连翘酯苷对基质金属蛋白酶活性抑制的研究
摘要
基质金属蛋白酶(MMPs)是一类Zn2+依赖的内肽酶家族,在正常和病理情况下的细胞外基质的重塑过程中发挥重要的作用。MMPs在调控细胞因子、生长因子、激素和细胞粘附分子受体等的合成和分泌中发挥重要作用,进而参与细胞凋亡、形态发生、增殖和发育等一系列病理和生理过程。当MMPs的调控机制失去平衡,MMPs活性异常升高时,就会导致一些病理过程的发生。因此,以MMPs为治疗靶点,寻找MMPs的拮抗药物,尤其是MMPs选择性抑制药物,已经成为筛选抗肿瘤药物的热点。
本论文通过从中药连翘中分离提取出能够抑制MMPs活性的单体化合物连翘酯苷。通过酶学方法和分子对接模拟方法检测其对MMPs活性的抑制作用,并从通过起其对细胞行为学的影响检测其在细胞学水平的影响,发现连翘酯苷对MMPs的抑制作用具有选择性,这将为新型的MMPs特异性抑制剂的开发提供思路。
The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidase involved in the degradation and remodeling the extracellular matrix proteins. The activities of these enzymes are well regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Chronic stimulation of MMPs activities due to an imbalance in the levels of MMPs and TIMPs has been implicated in the pathogenesis of a variety of diseases such as cancer, osteoarthritis, rheumatoid arthritis. The MMPs which have been proved top lay key roles in the processes of tumor growth, invasion, metastasis and angiogenesis,are frequently overexpressed in malignant tumors, and have been associated with an aggressive malignant phenotype and poor prognosis inpatients with cancer. Thus it is believed that the growth and metastasis can be controlled by MMP inhibitors (MMPIs) which are expected to be useful for the treatment of cancer. Nowadays, many of the inhibitors of matrix metalloproteinase have come into clinical trial, but to most of our disappointment, none was tested to be a candidate for further curing disease. The main problem of this is the weak selectivity of MMPIs, because a long time non-selectivity inhibition of MMPs may bring out some unexpected biological disorder. In conclusion, it is foremost meaningful to find a MMPI with a high selectivity, affinity and biological availability.
Forsythiaside belonging to the derivatives of caffeinic acid is the effective ingredient of the Chinese herb Forsythia. In this thesis, Forsythiaside has been obtained by using Macro-reticular resin D101 and HPLC, which have some obvious advantages: saving solve, time and having high performance. The inhibition ability to MMPs of forsythiaside was tested by monitoring the strength of fluorescent emissioning from the cleaved substrate of MMPs. Then we docked the forsythiaside to different MMPs providing a detailed inhibition mechanism. At last, we observed the affect of forsythiaside to HT1080 cells on the aspect of cell proliferation, 3-D culture, migration and invasion. All the results showed that forsythiaside has higher inhibition ability to MMP-13 than others with a dose-dependent way which is consistent to data of docking. The docking as well suggested that forsythiaside located the S1 and S1’pocket of MMPs. Forsythiaside can inhibit the migration and invasion of HT1080 cells in vitro. The specific mechanism to be further verified. As a result, the specific MMP inhibition of forsythiaside will not only greatly help us to find a leading more potent compound but provide a new strategy for MMPIs development.
We can continue studying in the future, it is investigated to pay attention to the inhibition of this new MMPI, at the same time, we should take attention to activities, toxicities, pharmacology, biological, pharmacokinetic and drug metabolism of forsythiaside further. On one hand facilitate our synthesizing and finding new MMP inhibitor, thus develop and regard MMP as the molecule target new medicine with independent intellectual property right; On the other hand explain the function mechanism of traditional Chinese medicine have benefits greatly from molecule level for us, and has offered basis for developing, synthesizing and transforming the medicine guide compound and guidance. Continue launching animal’s model experiment and clinical experiment in a situation that a large number of external experiments are ripe, the mechanism of treating tumour for traditional Chinese medicine studies opens up the new broad space.
本论文通过从中药连翘中分离提取出能够抑制MMPs活性的单体化合物连翘酯苷。通过酶学方法和分子对接模拟方法检测其对MMPs活性的抑制作用,并从通过起其对细胞行为学的影响检测其在细胞学水平的影响,发现连翘酯苷对MMPs的抑制作用具有选择性,这将为新型的MMPs特异性抑制剂的开发提供思路。
The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidase involved in the degradation and remodeling the extracellular matrix proteins. The activities of these enzymes are well regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Chronic stimulation of MMPs activities due to an imbalance in the levels of MMPs and TIMPs has been implicated in the pathogenesis of a variety of diseases such as cancer, osteoarthritis, rheumatoid arthritis. The MMPs which have been proved top lay key roles in the processes of tumor growth, invasion, metastasis and angiogenesis,are frequently overexpressed in malignant tumors, and have been associated with an aggressive malignant phenotype and poor prognosis inpatients with cancer. Thus it is believed that the growth and metastasis can be controlled by MMP inhibitors (MMPIs) which are expected to be useful for the treatment of cancer. Nowadays, many of the inhibitors of matrix metalloproteinase have come into clinical trial, but to most of our disappointment, none was tested to be a candidate for further curing disease. The main problem of this is the weak selectivity of MMPIs, because a long time non-selectivity inhibition of MMPs may bring out some unexpected biological disorder. In conclusion, it is foremost meaningful to find a MMPI with a high selectivity, affinity and biological availability.
Forsythiaside belonging to the derivatives of caffeinic acid is the effective ingredient of the Chinese herb Forsythia. In this thesis, Forsythiaside has been obtained by using Macro-reticular resin D101 and HPLC, which have some obvious advantages: saving solve, time and having high performance. The inhibition ability to MMPs of forsythiaside was tested by monitoring the strength of fluorescent emissioning from the cleaved substrate of MMPs. Then we docked the forsythiaside to different MMPs providing a detailed inhibition mechanism. At last, we observed the affect of forsythiaside to HT1080 cells on the aspect of cell proliferation, 3-D culture, migration and invasion. All the results showed that forsythiaside has higher inhibition ability to MMP-13 than others with a dose-dependent way which is consistent to data of docking. The docking as well suggested that forsythiaside located the S1 and S1’pocket of MMPs. Forsythiaside can inhibit the migration and invasion of HT1080 cells in vitro. The specific mechanism to be further verified. As a result, the specific MMP inhibition of forsythiaside will not only greatly help us to find a leading more potent compound but provide a new strategy for MMPIs development.
We can continue studying in the future, it is investigated to pay attention to the inhibition of this new MMPI, at the same time, we should take attention to activities, toxicities, pharmacology, biological, pharmacokinetic and drug metabolism of forsythiaside further. On one hand facilitate our synthesizing and finding new MMP inhibitor, thus develop and regard MMP as the molecule target new medicine with independent intellectual property right; On the other hand explain the function mechanism of traditional Chinese medicine have benefits greatly from molecule level for us, and has offered basis for developing, synthesizing and transforming the medicine guide compound and guidance. Continue launching animal’s model experiment and clinical experiment in a situation that a large number of external experiments are ripe, the mechanism of treating tumour for traditional Chinese medicine studies opens up the new broad space.
引文
1. Burgeson RE, Nimni ME. Collagen types, molecular structure and tissue distribution [J]. Clin Orthop Relat Res, 1992, 7 (282): 250-272
2. 刘秉慈, 许增禄. 细胞外基质. 生理科学进展, 1996, 27 (3): 63-71
3. Lynch CC, Matrisian LM. Matrix metalloproteinases in tumor-host cell communication [J]. Differentiation, 2002, 70 (9-10): 561-573
4. Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression[J]. Nature Reviews Cancer, 2002, 2: 161-174.
5. Overall CM, López-Otín C. Strategies for MMP inhibition in cancer: Innovation for the post-trial era[J]. Nature Reviews Cancer, 2002, 2: 657-672.
6. Nagase H, Woessner JF Jr. Matrix metalloproteinases. Biological Chemistry[J], 1999, 274(31): 21491-21494..
7. Rosenblum G, Meroueh SO, Kleifeld O, et al. Structural Basis for potent slow binding inhibition of human matrix metalloproteinase-2[J]. J Biol Chem, 2003, 278(29): 27009-27015
8. Visse R, Nagase H. Matrix Metalloproteinases and tissue Inhibitors of metalloproteinases: structure, function, and biochemistry[J]. Circ Res, 2003, (92): 827-830.
9. Kiynma R, Tamura Y, Watanabe F, et al. Homology modeling of gelatinase catalytic domain and docking simulations of novel sulfonamide inhibitors[J]. J Med Chem, 1999, 42(10): 1723-1738
10. Morgunova E, Tuttila A, Bergmann U, et al. Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed[J].Science, 1999, 284(5420): 1667-1670
11. Park HI, Ni J, Gerkema FE, et al. Identification and characterization of human endometase (matrix metalloproteinase-26) from endometrial tumor[J]. J Biol Chem, 2000, 275(27): 20540-25544
12. Pei D. CA-MMP: a matrix metalloproteinase with a novel cysteine array, but without the classic cysteine switch[J]. FEBS Lett, 1999, 457: 262-270
13. Stetler-Stevenson WG, Aznavoorian S, Liotta LA. Tumor cell interactions with the extracellular matrix during invasion and metastasis[J]. Annu. Rev Cell Biol, 1993, 9: 541-573
14. Sato H, Takino T, Okada Y, et al. A matrix metalloproteinase expressed on the surface of invasive tumor cells[J]. Nature, 1994, 370: 61-65
15. Chambers AF, Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis[J]. J Natl Cancer Inst, 1997, 89: 1260-1270
16. Johnson LL, Dyer R, Hupe DJ, et al. Matrix metahoproteinases[J]. Curr Opin Chem Bio, 1998, 2: 466-471
17. Nagase H, Woessner JF Jr. Matrix metalloproteinases[J]. J Biol Chem, 1999, 274(31): 21491-21494
18. Takino T, Sato H, Shinagawa A, et al. Identification of the second membrane-type matrix metalloproteinase (MT MMP-2) gene from a human placenta cDNA library[J]. J Biol Chem, 1995, 270(39): 23013-23020
19. Lohmander LS, Hoerrner LA, Lark MW, et al. Metalloproteinases, tissue inhibitor, and proteoglycan fragments in knee synovial fluid inhuman osteoarthritis[J]. Arthritis Rheum, 1993, 36(2): 181-189.
21. Mira E, Manes S, Lacalle RA, et al. Insulin-like growth factor I-triggered cell migration and invasion are mediated by matrix metalloproteinase-9[J]. Endocrenology, 1999, 140(4): 1657-1664
22. Martin DC, Fowlkes JL, Babic B, et al. Insulin-like growth factor II signaling in neoplastic proliferation is blocked by transgenic expression of the metalloproteinase inhibitor TIMP-1[J]. J Cell Biol. 1999, 146(4): 881-892
23. Manes S, Mira E, Barbacid MM, et al. Identification of insulin-like growth factor-binding protein-1 as a potential physiological substrate for human stromelysin-3[J]. J Bio Chem, 1997, 272(41): 25706-25712
24. Simon C, Goepfert H, Boyd D. Inhibition of the p38 mitogen-activated protein kinase by SB 203580 blocks PMA-induced Mr 92,000 type IV collagenase secretion and in vitro invasion[J]. Cancer Res, 1998, 58(6): 1135-1139
25. Nelson AR, Fingleton B, Rothenberg ML, et al. Matrix metalloproteinases: biologic activity and clinical implications[J]. J Clin Onco, 2000, 18(5): 1135-1149
26. Vacca A, Ribatti D, Presta M, et al. Bone marrow neovascularization, plasma cell angiogenic potential, and matrix metalloproteinase-2 secretion parallel progression of human multiple myeloma[J]. Blood, 1999, 93(9): 3064-3073
27. Ray JM, Stetler-Stevenson WG. The role of matrix metalloproteinases and their inhibitors in tumor invasion, metastasis and angiogenesis [J].Eur Respir J, 1994, 7(11): 2062-2072
28. Ivaska J, Heino J. Adhesion receptors and cell invasion: mechanisms of integrin-guided degradation of extracellular matrix [J]. Cell Mol Life Sci, 2000, 57(1): 16-24
29. O'Reilly MS, Holmgren L, Shing Y, et al. Angiogenesis: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma [J]. Cell, 1994, 79(2): 315-328
30. Giannelli G, Falk Marzillier J, Schiraldi O, et al. Induction of cell migration by matrix metalloproteinase-2 cleavage of laminin-5[J]. Science, 1997, 277(5352): 225-228
31. Malik N, Greenfield BW, Wahl AF, et al. Activation of human monocytes through CD40 induces matrix metalloproteinases[J]. J Immunol. 1996, 156: 3952-3960.
32. Schonbeck U, Mach F, Sukhova GK, et al. Regulation of matrix metalloproteinase expression in human vascular smooth muscle cells by T lymphocytes[J]. Circ Res, 1997, 81(3): 448-454.
33. Spinale FG, Coker ML, Heung LJ, et al. A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure[J]. Circulation, 2000, 102(16): 1944-1949.
34. Wassenaar A, Virschoor T, Kievit F, et al. CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts[J]. Clin Exp Immunol, 1999, 115: 161-167.
35. Mauviel A. Cytokine regulation of metalloproteinase gene expression[J]. J Cell Biochem. 1993, 53(4): 288-295.
36. Makela M, Salo T, Larjava H. MMP-9 from TNFα-stimulated keratinocytes binds to cell membranes and type I collagen: a cause for extended matrix degradation in inflammation[J]. Biochem Biophys Res Commun, 1998, 253: 325-335.
37. Nagase H, Woessner, JF, Jr. Matrix metalloproteinases[J]. J Biol Chem, 1999, 274: 21491-21494
38. Caterina J, Shi J, Krakora S, et al. Isolation, characterization, and chromosomal location of the mouse enamelysin gene[J]. Genomics, 1999, 62: 308-311
39. Simon C, Goepfet H, Boyd D. Inhibition of the p38 mitogen-activated protein kinase by SB 203580 blocks PMA-induced Mr 92,000 type IV collagenase secretion and in vitro invasion[J]. Cancer Res, 1998, 58: 1135-1139
40. Chau I, Rigg A, Cunningham D. Matrix metalloproteinase inhibitors-an emphasis on gastrointestinal malignancies[J]. Critical Reviews in Oncology/Hematology, 2003, 45: 151-176
41. Liu YE, Wang M, Greene J,et al. Preparation and characterization of recombinant tissue inhibitor of metalloproteinase-4 (TIMP-4)[J]. J Biol Chem. 1997, 272(33): 20479-20483.
42. Greene J, Wang M, Liu YE, et al. Molecular cloning and characterization of human tissue inhibitor of metalloproteinase-4[J]. J Biol Chem, 1996, 271(48): 30375-30380.
43. Hayakawa T, Yamashita K, Ohuchi E, et al. Cell growth promoting activity of tissue inhibitor of metalloproteinases-2 (TIMP-2) [J]. J Cell Sci, 1994, 107(9): 2373-2379.
44. Hayakawa T, Yamashita K, Tanzawa K, et al. Growth promoting activity of tissue inhibitor of metalloproteinases-1 (TIMP-1) for a wide range of cells: a possible new growth factor in serum[J]. FEBS Lett, 1992, 298(1): 29-32.
45. Thorgeirsson UP, Yoshiji H, Sinha CC, et al. Breast cancer, tumor neovasculature and the effect of tissue inhibitor of metalloproteinases-1 (TIMP-1) on angiogenesis[J]. In Vivo, 1996, 10(2): 137-144.
46. Baker AH, Zaltsman AB, George SJ, et al. Divergent effects of tissue inhibitor of metalloproteinase-1, -2 or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis[J]. J Clin Invest, 1998, 101: 1478 -1487.
47. http://merops.sanger.ac.uk/
48. Kontogiorgis CA, Papaioannou P, Hadjipavlou-Litina DJ. Matrix metalloproteinase inhibitors: a review on pharmacophore mapping and (Q) SARs results[J]. Curr Med Chem, 2005, 12: 339-355
49. Johansson N, Ala-aho R, Uitto V, et al. Expression of collagenase-3 (MMP-13) and collagenase-l (MMP-l) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase [J]. J Cell Sci, 2000, 113(2): 227-235.
50. 孔灵玲, 方伟岗, 由江峰, 等. 可调控型反义基质金属蛋白酶-9表达抑制人黑色素瘤细胞侵袭转移表型的研究[J]. 中华病理学杂志, 2003, 32(2): 137-141
51. 杨鹏, 张金桐, 黄晋玲, 等. 山西省野生油脂植物连翘资源的调查及种籽油提取与分析[J]. 山西农业大学学报, 1996, 16(4): 394-396
52. Nishibe S, Okabe K, Tsukamoto H, et al. The structure of forsythiaside isolated from forsythia suspense[J]. Chem Pharm Bull,1982, 30(3): 1048-1050
53. Nishibe S, Okabe K, Tsukamoto H, et al. Studies on the Chinese crude drug “Forsythiae Fructus”Ⅵ [J]. Chem Pharm Bull, 1982, 30(12): 4548-4553
54. 梁文藻. 连翘成分分析 I 连翘甙和连翘脂素的分离鉴定和测定[J]. 药物分析杂志. 1985. 5(1): 58
55. 匡海学, 张宁, 陆志博. 青连翘抗菌活性成分的研究[J]. 中药通报, 1988, 13(7): 32-34
56. 贺铭, 王教才. 连翘子精油的化学成分研究(第一报)[J]. 陕西新医药, 1982, 11(8): 53-55
57. 朱淑云, 杨建雄, 李发荣. 连翘叶提取物对小鼠氧化损伤的保护作用[J]. 中药药理与临床, 2004, 20(1): 18-20
58. 孔杰, 姚健, 达文燕等. 连翘挥发油化学成分的研究[J]. 西北师范大学学报(自然科学版), 2001, 37(4): 77-81
59. 张海燕. 连翘化学成分及药理活性的研究进展[J]. 中药材, 2000, 23(10): 298-307
60. 张海燕. 连翘化学成分及药理活性的研究进展[J]. 中药材, 2000, 23(10): 657-659
61. 高淑娟, 戴锡珍, 要华民, 等. 几种清热解毒中药抗内毒素作用的比较实验[J]. 天津中医, 1992, (3): 42
62. 丁冈, 刘延泽, 中药连翘及其同属植物的研究近况[J]. 中药材, 1994, 17(10): 42-44
63. 于起福, 孙非. 四种中草药水煎剂抗柯萨奇 B5 病毒的细胞学实验研究[J]. 吉林中医药, 1995, 1: 35
64. Stote RH, Karplus M. Zinc binding in proteins and solution: a simple but accurate nonbonded representation[J]. Proteins, 1995, 23(1): 12-31.
65. Hu X, Balaz S, Shelver WH. Docking studies of matrix metalloproteinase inhibitors: zinc parameter optimization to improve the binding free energy prediction[J]. Journal of Molecular Graphics and Modelling. 2003, 22(2): 115-126
2. 刘秉慈, 许增禄. 细胞外基质. 生理科学进展, 1996, 27 (3): 63-71
3. Lynch CC, Matrisian LM. Matrix metalloproteinases in tumor-host cell communication [J]. Differentiation, 2002, 70 (9-10): 561-573
4. Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression[J]. Nature Reviews Cancer, 2002, 2: 161-174.
5. Overall CM, López-Otín C. Strategies for MMP inhibition in cancer: Innovation for the post-trial era[J]. Nature Reviews Cancer, 2002, 2: 657-672.
6. Nagase H, Woessner JF Jr. Matrix metalloproteinases. Biological Chemistry[J], 1999, 274(31): 21491-21494..
7. Rosenblum G, Meroueh SO, Kleifeld O, et al. Structural Basis for potent slow binding inhibition of human matrix metalloproteinase-2[J]. J Biol Chem, 2003, 278(29): 27009-27015
8. Visse R, Nagase H. Matrix Metalloproteinases and tissue Inhibitors of metalloproteinases: structure, function, and biochemistry[J]. Circ Res, 2003, (92): 827-830.
9. Kiynma R, Tamura Y, Watanabe F, et al. Homology modeling of gelatinase catalytic domain and docking simulations of novel sulfonamide inhibitors[J]. J Med Chem, 1999, 42(10): 1723-1738
10. Morgunova E, Tuttila A, Bergmann U, et al. Structure of human pro-matrix metalloproteinase-2: activation mechanism revealed[J].Science, 1999, 284(5420): 1667-1670
11. Park HI, Ni J, Gerkema FE, et al. Identification and characterization of human endometase (matrix metalloproteinase-26) from endometrial tumor[J]. J Biol Chem, 2000, 275(27): 20540-25544
12. Pei D. CA-MMP: a matrix metalloproteinase with a novel cysteine array, but without the classic cysteine switch[J]. FEBS Lett, 1999, 457: 262-270
13. Stetler-Stevenson WG, Aznavoorian S, Liotta LA. Tumor cell interactions with the extracellular matrix during invasion and metastasis[J]. Annu. Rev Cell Biol, 1993, 9: 541-573
14. Sato H, Takino T, Okada Y, et al. A matrix metalloproteinase expressed on the surface of invasive tumor cells[J]. Nature, 1994, 370: 61-65
15. Chambers AF, Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis[J]. J Natl Cancer Inst, 1997, 89: 1260-1270
16. Johnson LL, Dyer R, Hupe DJ, et al. Matrix metahoproteinases[J]. Curr Opin Chem Bio, 1998, 2: 466-471
17. Nagase H, Woessner JF Jr. Matrix metalloproteinases[J]. J Biol Chem, 1999, 274(31): 21491-21494
18. Takino T, Sato H, Shinagawa A, et al. Identification of the second membrane-type matrix metalloproteinase (MT MMP-2) gene from a human placenta cDNA library[J]. J Biol Chem, 1995, 270(39): 23013-23020
19. Lohmander LS, Hoerrner LA, Lark MW, et al. Metalloproteinases, tissue inhibitor, and proteoglycan fragments in knee synovial fluid inhuman osteoarthritis[J]. Arthritis Rheum, 1993, 36(2): 181-189.
21. Mira E, Manes S, Lacalle RA, et al. Insulin-like growth factor I-triggered cell migration and invasion are mediated by matrix metalloproteinase-9[J]. Endocrenology, 1999, 140(4): 1657-1664
22. Martin DC, Fowlkes JL, Babic B, et al. Insulin-like growth factor II signaling in neoplastic proliferation is blocked by transgenic expression of the metalloproteinase inhibitor TIMP-1[J]. J Cell Biol. 1999, 146(4): 881-892
23. Manes S, Mira E, Barbacid MM, et al. Identification of insulin-like growth factor-binding protein-1 as a potential physiological substrate for human stromelysin-3[J]. J Bio Chem, 1997, 272(41): 25706-25712
24. Simon C, Goepfert H, Boyd D. Inhibition of the p38 mitogen-activated protein kinase by SB 203580 blocks PMA-induced Mr 92,000 type IV collagenase secretion and in vitro invasion[J]. Cancer Res, 1998, 58(6): 1135-1139
25. Nelson AR, Fingleton B, Rothenberg ML, et al. Matrix metalloproteinases: biologic activity and clinical implications[J]. J Clin Onco, 2000, 18(5): 1135-1149
26. Vacca A, Ribatti D, Presta M, et al. Bone marrow neovascularization, plasma cell angiogenic potential, and matrix metalloproteinase-2 secretion parallel progression of human multiple myeloma[J]. Blood, 1999, 93(9): 3064-3073
27. Ray JM, Stetler-Stevenson WG. The role of matrix metalloproteinases and their inhibitors in tumor invasion, metastasis and angiogenesis [J].Eur Respir J, 1994, 7(11): 2062-2072
28. Ivaska J, Heino J. Adhesion receptors and cell invasion: mechanisms of integrin-guided degradation of extracellular matrix [J]. Cell Mol Life Sci, 2000, 57(1): 16-24
29. O'Reilly MS, Holmgren L, Shing Y, et al. Angiogenesis: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma [J]. Cell, 1994, 79(2): 315-328
30. Giannelli G, Falk Marzillier J, Schiraldi O, et al. Induction of cell migration by matrix metalloproteinase-2 cleavage of laminin-5[J]. Science, 1997, 277(5352): 225-228
31. Malik N, Greenfield BW, Wahl AF, et al. Activation of human monocytes through CD40 induces matrix metalloproteinases[J]. J Immunol. 1996, 156: 3952-3960.
32. Schonbeck U, Mach F, Sukhova GK, et al. Regulation of matrix metalloproteinase expression in human vascular smooth muscle cells by T lymphocytes[J]. Circ Res, 1997, 81(3): 448-454.
33. Spinale FG, Coker ML, Heung LJ, et al. A matrix metalloproteinase induction/activation system exists in the human left ventricular myocardium and is upregulated in heart failure[J]. Circulation, 2000, 102(16): 1944-1949.
34. Wassenaar A, Virschoor T, Kievit F, et al. CD40 engagement modulates the production of matrix metalloproteinases by gingival fibroblasts[J]. Clin Exp Immunol, 1999, 115: 161-167.
35. Mauviel A. Cytokine regulation of metalloproteinase gene expression[J]. J Cell Biochem. 1993, 53(4): 288-295.
36. Makela M, Salo T, Larjava H. MMP-9 from TNFα-stimulated keratinocytes binds to cell membranes and type I collagen: a cause for extended matrix degradation in inflammation[J]. Biochem Biophys Res Commun, 1998, 253: 325-335.
37. Nagase H, Woessner, JF, Jr. Matrix metalloproteinases[J]. J Biol Chem, 1999, 274: 21491-21494
38. Caterina J, Shi J, Krakora S, et al. Isolation, characterization, and chromosomal location of the mouse enamelysin gene[J]. Genomics, 1999, 62: 308-311
39. Simon C, Goepfet H, Boyd D. Inhibition of the p38 mitogen-activated protein kinase by SB 203580 blocks PMA-induced Mr 92,000 type IV collagenase secretion and in vitro invasion[J]. Cancer Res, 1998, 58: 1135-1139
40. Chau I, Rigg A, Cunningham D. Matrix metalloproteinase inhibitors-an emphasis on gastrointestinal malignancies[J]. Critical Reviews in Oncology/Hematology, 2003, 45: 151-176
41. Liu YE, Wang M, Greene J,et al. Preparation and characterization of recombinant tissue inhibitor of metalloproteinase-4 (TIMP-4)[J]. J Biol Chem. 1997, 272(33): 20479-20483.
42. Greene J, Wang M, Liu YE, et al. Molecular cloning and characterization of human tissue inhibitor of metalloproteinase-4[J]. J Biol Chem, 1996, 271(48): 30375-30380.
43. Hayakawa T, Yamashita K, Ohuchi E, et al. Cell growth promoting activity of tissue inhibitor of metalloproteinases-2 (TIMP-2) [J]. J Cell Sci, 1994, 107(9): 2373-2379.
44. Hayakawa T, Yamashita K, Tanzawa K, et al. Growth promoting activity of tissue inhibitor of metalloproteinases-1 (TIMP-1) for a wide range of cells: a possible new growth factor in serum[J]. FEBS Lett, 1992, 298(1): 29-32.
45. Thorgeirsson UP, Yoshiji H, Sinha CC, et al. Breast cancer, tumor neovasculature and the effect of tissue inhibitor of metalloproteinases-1 (TIMP-1) on angiogenesis[J]. In Vivo, 1996, 10(2): 137-144.
46. Baker AH, Zaltsman AB, George SJ, et al. Divergent effects of tissue inhibitor of metalloproteinase-1, -2 or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis[J]. J Clin Invest, 1998, 101: 1478 -1487.
47. http://merops.sanger.ac.uk/
48. Kontogiorgis CA, Papaioannou P, Hadjipavlou-Litina DJ. Matrix metalloproteinase inhibitors: a review on pharmacophore mapping and (Q) SARs results[J]. Curr Med Chem, 2005, 12: 339-355
49. Johansson N, Ala-aho R, Uitto V, et al. Expression of collagenase-3 (MMP-13) and collagenase-l (MMP-l) by transformed keratinocytes is dependent on the activity of p38 mitogen-activated protein kinase [J]. J Cell Sci, 2000, 113(2): 227-235.
50. 孔灵玲, 方伟岗, 由江峰, 等. 可调控型反义基质金属蛋白酶-9表达抑制人黑色素瘤细胞侵袭转移表型的研究[J]. 中华病理学杂志, 2003, 32(2): 137-141
51. 杨鹏, 张金桐, 黄晋玲, 等. 山西省野生油脂植物连翘资源的调查及种籽油提取与分析[J]. 山西农业大学学报, 1996, 16(4): 394-396
52. Nishibe S, Okabe K, Tsukamoto H, et al. The structure of forsythiaside isolated from forsythia suspense[J]. Chem Pharm Bull,1982, 30(3): 1048-1050
53. Nishibe S, Okabe K, Tsukamoto H, et al. Studies on the Chinese crude drug “Forsythiae Fructus”Ⅵ [J]. Chem Pharm Bull, 1982, 30(12): 4548-4553
54. 梁文藻. 连翘成分分析 I 连翘甙和连翘脂素的分离鉴定和测定[J]. 药物分析杂志. 1985. 5(1): 58
55. 匡海学, 张宁, 陆志博. 青连翘抗菌活性成分的研究[J]. 中药通报, 1988, 13(7): 32-34
56. 贺铭, 王教才. 连翘子精油的化学成分研究(第一报)[J]. 陕西新医药, 1982, 11(8): 53-55
57. 朱淑云, 杨建雄, 李发荣. 连翘叶提取物对小鼠氧化损伤的保护作用[J]. 中药药理与临床, 2004, 20(1): 18-20
58. 孔杰, 姚健, 达文燕等. 连翘挥发油化学成分的研究[J]. 西北师范大学学报(自然科学版), 2001, 37(4): 77-81
59. 张海燕. 连翘化学成分及药理活性的研究进展[J]. 中药材, 2000, 23(10): 298-307
60. 张海燕. 连翘化学成分及药理活性的研究进展[J]. 中药材, 2000, 23(10): 657-659
61. 高淑娟, 戴锡珍, 要华民, 等. 几种清热解毒中药抗内毒素作用的比较实验[J]. 天津中医, 1992, (3): 42
62. 丁冈, 刘延泽, 中药连翘及其同属植物的研究近况[J]. 中药材, 1994, 17(10): 42-44
63. 于起福, 孙非. 四种中草药水煎剂抗柯萨奇 B5 病毒的细胞学实验研究[J]. 吉林中医药, 1995, 1: 35
64. Stote RH, Karplus M. Zinc binding in proteins and solution: a simple but accurate nonbonded representation[J]. Proteins, 1995, 23(1): 12-31.
65. Hu X, Balaz S, Shelver WH. Docking studies of matrix metalloproteinase inhibitors: zinc parameter optimization to improve the binding free energy prediction[J]. Journal of Molecular Graphics and Modelling. 2003, 22(2): 115-126