the inflammatory process of periodontal diseases, thereby to provide an e
xperimental basis for the application of the gallnut extract in the prevention and treatment of periodontal diseases.
1. The effect of the gallnut water extract on the activity of Pg-LPS
The effect of the gallnut water extract on the activity and the degradation of Pg-LPS were measured by Using ESD-99 LPS detection system and dynamic turbidity method. The results showed that all 5 concentrations of the extract used in the experiment were effective in inhibiting the activity of the Pg-LPS and its effect on the degradation was time- and dose-dependent.
2. The effect of gallnut water extract on the structure of Pg-LPS
The effect of gallnut water extract on the structure of Pg-LPS was observed with transmission electron microscope. The results revealed that the normal structure
of the Pg-LPS lost, only reticular or short strip like fragments could be seen. This indicated that the inhibition of Pg-LPS activity by the gallnut water extract was not a temporary effect, but a persistent one through the destruction of Pg-LPS structure.
3. The effect of the gallnut water extract on the IL-1β activity induced by Pg-LPS
The effect of the gallnut water extract on the activity of IL-1β secreted by the Pg-LPS pre-treated human peripheral monocytes was examined by radioimmunoassay. The results showed that gallnut water extract could significantly inhibit the activity of IL-1β induced by Pg-LPS, and the effect was dose-dependent. These results indicated that the gallnut extract exerted its anti-inflammatory effect by inhibiting the activity of IL-1β .
4. The effect of the gallnut water extract on the synthesis of PGE2 and LTB4 in human periodontal ligament cells
The effect of the gallnut water extract on the synthesis of PGE2 and LTB4 in human periodontal ligament cells was examined by enzyme linked immunosorbent compitition assay and high-performance liquid chromatography. The results showed that the gallnut water extract had anti-inflammatory effect and could markedly reduce the synthesis and release of PGE2 and LTB4 by human periodontal ligament cells with a dose-dependent manner. These results indicated that the anti-inflammatory effect of gallnut extract might be due to its inhibition to epoxidase and lipoxidase. However, the inhibitory effects at the five concentrations were lower than that of 100ng/ml of Indomethacin
5. The effect of the gallnut water extract on collagenase in periodontal inflammation
The effect of the gallnut water extract on collagenase in periodontal inflammation was examined by Using hydroxyproline detection method. The experiments showed that the gallnut extract had the significant dose-dependent inhibitory effect on the activity of collagenase. However, the inhibitory effect of gallnut extract below 100ug/ml was less than that of 5ug/ml doxycycline. These results indicated that gallnut waster extract could block the connective tissues destruction made by collagenase, thus inhibiting or slowering the progress of periodontal inflammation.
引文
1．牛忠英，史俊南，肖明振．IL-1β与尖周病、牙周病的关系．国外医学．口腔医学分册，1992；19：7．
2．李德懿主编．牙周病微生物学．第一版．天津：天津科技翻译出版公司出版 1994
3. Mayer H, Salimath OV, Holst O et al. Unusual lipid A types in phototrophic bacteria and related species. Rew Infect Dis 1984;6:542-545
4．焦炳华主编．分子内毒素学．第一版．上海科学技术文献出版社 1995
5. Bayston KF, Cohen J. Bacterial endotoxin and current concepts in the diagnosis and treatment of endotoxin. J Med Micrbiol 1990;31:73-83
6. Mitov IG, Terziiski DG. Immunoprophyiaxia and immunotherapy of gramnegative sepsis and shock with antiboodies to core glycolipids and lipids A of bacterial lipopolysaccharides. Infection 1991;19:383-390
7. Matthew J, David A. Endotoxin signal transduction in macrophage. J Leukoc Biolo, 1996; 60: 8-26.
8. Viriyakosol S, Kirkland T. Knowledge of cellular receptors for bacterial endotoxin. Clin Inflect Dis, 1995; 21: s190-s195.
9. Ulevitch RT, Tobias PS. Receptor-dependent mechanisms of cell stimulation by bacterial endotoxin. Ann Rev Immunol, 1995; 13:437-457.
10. Tobias PS, Soldau K, Ulevitch RJ. et al. Identification of a lipid A binding site in the acute phase reactant lipopolysaccharide binding protein. J Biol Chem, 1989;264:10867
11．姜伟主编．内毒素相关疾病治疗与检测．第一版。沈阳．辽宁科学技术出版社 1999
12. Tobias PS, Soldank, Gegner JA et al. Lipopolysaccharide binding
protein-mediated complexation of lipopolysaccharide with soluble CD14. J Biol Chem 1995;270(18):10482-10488
13．侯本祥．第四军医大学博士论文．西安．1992；34
14. Frank AR, Gloria JR, Suzanne MM. Effects ofporphyromonas gingivalis and Escherichia coli ipopolysaccharides on mononuclear phagocyte. Infect Immun 1997;65(8):3248-3254
15. Reddi K, Poles S, Nair S et al. Lipid A-associated proteins from periodontopathogenic bacteria induce interleukin-6 production by human gingival fibroblasts and monocytes. FEMS Immunol Med Microbiol 1995; 11(2): 137-144
16. Schytte BIJ, Helgeland K, Hvattum E et al. Lipopolysaccharide from Actinobacillus actinomycetemcomitans stimulates prodution of interleukin-1β, tumor necrosis factor-α, interleukin-6 and interleukin-lreceptor antagonist in human whole blood. J Periodont Res 1999;34(1):34-40
17. Lucus RM. Et al. J Periodont, 1979;50:20
18. Morioka M, Hinode D, Nagata A et al. Cytotoxicity of porphyromonas gingivalis toward cultured human gingivalis fibroblast. Oral Microbiol Immunol 1993;8(4):203-207
19．张郁。口腔厌氧菌内毒素对人牙髓、牙周膜成纤维细胞和骨细胞的生物学效应及其相关临床研究．西安．第四军医大学博士论文 1992；6
20. Layman DL, Diedrich DL. Growth inhibitory effect of endotoxin from Bacteroides gingivals and intermedius on human gingival fibroblast in vitro. J Periodontol 1987;58:387-392
21. Agarwal S, Chandra CS, Piesco NP et al. Regulation of periodontal ligament cell functions by interleukin-1beta. Infect Immun 1998;66(3):932-937
22．荫俊，等．牙髓紫卟啉菌内毒素对炎症性细胞因子的介导作用．微生物学通报 1999；26(1)；37
23．侯艳蓉，梁傥，罗财成，等。白细胞介素-1加速兔牙齿移动的实验研究．中华口腔医学杂志．1997；32：46
24. McFarlane CG, Reynolds JJ, Meikle MC. The release of interleukin-1 beta, tumor necrosis factor-alpha and interferon-gamma by cultured peripheral blood mononuclear cells from patients with periodontitis. J Periodontal Res, 1990, 25(24):207
25. Feldner BD,Reinhardt RA, Garbin CP, et al. Histological evaluation of interleukin-1 beta and collagen in gingival tissue from untreated adult periodontitis. J Periodont Res 1994;29:54
26. Hoenig JF, Rordorf_Adam C, Siegmund C, et al. Measurement of interleukin 1 alpha and 1 beta (IL-1 alpha and IL-1 beta) in human cystic lesions of the jaw. Implications for the pathogenesis of radicular cysts. J Periodont Res, 1990, 25(4): 156
27．李德懿，石明德，李惠源，等．器官培养中骨吸收因子的相互作用。中华口腔医学杂志 1997；32：306
28．北京医学院主编．生物化学．北京．人民卫生出版社 1981：328
29．陈铁楼，周以钧，吴织芬等．牙周病患者牙龈组织中PGE_2放免测定、免疫组化定量分析．牙体牙髓牙周病学杂志 1995；5(3)：141
30．周以钧，韩桃娟，陈铁楼等．牙周病患者牙龈组织中前列腺素E_2水平研究．实用口腔医学杂志 1992；8(2)：136
31．周以钧，袁乃梅，陈铁楼等．牙周炎患者龈沟液和牙龈组织中PGE_2水平相关性研究．实用口腔医学杂志 1994；10(3)：160
32．彭彬，樊明文．白三烯与牙髓炎．国外医学口腔医学分册 1993；20：1
33. Dahlen SE, Hedqvist P, Hammarstrom S, et al. Leukotrienes are potent constrictors of human bronchi. Nature 1980;288:484
34. White RK, Montgomery S. Leukotrienes: inflammatory mediators--a review. Oral Surg 1986;61:514
35．周学东主编．口腔生物化学．成都．四川大学出版社．2002
36. Lee W, Aitken S, Sodek J, et al. Evidence of a direct relationship between neutrophil collagenase activity and periodontal tissue destruction in vivo: role of active enzyme in human periodontitis. J Periodont Res 1995;30(1):23
37. Romanelli R, Mancini Si, Laschinger C, et al. Activation of neutrophil collagenase in periodontitis. Infect Immun 1999;67(5):2319
38. Ingman T, Tervahartiala T, Ding Y, et al. Matrix metalloproteinases and their inhibitors in gingival crevicular fluid and saliva of periodontitis patients. J Clin Periodontol 1996;23(12): 1127
39．李顺保．内毒素血症的中医药研究进展．中医杂志1996；(1)：53
40．邓文龙，徐嘉红，王文烈，等．热毒平的抗内毒素作用研究．中药药理与临床 1995；(2)：16
41．邓文龙．中医解毒实质的研究及内毒素性疾病的中医药防治．中药药理与临床 1992；(4)：40
42．鲍朝辉，黄启福，许文忠．四君子汤对内毒素激活白细胞诱发肝匀浆脂质过氧化物的影响．北京中医药大学学报 1994；(2)：61
43．张海男，黎杏群，李学文．清肝泻火汤对内毒素诱发家兔肝火证的疗效和机理。中国中西医结合杂志 1996；(2)：95
44．王道生，陈星织，刘世增，等。五种六神丸对大肠杆菌内毒素解毒作用的比较。中成药研究 1995；(7)：28
45．王道生，藏玉英，赵文宝，等．消炎解毒丸对大肠杆菌内毒素的解毒作用．中
成药研究 1982；(10)：31
46．戴锡珍，高淑娟。黄连解毒汤体外抗内毒素作用的实验研究．天津中医 1999；16(3)19
47. Rocksen D, lilliehook B, larsson R, et al. Differential anti-inflammatory and anti-oxidative effects of dexamethasone and N-acetylcysteine in endotoxin-induced lung inflammation. Clin Exp Immunol, 2000; 122(2): 249
48. Dinarello CA. Biologic basis for interleukin-1 in disease. Blood, 1996;87(6)2095
49. Saha N, Moldovan F, Tarlif G, et al. Interleukin-lbeta-converting enzyme/caspase-1 in human osteoarthritic tissues: localization and role in the maturation of interleukin-lbeta and interleukin-18. Arthritis Rherum, 1999;42(8): 1577
50. Bresnihan B, Alvaro-Gracia JM, Cobby M, et al. Treatment of rheumatoid arthritis with recombinant human interleukin-1 receptor antagonist. Arthritis Rherum, 1998;41(12):2196
51. Jeffcoat MK, Reddy MS, Haigh S, et al. A comparison of topical ketorolac, systemic flurbiprofen, and placebo for the inhibition of bone loss in adult periodontitis. J Periodontol 1995;66(5):329
52. Pruzanski W, Stefanski E, Vadas P, et al. Chemically modified non-antimicrobial tetracyclines inhibit activity of phospholipases A2. J Rheumatol, 1998;25(9): 1807
53. Lee HM, Golub LM, Chan D, et al. alpha 1-Proteinase inhibitor in gingival crevicular fluid of humans with adult periodontitis: serpinolytic inhibition by doxycycline. J Periodont Res 1997;32(1):9
54. Greenwald RA, Golub LM, Ramamurthy NS, et al. In vitro sensitivity of the
three mammalian collagenases to tetracycline inhibition: relationship to bone and cartilage degradation. Bone, 1998;22(1):33
55. Ashley RA. Clinical trials of a matrix metalloproteinase inhibitor in human periodontal disease. SDD Clinical Research Team. Ann N Y Acad Sci, 1999;878:335
56. Golub LM, Evans RT, Mcnamara TF, et al. A non-antimicrobial tetracycline inhibits gingival matrix metalloproteinases and bone loss in Porphyromonas gingivalis-induced periodontitis in rats. Ann N Y Acad Sci, 1994;732:96
57．王文俊，等．大黄素丹参素对单核细胞分泌炎性细胞因子的调节．中国免疫学杂志，1995；11(6)：370-373
58. James SS, Hana Haleem-Smith, Jorge Gomer. Tyrosine phosphorlation of vavo stimulates IL-6 production in mast cell by a Rat/c-jun N-termi-nal kinase-dependent pathway. J Immunology, 1999,16:1767-1772
59．徐志鸿，魏克立，俞帆．牙周败毒饮的实验研究．1982，17(3)：177．
60．徐志鸿，孙小平，华红，等．固齿健周煎剂对老年小鼠的抗氧化作用．中华口腔医学杂志，1998，33(4)：219．
61．周黎安，赵瑞芳，周以钧．青少年牙周炎患者服用固齿膏的疗效观察．牙体牙髓牙周病学杂志，1993，3(4)：217。
62．宋红，赵瑞芳，周以钧．固齿膏对牙周炎龈沟液中IL-8的影响．中华口腔医学杂志，1997，32(6)：372．
63．樊明文，彭彬，罗玲莉．固齿散对体外培养人牙龈成纤维细胞的影响．中华口腔医学杂志，1995，30(5)：298。
64．曹进．茶色素抗菌抗病毒作用的研究进展．中草药，1998；29(9)：636-639．
65. Takada H,Mihara J,Morisako I,Hamada S. Induction of interleukin-1 and -6 in human gingival fibroblast cultures stimulated with bacteroides
lipopolysaccharides. Infect Immun, 1991; 59:295-301.
66. Chong-Pyoung Chung, Joon-Bong Park, Ki-Hwan Bae. Pharmacological effects of methanolic extract from the root of Scutellaria baicalensis and its flavonoids on human gingival fibroblast. Planta Med, 1995; 61(3): 150-153.
67．含桂皮提取物抗牙周炎的牙膏[英]／CPI(B)1992，(9211)：50 J0 4029-933-A．《国外医药》植物药分册，1993；8(1)：45．
68．治疗牙周疾病的草药合剂--W0 9735599-A_1，《国外医药》植物药分册，1999；14(1)：38．
69．含芦荟和叶绿素混合物具有抗炎作用的牙膏[英]／CPI(B)1994，(9411)：102．《国外医药》植物药分册，1995；10(4)：188。
70．银杏叶提取物在口腔保健上的应用．W0 9818479-A_1，《国外医药》植物药分册，2000；15(1)：39
71. Ho Ky, Huang JS, Tsai CC, et al. Antioxidant activity of tannin components from Vaccinium vitis-idaea L. J Pharm Pharmacol, 1999; 51 (9): 1075-1078
72．刘国廉．五倍子抗菌作用研究 中华医学杂志；1965；51(4)：245-247
73．吴玉林．五倍子用于拔牙后出血。现代应用药学，1988，10(4)：41．
74．李广勋等．中药药理与临床．天津科技出版公司 1992：272
75．傅乃武、郭蓉、刘福成．柯子鞣质和五倍子鞣酸抑制体内亚硝胺生成和对抗活性氧的作用 中草药 1992；23(11)：585-589
76．何敏．五倍子临床近况研究，中医药学报．1990。(4)：42
77．熊炎斌、李德玲等．几种植物药对变链菌生长、产酸、粘附影响的体外实验．中华口腔医学杂志 1997；32(5)：261
78．王艳，唐荣银；中药五倍子防龋的动物实验．牙体牙髓牙周病学杂志．2001；11(3)：172-174
79. Wu-yuan CD; Chen CY; Wu RT. Gallotannins inhibit growth, water-insoluble
glucan synthesis, and aggregation of mutans streptococci. J Dent Res, 1988; 67:51-55
80. Ooshima T, Minami T, Aono W, et al. Oolong tea polypHenols inhibit experimental dental caries in SPF rats infected with mutans streptococci. Caries Res, 1993; 27(2): 124-129
81．王艳 中药五倍子防龋的实验研究[学位论文] 第四军医大学口腔医学院 2001
82．朱秀丽，陈强，唐荣银，等．五倍子对5种常见牙周细菌抑制作用的体外研究．牙体牙髓牙周病学杂志，2002；12(5)：255
