纳豆激酶抗血栓作用机制的实验研究
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摘要
目的:研究纳豆激酶抗血栓作用的机制。方法:以肾上腺素加冰浴制备大鼠急性血瘀模型,观察纳豆激酶对大鼠血液流变学的影响;分别采用ADP、凝血酶和花生四烯酸诱导大鼠血小板聚集,用比浊法测定不同时间点的聚集率及最大聚集率;采用Fluo-3/AM钙离子荧光指示剂,流式细胞仪测定纳豆激酶对凝血酶诱导的大鼠血小板胞浆游离钙离子浓度的影响;放免法测定血浆TXB2、6-Keto-PGF1α及ET-1的含量,硝酸还原酶法测定血浆NO含量,酶标法测定人血浆GMP-140和vWF的含量;通过Real Time PCR方法观察纳豆激酶对大鼠主动脉t-PA、PAI-1及UK基因表达的影响。
结果:纳豆激酶能明显降低血瘀模型大鼠的全血低、中、高切粘度,血浆粘度,红细胞压积,纤维蛋白原,红细胞聚集指数及电泳指数;明显抑制ADP、凝血酶、花生四烯酸诱导的血小板聚集,其ED50分别为1431.9 IU/kg、1055.3 IU/kg和1390.8 IU/kg;纳豆激酶1000～2000IU/kg能显著抑制凝血酶诱导的血小板胞浆游离钙离子浓度的升高;降低血浆TXB2含量(P<0.05或P<0.01),升高6-Keto-PGF1α含量(P<0.05),使TXB2/6-Keto-PGF1α比值明显降低(P<0.05或P<0.01);降低血浆ET-1含量(P<0.05或P<0.01),增加NO含量(P<0.05或P<0.01),明显降低ET-1/NO比值(P<0.01);在体外,纳豆激酶40IU/mL～80IU/mL作用下能明显降低人血浆GMP-140和vWF含量(P<0.05或P<0.01);纳豆激酶1000～2000IU/kg可明显增加t-PA mRNA和UK mRNA的表达(P<0.05或P<0.01),但对PAI-1 mRNA的表达无明显性影响(P>0.05)。
结论:纳豆激酶抗血栓作用机制可能与改善血液流变学,抑制血小板聚集,包括抑制血小板胞浆游离钙离子浓度,降低TXB2和ET-1含量,升高6-Keto-PGF1α和NO含量,降低GMP-140和vWF水平,以及通过促进t-PA和UK基因表达而提高机体的纤溶活性有关。
Objective: To study the antithrombotic mechanisms of nattokinase.
Methods: A model of acute blood stasis was established by means of giving rats an injection of adrenalin and making it swim in ice-cold water, the change of hemorheology was observed after the rats took nattokinase for 10 days; Rat platelet aggregation induced by ADP,thrombin and arachidonic acid, was measured by turbidimetric method at different time. The cytosolic free calcium concentration [Ca2+]i in rat platelets was measured with Fluo-3/ AM and the flowcytometer. The levels of TXB2, 6-Keto-PGF1α, NO and ET-1 in rat plasma and the levels of GMP-140 and vWF in human plasma were measured. Expression of t-PA, UK and PAI-1 mRNA in rat aorta tissue was examined by Real Time PCR.
Results: Nattokinase could markedly decrease the whole blood viscosity of low, middle, and high shear rate, plasma viscosity, hematocrit, fibrinogen, erythrocyte aggregation index and electrophoresis index. Nattokinase could significantly inhibit platelet aggregation induced by ADP,thrombin and arachidonic acid , and the values of ED50 (median effective dose) were 1431.9 IU/kg for ADP, 1055.3 IU/kg for thrombin and 1390.8 IU/kg for arachidonic acid, respectively. Nattokinase could decrease the [Ca2+]i in rat platelets induced by thrombin. The levels of 6-Keto-PGF1αand NO were increased, while the levels of TXB2 , ET-1, and the ratios of TXB2/6-Keto-PGF1αand ET-1/NO were reduced. And the levels of GMP-140 and vWF in human plasma were also decreased. Nattokinase could significantly increase the mRNA expression of t-PA and UK, but not influence the mRNA expression of PAI-1.
Conclusion: Nattokinase had the effect of anti- thrombosis, and its mechanisms might be associated with the improvement of hemorheology in rats of acute blood stasis, inhibition of platelet aggregation, including the reductions of [Ca~(2+)]i in platelets, TXB2 and ET-1, increases of 6-Keto-PGF1αand NO, decreases of GMP-140 and vWF, and enhancement of fibrinolytic activity by promoting the gene expression of t-PA and UK.
结果:纳豆激酶能明显降低血瘀模型大鼠的全血低、中、高切粘度,血浆粘度,红细胞压积,纤维蛋白原,红细胞聚集指数及电泳指数;明显抑制ADP、凝血酶、花生四烯酸诱导的血小板聚集,其ED50分别为1431.9 IU/kg、1055.3 IU/kg和1390.8 IU/kg;纳豆激酶1000～2000IU/kg能显著抑制凝血酶诱导的血小板胞浆游离钙离子浓度的升高;降低血浆TXB2含量(P<0.05或P<0.01),升高6-Keto-PGF1α含量(P<0.05),使TXB2/6-Keto-PGF1α比值明显降低(P<0.05或P<0.01);降低血浆ET-1含量(P<0.05或P<0.01),增加NO含量(P<0.05或P<0.01),明显降低ET-1/NO比值(P<0.01);在体外,纳豆激酶40IU/mL～80IU/mL作用下能明显降低人血浆GMP-140和vWF含量(P<0.05或P<0.01);纳豆激酶1000～2000IU/kg可明显增加t-PA mRNA和UK mRNA的表达(P<0.05或P<0.01),但对PAI-1 mRNA的表达无明显性影响(P>0.05)。
结论:纳豆激酶抗血栓作用机制可能与改善血液流变学,抑制血小板聚集,包括抑制血小板胞浆游离钙离子浓度,降低TXB2和ET-1含量,升高6-Keto-PGF1α和NO含量,降低GMP-140和vWF水平,以及通过促进t-PA和UK基因表达而提高机体的纤溶活性有关。
Objective: To study the antithrombotic mechanisms of nattokinase.
Methods: A model of acute blood stasis was established by means of giving rats an injection of adrenalin and making it swim in ice-cold water, the change of hemorheology was observed after the rats took nattokinase for 10 days; Rat platelet aggregation induced by ADP,thrombin and arachidonic acid, was measured by turbidimetric method at different time. The cytosolic free calcium concentration [Ca2+]i in rat platelets was measured with Fluo-3/ AM and the flowcytometer. The levels of TXB2, 6-Keto-PGF1α, NO and ET-1 in rat plasma and the levels of GMP-140 and vWF in human plasma were measured. Expression of t-PA, UK and PAI-1 mRNA in rat aorta tissue was examined by Real Time PCR.
Results: Nattokinase could markedly decrease the whole blood viscosity of low, middle, and high shear rate, plasma viscosity, hematocrit, fibrinogen, erythrocyte aggregation index and electrophoresis index. Nattokinase could significantly inhibit platelet aggregation induced by ADP,thrombin and arachidonic acid , and the values of ED50 (median effective dose) were 1431.9 IU/kg for ADP, 1055.3 IU/kg for thrombin and 1390.8 IU/kg for arachidonic acid, respectively. Nattokinase could decrease the [Ca2+]i in rat platelets induced by thrombin. The levels of 6-Keto-PGF1αand NO were increased, while the levels of TXB2 , ET-1, and the ratios of TXB2/6-Keto-PGF1αand ET-1/NO were reduced. And the levels of GMP-140 and vWF in human plasma were also decreased. Nattokinase could significantly increase the mRNA expression of t-PA and UK, but not influence the mRNA expression of PAI-1.
Conclusion: Nattokinase had the effect of anti- thrombosis, and its mechanisms might be associated with the improvement of hemorheology in rats of acute blood stasis, inhibition of platelet aggregation, including the reductions of [Ca~(2+)]i in platelets, TXB2 and ET-1, increases of 6-Keto-PGF1αand NO, decreases of GMP-140 and vWF, and enhancement of fibrinolytic activity by promoting the gene expression of t-PA and UK.
引文
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[14] Suzuki Y, Kondo K, Ichise J, et al. Dietary supplementation with fermented soybeans suppresses intimal thickening [ J ]. Nutrition, 2003, 19(3):261-264.
[15]郭婷,姚文兵,高向东. 纳豆激酶的纤溶活性及其机理研究[J].中国生化药物杂志,2004,25(4),226-228.
[16] Yoshikazu Yuki , Nakagawa T , Fujita M, et al . A Sandwich enzyme-linked immunosorbent asay for nattokinase[ J ]. Biosci Biotech Biochem, 1994 ,58(2) : 366-70.
[17] Kyu-sil Choi , Sandra L , Fitzpatrick , et al . Regulation of plasmin dependent fibrin clot lysis by annexin Ⅱ hetero-tetramer[ J ]. J of Biological Chemidtrg , 2001 , 276 (2) :25212～25221. [18 ] Chavakis T ,Robin A , Pixley I , et al . A novel antithronbotic role for high molecular weight kininogen as inhibitor of plasminogen activator inhibitor function [ J ]. J Biol Chem, 2002 ,277(36) :32677～32682. [19 ] Urono T , Hayato I , Kazuo U , et al . The profibrinolytic enzyme subtilisin NATpurified from bacillus subtilis cleaves and inactivates plasminogen activator inhibitor type-1[ J ]. J Biol Chem , 2001 ,276 (27) :24690-24696.
[20] Fujita M, Hong K, Ito Y, et al.Transport of nattokinase across the rat intestinal tract[J]. Biol Pharm Bull , 1995,18(9):1194–1196.
[21] Sumi H. Single oral dose toxicity study of nattokinase(NSKFD) powder in rats[J ] . Japan Bio Science Laboratory ,1999 ,26 ,February.
[22] 胡景柱,夏寿华,吴林.纳豆冻干粉的安全性研究[J].安徽农业科学,2000,28(3):380–381.
[ 2 ]Rosenberg RD , Aird WC. Vascular-bed-specific hemostasis and Hypercoagulable states [ J ]. N Engl J Med , 1999 , 340 (20) :1555-64.
[ 3 ]Repo H , Harlan JM. Mechanisms and consequences of phagocyte adhesion to endothelium [ J ]. Ann Med , 1999 , 31 (3) :156-65.
[ 4 ]Cines DB , Pollak ES , Buck CA. Endothelial cells in physiology and in the pathophysiology of vascular disorders [ J ]. Blood , 1998 , 91 (10) :3527-61.
[ 5 ]Gross PL , Aird WC. The endothelium and thrombosis [ J ]. Semin Thromb Hemost , 2000 , 26 (5) :463-78.
[ 6 ]Brass L. Fifty (or more) ways to leave your platelets (in a thrombus) [ J ].Arterioscler Thromb Vasc Biol , 2004, 24(6):989-91.
[ 7 ]Nieswandt B, Watson SP.Platelet-collagen interaction:is GPⅥ the central receptor[ J ]? BIood, 2003, 102: 449-61.
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