地榆提取物对基质金属蛋白酶的抑制及其对细胞行为影响的研究
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摘要
基质金属蛋白酶(MMPs)是一类由细胞分泌的Zn2+依赖的内肽酶家族,参与许多基质成分的降解。MMPs在正常生理条件下的细胞外基质的重塑过程中发挥着重要作用: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,进而参与细胞凋亡、形态发生、增殖和发育等一系列生理过程。同时MMPs在病理情况下也起着重要作用:与肿瘤的生长、浸润、转移、肿瘤血管新生及细胞凋亡密切联系,而且MMPs在关节炎、中风以及心血管等重大疾病中常过度表达。因此,开发以MMPs为靶点的抑制剂对于治疗这些疾病具有广阔的前景。
本论文首先从大肠杆菌中表达纯化并复性了MMPs,然后以MMP-3、13、14为分子靶点,以我国丰富的中药资源为基础,建立中药文库,应用生物化学、酶学和细胞生物学的方法对中药文库进行大规模筛选和鉴定,以期筛选出具有理想效果的天然产物抑制剂,从而开发出以MMPs为靶点的新药。实验结果表明,我们获得了高纯度和高活性的目的蛋白,通过酶学方法,我们从271种中药中筛选出了对MMPs有明显抑制作用的中药——地榆,进一步用细胞生物学的方法证明了地榆水煮液能够很好地抑制肿瘤细胞行为,并用分子对接模拟方法模拟了地榆的主要成分与MMPs的相互作用方式,分析其结合位点。这些结果表明,中药文库的筛选体系的建立为基质金属蛋白酶抑制剂的开发开辟了新的途径,为中药理论的研究提供了实验依据。
MMPs are a family of zinc endopeptidase, and it is dispensable for the degradation process of extracellular matrix (ECM) and connective tissue proteins. Based on their substrate specificity and primary structure, the MMPs family can be subdivided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane type (MT) and heterogeneous subgroup. MMPs can degrade ECM components, other proteases, clotting factors, chemotactic molecules, latent growth factors, growth-factor binding proteins, adhesion molecules, cell surface receptors etc. So it plays an important role in the physiological and pathological conditions. The expression and activation of MMPs are directly correlated with the growth, invasion and diversion of tumor. MMPs are often expressed excessively in the malignant tumor. 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 incomplete understanding of the function of MMPs, non-specific and side effects of MMPs inhibitors. Along with the continuous deepening of the understanding of MMPs function, the researching and screening MMPs inhibitors with high specificity and low toxicity are becoming increasingly important. According to the reports, currently, some research institutions have isolated new and potentially beneficial compounds which have been shown to exhibit some degree of MMPI activities from herbs, plants, fruits, and other agriculture products, but they are far less potent and specific than the TIMP family. These natural compounds include long chain fatty acids, epigallocatechin gallate (EGCG) and other polyphenols, flavonoids, and a variety of other natural compounds. Kim et al. have found nearly 90 extracts of oriental medicinal herbs, which are used as prescriptions for cancer treatment, such as Baicalin, cinnamon, Euonymus alatus and Magnolia, which all have strong inhibitory effects on MMPs. Recently, they found a new matrix metalloproteinase inhibitor, caffeic acid, from methanol extracts of Euonymus alatus. In our previous study, we proved that one of the components of Qing-Kai-Ling, Fructus gardeniae, strongly inhibits MMP activity.
Traditional Chinese medicine (TCM) has a long history of development and application. Over the past decades,Chinese herbal medicines (CHM) and their commercial derivatives are said to possess numerous properties and as a consequence are used to treat a variety of diseases including arthritis, cancer, asthma and allergies,and these medicines with anti-inflammation and anti-tumor properties are extensively used in Chinese clinical practice. However, the molecular mechanism of these herbal medicines has not been fully recognized due to the lack of definitive information of active ingredients. Therefore, it is of great significance to do such a work.The purpose of this thesis is based on Chinese herbal medicine and expression, purification and refolding of matrix metalloproteinase,we intend to apply enzymology methods, screen MMPs inhibitors with high specificity and low toxicity, develop new drug targeting to MMPs. wec purified three kinds of MMPs, which were MMP-3、MMP-13 and MMP-14. Then, we used DQ-gelatin as substrate to study the inhibitory activities of sanguisorba officinalis to MMPs by fluorescent assays in vitro. Then we investigated the inhibitory effects of a number of Chinese medicines on MMPs,and find that Sanguisorba extract significantly inhibits the activities of MMP-2, MMP-3, MMP-9 and MMP-13. To evaluate the effect of this aqueous extract on B16 cell viability and migration, we used the MTT cell viability assay and wound-healing assay in which cells was induced to migrate by physical wounding of cells plated on plastic plates. We found that cell viability was not affected in concentrations ranging from 0 to 200μg/ml by the aqueous extract. However, the migration ability of B16 cells treated by aqueous extract was inhibited as compared with untreated cells, and the quantitative data revealed that aqueous extract could inhibit B16 cells migration in a dose-dependent manner and the effect was significantly. Because aqueous extracts also can strongly inhibit MMPs (MMP-2, MMP-3, MMP-9 and MMP-13) activity, we surmise that MMP inhibition may change cell morphology, which may in turn lead to an alteration of cell behavior and function. Then we docked the major components of sanguisorba officinalis(ziyu-glycoside I,ziyu-glycoside II) to different MMPs. The results of molecular simulation and the enzymatic data are comparable, and have a high credibility of experimental results,However, ziyu-glycoside I, ziyu-glycoside II are just two main component of sanguisorba officinalis and its other known or unknown ingredients may also inhibit MMPs. The simulation results of this experiment for further studying the active ingredients of sanguisorba officinalis which inhibit MMPs activity provide a basis, and provide a help for the understanding of which sanguisorba officinalis inhibit mechanism of MMPs. In this paper, we found that Sanguisorba officinalis (L.) strongly inhibited MMPs activities, and had no affect on B16 cell viability but inhibited cell migration ability. So we may presume that its ability of anti-tumor is correlated with its ability of inhibiting the activities of MMPs. It explained that why Sanguisorba had the ability of anti-tumor in molecular level. This means that its extract contains effective MMP-inhibitory compounds. We may abstract MMP inhibitor from Sanguisorba officinalis (L.), and we can explore new traditional Chinese medicines targating to MMPs.
本论文首先从大肠杆菌中表达纯化并复性了MMPs,然后以MMP-3、13、14为分子靶点,以我国丰富的中药资源为基础,建立中药文库,应用生物化学、酶学和细胞生物学的方法对中药文库进行大规模筛选和鉴定,以期筛选出具有理想效果的天然产物抑制剂,从而开发出以MMPs为靶点的新药。实验结果表明,我们获得了高纯度和高活性的目的蛋白,通过酶学方法,我们从271种中药中筛选出了对MMPs有明显抑制作用的中药——地榆,进一步用细胞生物学的方法证明了地榆水煮液能够很好地抑制肿瘤细胞行为,并用分子对接模拟方法模拟了地榆的主要成分与MMPs的相互作用方式,分析其结合位点。这些结果表明,中药文库的筛选体系的建立为基质金属蛋白酶抑制剂的开发开辟了新的途径,为中药理论的研究提供了实验依据。
MMPs are a family of zinc endopeptidase, and it is dispensable for the degradation process of extracellular matrix (ECM) and connective tissue proteins. Based on their substrate specificity and primary structure, the MMPs family can be subdivided into six groups: collagenases, gelatinases, stromelysins, matrilysins, membrane type (MT) and heterogeneous subgroup. MMPs can degrade ECM components, other proteases, clotting factors, chemotactic molecules, latent growth factors, growth-factor binding proteins, adhesion molecules, cell surface receptors etc. So it plays an important role in the physiological and pathological conditions. The expression and activation of MMPs are directly correlated with the growth, invasion and diversion of tumor. MMPs are often expressed excessively in the malignant tumor. 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 incomplete understanding of the function of MMPs, non-specific and side effects of MMPs inhibitors. Along with the continuous deepening of the understanding of MMPs function, the researching and screening MMPs inhibitors with high specificity and low toxicity are becoming increasingly important. According to the reports, currently, some research institutions have isolated new and potentially beneficial compounds which have been shown to exhibit some degree of MMPI activities from herbs, plants, fruits, and other agriculture products, but they are far less potent and specific than the TIMP family. These natural compounds include long chain fatty acids, epigallocatechin gallate (EGCG) and other polyphenols, flavonoids, and a variety of other natural compounds. Kim et al. have found nearly 90 extracts of oriental medicinal herbs, which are used as prescriptions for cancer treatment, such as Baicalin, cinnamon, Euonymus alatus and Magnolia, which all have strong inhibitory effects on MMPs. Recently, they found a new matrix metalloproteinase inhibitor, caffeic acid, from methanol extracts of Euonymus alatus. In our previous study, we proved that one of the components of Qing-Kai-Ling, Fructus gardeniae, strongly inhibits MMP activity.
Traditional Chinese medicine (TCM) has a long history of development and application. Over the past decades,Chinese herbal medicines (CHM) and their commercial derivatives are said to possess numerous properties and as a consequence are used to treat a variety of diseases including arthritis, cancer, asthma and allergies,and these medicines with anti-inflammation and anti-tumor properties are extensively used in Chinese clinical practice. However, the molecular mechanism of these herbal medicines has not been fully recognized due to the lack of definitive information of active ingredients. Therefore, it is of great significance to do such a work.The purpose of this thesis is based on Chinese herbal medicine and expression, purification and refolding of matrix metalloproteinase,we intend to apply enzymology methods, screen MMPs inhibitors with high specificity and low toxicity, develop new drug targeting to MMPs. wec purified three kinds of MMPs, which were MMP-3、MMP-13 and MMP-14. Then, we used DQ-gelatin as substrate to study the inhibitory activities of sanguisorba officinalis to MMPs by fluorescent assays in vitro. Then we investigated the inhibitory effects of a number of Chinese medicines on MMPs,and find that Sanguisorba extract significantly inhibits the activities of MMP-2, MMP-3, MMP-9 and MMP-13. To evaluate the effect of this aqueous extract on B16 cell viability and migration, we used the MTT cell viability assay and wound-healing assay in which cells was induced to migrate by physical wounding of cells plated on plastic plates. We found that cell viability was not affected in concentrations ranging from 0 to 200μg/ml by the aqueous extract. However, the migration ability of B16 cells treated by aqueous extract was inhibited as compared with untreated cells, and the quantitative data revealed that aqueous extract could inhibit B16 cells migration in a dose-dependent manner and the effect was significantly. Because aqueous extracts also can strongly inhibit MMPs (MMP-2, MMP-3, MMP-9 and MMP-13) activity, we surmise that MMP inhibition may change cell morphology, which may in turn lead to an alteration of cell behavior and function. Then we docked the major components of sanguisorba officinalis(ziyu-glycoside I,ziyu-glycoside II) to different MMPs. The results of molecular simulation and the enzymatic data are comparable, and have a high credibility of experimental results,However, ziyu-glycoside I, ziyu-glycoside II are just two main component of sanguisorba officinalis and its other known or unknown ingredients may also inhibit MMPs. The simulation results of this experiment for further studying the active ingredients of sanguisorba officinalis which inhibit MMPs activity provide a basis, and provide a help for the understanding of which sanguisorba officinalis inhibit mechanism of MMPs. In this paper, we found that Sanguisorba officinalis (L.) strongly inhibited MMPs activities, and had no affect on B16 cell viability but inhibited cell migration ability. So we may presume that its ability of anti-tumor is correlated with its ability of inhibiting the activities of MMPs. It explained that why Sanguisorba had the ability of anti-tumor in molecular level. This means that its extract contains effective MMP-inhibitory compounds. We may abstract MMP inhibitor from Sanguisorba officinalis (L.), and we can explore new traditional Chinese medicines targating to MMPs.
引文
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[2] Lynch CC, Matrisian LM. Matrix metalloproteinases in tumor-host cell communication [J]. Differentiation, 2002, 70 (9-10): 561-573
[3] Sternlicht M D, Werb Z. How matrix metalloproteinases regulate cell behavior [J]. Annu Rev Cell Dev Biol, 2001, 17: 463-516.
[4] Sang QX, Jin Y, Newcomer RG, Monroe SC, Fang X, Hurst DR, et al. Matrix metalloproteinase inhibitors as prospective agents for the prevention and treatment of cardiovascular and neoplastic diseases [J]. Curr Top Med Chem. 2006, 6: 289–316.
[5] Hu J, Van den Steen PE, Sang QX, et al. Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases [J]. Nat Rev Drug Discov. 2007, 6(6): 480-498
[6] Mikala Egeblad, Zena Werb. New functions for the matrix metalloproteinases in cancer progression [J]. Nature Reviews Cancer. 2002, 2(3): 161-174.
[7] Overall, C.M, Lopez-Otin, C. Strategies for MMP inhibition in cancer: innovations for the post-trial era [J]. Nat Rev Cancer. 2002, 2 (2): 657-672.
[8] Raffetto JD, Khalil RA. Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease [J]. Biochem Pharmacol. 2008 Jan 15; 75 (2): 346-59. Epub 2007 Jul 7
[9] 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.
[10] Suzuki K, Enghild JJ, Morodomi T, Salvesen G, Nagase H. Mechanisms of activation of tissue procollagenase by matrix metalloproteinase 3 (stromelysin) [J]. Biochemistry. 1990, 29: 10261–10270.
[11] Uría J. A., López-Otín C. Matrilysin-2, a new matrix metalloproteinase expressed in human tumors and showing the minimal domain organization required for secretion, latency, and activity [J]. Cancer Res, 2000, 60: 4745-4751.
[12] Pepper MS. Extracellular proteolysis and angiogenesis [J]. Thromb Haemost. 2001; 86: 346–355.
[13] Shipley JM, Wesselschmidt RL, Kobayashi DK, Ley TJ, Shapiro SD. Metalloelastase is required for macrophage-mediated proteolysis and matrixinvasion in mice [J]. Proc Natl Acad Sci U S A. 1996; 93: 3942–3946.
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