扇贝糖胺聚糖对血管内皮细胞损伤的保护作用与机理研究
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摘要
目的：
研究扇贝裙边提取物—糖胺聚糖(SS-GAG)对内皮细胞损伤的保护作用，探讨SS-GAG的抗动脉粥样硬化(AS)作用机制。
方法：
1 采用体外培养人脐静脉内皮细胞(HUVEC)，建立氧自由基、多聚阳离子诱导的HUVEC的损伤模型，通过MTT比色法研究SS-GAG对HUVEC损伤和增殖活性抑制的影响。
2．采用Fenton体系造成体外培养HUVEC的氧化损伤，用化学方法观察SS-GAG对HUVEC损伤后乳酸脱氢酶(LDH)渗出的影响。
3．应用放射免疫方法，观察SS-GAG对Fenton体系所引发的HUVEC损伤后内皮素及血管紧张素Ⅱ分泌改变的影响。
4．运用酶联免疫吸附测定(ELISA)法，观察HUVEC受到Fenton体系所致的氧化损伤后，血管内皮细胞粘附分子-1(VCAM-1)表达的改变，以及SS-GAG对此改变的影响。
5．运用酶联免疫吸附测定(ELISA)法，观察HUVEC经Fenton体系氧化损伤后，血小板衍生生长因子B链(PDGF-BB)表达的改变，观察SS-GAG对此改变有否影响。
结果：
1．分别经Fenton体系、多聚赖氨酸作用的损伤对照组，内皮细胞增殖活性明显下降(P＜0.01)。而细胞经不同浓度的SS-GAG预先处理后，细胞活性的损伤明显减轻(P＜0.05)，高剂量的SS-GAG保护组内皮细胞增殖活性明显高于低剂量组(P＜0.05)。
2．经Fenton体系介导的氧自由基损伤后，HUVEC培养液中LDH渗出明显增多，经SS-GAG预处理后，LDH渗出量明显减少(P＜0.01)。
3．SS-GAG保护组与经氧自由基损伤的损伤对照组对比，内皮素的分泌明显减少(P＜0.01)，且随SS-GAG剂量增加而降低；SS-GAG保护组与经氧自由基损伤的损伤对照组对比，血管紧张素Ⅱ的分泌也明显减少(P＜0.01)，高剂量的SS-GAG组的血管紧张素Ⅱ的分泌量低于低剂量组(P＜0.01)。
4．经SS-GAG保护的HUVEC表达VCAM-1的量，较氧自由基损伤的损伤
中文摘要
对照组明显减少(P<0.01)。
:5 55一GAG保护的HUvEC表达PDGF一BB的量，较Fenion体系作用的损伤
对照组明显减少(P 结论:
s$GAG可减少Fenton体系及多聚赖氨酸介导的氧自由基及多舞阳离子对
血管内皮细胞损伤，抑制血管内皮细胞对内皮素和血管紧张素n的分泌，减少血
管内皮细胞VCAM一1、PDGF一BB的表达。表明55一GAG对血管内皮细胞的损伤
有良好的保护作用，提示 55一GAG的抗动脉粥样硬化机制可能与其保护内皮细
胞免受损伤、抑制损伤的血管内皮细胞分泌内皮素和血管紧张素n、使vCAM‘l、
PDGF一BB的表达增强有关。
Objective To investigation the protective action of the extract of scallop skirt-glycosaminoglycan(SS-GAG) on the endothelial cell , and to discuss it's mechanism of anti-atherosclerosis(AS).
Methods 1. The endothelial cell of human umbilical vein had been cultured in vitro, and induced by oxygen-derived free radidicals or polycatholyte , then the damage model had been established. MTT chromatometry was used to study the anti-effect of SS-GAG on the endothelial cell damage and proliferation activity.
2. Oxidative damage to vascular endothelial cell was caused by Fenton reaction, to observe the effect of SS-GAG on the lactate dehydrogenase(LDH) secret of the endothelial cell.
3. To observe the action of SS-GAG on the secret of endothelins and angiotensin II in the Fenton architecture by the means of radio-immunity.
4. The effect on the expression of vascular cell adhesiveness molecul-1(VCAM-1) and platelet derivation growth factor(PDGF-BB) had been observed by the means of ELISA after the endothelial cell oxidizing damage had been caused by Fenton architecture.
Results 1.In positive groups, polylysine, damaged by Fenton reaction , the endothelial cell proliferation dereased remarkably (P<0.01) . While pretreated by SS-GAG, cell proliferation damages lowered obviously (P < 0.05 ) , and cell proliferation in large dose SS-GAG protective control groups was higher than that in less dose control groups (P<0.05) .
2. LDH excretion of the endothelial in HUVEC cellculture medium increased remarkably when the endothelial cell damaged by oxygen-derived free radidicals induced by Fenton reaction, but it decreased remarkably when pretreated by SS-GAG
(P<0.01) .
3. Compare with positive control group damaged by oxygen-derived free radidicals , the excretion of endothelins decreased remarkably in SS-GAG group (P<0.01) , and decreased with increase of SS-GAG dose. After damaged by oxygen-derived free radidicals, the excretion of angiotensin II in HUVEC rised obviously (P<0.01) .
Anpotensin II excretion in large dose SS-GAG protective control groups was less than that in small dose control groups (P<0.01) .
4. Compare with positive control group, the number of VCAM-1 expression descended obviously in SS-GAG groups (P<0.01) .
5. Compare with positive control group, the number of PDGF-BB expression descended obviously in SS-GAG groups (P<0.05) .
Conclusion SS-GAG can reduce the endothelial cell damage caused by oxygen-derived free radidicals or polycatholyte, can restrain the excretion of endothelins and angiotensin II, and reduce the expression of VCAM-1 and PDGF-BB. These indicate SS-GAG has satisfactory protection effect on the endothelial cell damage, and suggest the antiatherosclerosis mechanism of SS-GAG may has relation to its protection on the endothelial cell, its inhibition of endothelins and angiotensin II excretion, and reduction of VCAM-1 and PDGF-BB expression.
研究扇贝裙边提取物—糖胺聚糖(SS-GAG)对内皮细胞损伤的保护作用，探讨SS-GAG的抗动脉粥样硬化(AS)作用机制。
方法：
1 采用体外培养人脐静脉内皮细胞(HUVEC)，建立氧自由基、多聚阳离子诱导的HUVEC的损伤模型，通过MTT比色法研究SS-GAG对HUVEC损伤和增殖活性抑制的影响。
2．采用Fenton体系造成体外培养HUVEC的氧化损伤，用化学方法观察SS-GAG对HUVEC损伤后乳酸脱氢酶(LDH)渗出的影响。
3．应用放射免疫方法，观察SS-GAG对Fenton体系所引发的HUVEC损伤后内皮素及血管紧张素Ⅱ分泌改变的影响。
4．运用酶联免疫吸附测定(ELISA)法，观察HUVEC受到Fenton体系所致的氧化损伤后，血管内皮细胞粘附分子-1(VCAM-1)表达的改变，以及SS-GAG对此改变的影响。
5．运用酶联免疫吸附测定(ELISA)法，观察HUVEC经Fenton体系氧化损伤后，血小板衍生生长因子B链(PDGF-BB)表达的改变，观察SS-GAG对此改变有否影响。
结果：
1．分别经Fenton体系、多聚赖氨酸作用的损伤对照组，内皮细胞增殖活性明显下降(P＜0.01)。而细胞经不同浓度的SS-GAG预先处理后，细胞活性的损伤明显减轻(P＜0.05)，高剂量的SS-GAG保护组内皮细胞增殖活性明显高于低剂量组(P＜0.05)。
2．经Fenton体系介导的氧自由基损伤后，HUVEC培养液中LDH渗出明显增多，经SS-GAG预处理后，LDH渗出量明显减少(P＜0.01)。
3．SS-GAG保护组与经氧自由基损伤的损伤对照组对比，内皮素的分泌明显减少(P＜0.01)，且随SS-GAG剂量增加而降低；SS-GAG保护组与经氧自由基损伤的损伤对照组对比，血管紧张素Ⅱ的分泌也明显减少(P＜0.01)，高剂量的SS-GAG组的血管紧张素Ⅱ的分泌量低于低剂量组(P＜0.01)。
4．经SS-GAG保护的HUVEC表达VCAM-1的量，较氧自由基损伤的损伤
中文摘要
对照组明显减少(P<0.01)。
:5 55一GAG保护的HUvEC表达PDGF一BB的量，较Fenion体系作用的损伤
对照组明显减少(P
s$GAG可减少Fenton体系及多聚赖氨酸介导的氧自由基及多舞阳离子对
血管内皮细胞损伤，抑制血管内皮细胞对内皮素和血管紧张素n的分泌，减少血
管内皮细胞VCAM一1、PDGF一BB的表达。表明55一GAG对血管内皮细胞的损伤
有良好的保护作用，提示 55一GAG的抗动脉粥样硬化机制可能与其保护内皮细
胞免受损伤、抑制损伤的血管内皮细胞分泌内皮素和血管紧张素n、使vCAM‘l、
PDGF一BB的表达增强有关。
Objective To investigation the protective action of the extract of scallop skirt-glycosaminoglycan(SS-GAG) on the endothelial cell , and to discuss it's mechanism of anti-atherosclerosis(AS).
Methods 1. The endothelial cell of human umbilical vein had been cultured in vitro, and induced by oxygen-derived free radidicals or polycatholyte , then the damage model had been established. MTT chromatometry was used to study the anti-effect of SS-GAG on the endothelial cell damage and proliferation activity.
2. Oxidative damage to vascular endothelial cell was caused by Fenton reaction, to observe the effect of SS-GAG on the lactate dehydrogenase(LDH) secret of the endothelial cell.
3. To observe the action of SS-GAG on the secret of endothelins and angiotensin II in the Fenton architecture by the means of radio-immunity.
4. The effect on the expression of vascular cell adhesiveness molecul-1(VCAM-1) and platelet derivation growth factor(PDGF-BB) had been observed by the means of ELISA after the endothelial cell oxidizing damage had been caused by Fenton architecture.
Results 1.In positive groups, polylysine, damaged by Fenton reaction , the endothelial cell proliferation dereased remarkably (P<0.01) . While pretreated by SS-GAG, cell proliferation damages lowered obviously (P < 0.05 ) , and cell proliferation in large dose SS-GAG protective control groups was higher than that in less dose control groups (P<0.05) .
2. LDH excretion of the endothelial in HUVEC cellculture medium increased remarkably when the endothelial cell damaged by oxygen-derived free radidicals induced by Fenton reaction, but it decreased remarkably when pretreated by SS-GAG
(P<0.01) .
3. Compare with positive control group damaged by oxygen-derived free radidicals , the excretion of endothelins decreased remarkably in SS-GAG group (P<0.01) , and decreased with increase of SS-GAG dose. After damaged by oxygen-derived free radidicals, the excretion of angiotensin II in HUVEC rised obviously (P<0.01) .
Anpotensin II excretion in large dose SS-GAG protective control groups was less than that in small dose control groups (P<0.01) .
4. Compare with positive control group, the number of VCAM-1 expression descended obviously in SS-GAG groups (P<0.01) .
5. Compare with positive control group, the number of PDGF-BB expression descended obviously in SS-GAG groups (P<0.05) .
Conclusion SS-GAG can reduce the endothelial cell damage caused by oxygen-derived free radidicals or polycatholyte, can restrain the excretion of endothelins and angiotensin II, and reduce the expression of VCAM-1 and PDGF-BB. These indicate SS-GAG has satisfactory protection effect on the endothelial cell damage, and suggest the antiatherosclerosis mechanism of SS-GAG may has relation to its protection on the endothelial cell, its inhibition of endothelins and angiotensin II excretion, and reduction of VCAM-1 and PDGF-BB expression.
引文
1. Raince EW, et al Smooth muscle cells and the patho gcncsis of the lesions of atherosclcro is Br Heart J, 1993, 69 (suppl): 30
2. Ehrlich J. et al.Chemistry and pharmancology of heparim J Pharm sci 1973,662 :L517
3．张均田．现代药理实验方法．北京：北京医科大学中国协和医科大学联合出版社．1998：1135
4．边兴艳．MTT比色法及其应用．国外医学．临床生物化学与检验学分册 1998 19 83-85
5. Deng,-z, HUANG-W, Jin-Y. the influence of vascular endothelial growth factor and its receptor on the growth of lartyngeal cancet cell Lin-Chuang- Er-BI-Yan-Hou-Ke-za-zahi. 1998Aug; 12:358-61
6. Morgan DML,Clover J,Pearson JD.Effect of synthetic poiycations on leucine incor-poration, lactate dehydrogenase release and morphology of human umbilical vein endothelial cells.Semin Thromb Hemost, 1991,17(supp11):231
8. Mathew V, Lerman A,, Clinical implications of a sandwich enzw me immunoassay for big endothert Clin Chem, 1997, 43: 9-10
9. Gadipathi NR.Berk BC.Active oxygen species stimylatc.vascular smooth musclc cell growth and proto oncogenc expression.Circ Rrowth and proto oncogene expression Ciec Res. 1992.70(3);593
10．齐爱东，吴葆杰，周序彬，内皮素的研究进展．生理科学进展，1992，3(1)：46-51
11. Grafe M.Auch-Schwelk W.Zakrzew icz A.et al Ang iotensin 11-induced leukoey teadhesion on hum an xironary endothelial cdlls is mdiated by E-se-leukoey adhesion on hum an conronary endorhelial cells is mediated by E-se-lectin Cire Res. 1997.81:804-811
12. Hernandez-P resa M ,Bustos C,Ortego M,et al ,Angiotensin-convertingenzyme inhibtor prevents arterial nuclear factor KB activation ,monocy tedhem attractant protein-1 expression and macrophage infiltration in a rabbit model of early accelerated athe rosclerosis[J].Circulation,1997,95:1532-1541
13．周华东综述．动脉粥样硬化的发生与细胞间粘附分子．国外医学老年医学分册，1997，18(4)：152
14．张进虎综述．粘附分子与冠心病．心血管病学进展，1997，18(1)：38
15. Hwang SJ, Ballaantyne CM, Shrrett AR, etal Circulating adhesion molecules VCAM ICAMI andselectin in carotid atherosclerosi and incident coronary heart disease cases:the atherosclerosis risk in communities (ARIC) study Circulation11997, 96 (12): 4219
16. Gornas P, Groski A The rile of cell adhesion molecules in the pathogenesis of coronary artery disease Pol Arch Med Mewn 1996, 96 (2): 183
17. Khan BV, Harrison DG Nitric oxide regulates vascularcell adhesion molecule lgene expression and redoxsensitie events in human vascular endothlialcell Proc Natl Acad Sei USA,1996, 93 (17): 9114
18．葛璐璐，张薇，戴云，等．大蒜精油抑制白细胞介素-1a诱导的单核细胞-内皮细胞粘附及机理探讨{1}．中国中西医结合杂志，1999，19(3)：152-154
19．严鸣，龚肖崎，张亚霖，等．白细胞与内皮细胞粘附分子的调节{J}．中国病理生理杂志，1993，9(2)：272-275
20. O'Brien KD, Allen MD, Mcddonald TO, et al. Vascular cell adhesion molecule-1 is expressed in human coronary atherosclerotic plagues: implication for. The mode of pro-gression of advanced coronary atherosclerosis. J. Clin. Invest, 993,92(2):945-951
21. Li H, Crbuisky MI, Gimbrone MA Jr, et al.Inducible expression of vascular cell adhesion molecule-1 by vascular smooth muscle cells in vitro and within rabbit atheroma.Am J Pathol,1993,143:1551-1559
22. Amberger A, Maczek C, Jurgens G, et al.Co-expression of ICAM-1, VCAM-1, ELAM-1, and Hsp60 in human arterial and venous endothelial cells in response to ey-tokines and oxidized low-density lipoproteins.Cell Stress Chaperones,1997, 2:94-103
23. Crrotendorst G, Seppa, H.E.J, Kleinman, HK, and Martin, G. Attachment of smooth cells to collagen and their migration toward platelet-derived growth factor. Proc. Natl. Acad. Sci. U.S.A. 78:3669,1981
24. Waters D ,et al.Effects of monotherapy with an HMGCOA reductase inhibitor on the progression of coronary atherosclerosis as assessed by serial quantita tive arteriography, The Canadian Atheroscleros is Intervention rial,Circulation, T, 1994,89:959
25. Srtoo-Rahm A, Hultgardh-Nilsson A, Regnstrom J, et al. Native and oxidized LDL enhances production of PDGFAA and the surface exptession of PDGF receptors in cultued hum an sm oth muscle cells.Arteriosler Thromb.1992.12:1099
2. Ehrlich J. et al.Chemistry and pharmancology of heparim J Pharm sci 1973,662 :L517
3．张均田．现代药理实验方法．北京：北京医科大学中国协和医科大学联合出版社．1998：1135
4．边兴艳．MTT比色法及其应用．国外医学．临床生物化学与检验学分册 1998 19 83-85
5. Deng,-z, HUANG-W, Jin-Y. the influence of vascular endothelial growth factor and its receptor on the growth of lartyngeal cancet cell Lin-Chuang- Er-BI-Yan-Hou-Ke-za-zahi. 1998Aug; 12:358-61
6. Morgan DML,Clover J,Pearson JD.Effect of synthetic poiycations on leucine incor-poration, lactate dehydrogenase release and morphology of human umbilical vein endothelial cells.Semin Thromb Hemost, 1991,17(supp11):231
8. Mathew V, Lerman A,, Clinical implications of a sandwich enzw me immunoassay for big endothert Clin Chem, 1997, 43: 9-10
9. Gadipathi NR.Berk BC.Active oxygen species stimylatc.vascular smooth musclc cell growth and proto oncogenc expression.Circ Rrowth and proto oncogene expression Ciec Res. 1992.70(3);593
10．齐爱东，吴葆杰，周序彬，内皮素的研究进展．生理科学进展，1992，3(1)：46-51
11. Grafe M.Auch-Schwelk W.Zakrzew icz A.et al Ang iotensin 11-induced leukoey teadhesion on hum an xironary endothelial cdlls is mdiated by E-se-leukoey adhesion on hum an conronary endorhelial cells is mediated by E-se-lectin Cire Res. 1997.81:804-811
12. Hernandez-P resa M ,Bustos C,Ortego M,et al ,Angiotensin-convertingenzyme inhibtor prevents arterial nuclear factor KB activation ,monocy tedhem attractant protein-1 expression and macrophage infiltration in a rabbit model of early accelerated athe rosclerosis[J].Circulation,1997,95:1532-1541
13．周华东综述．动脉粥样硬化的发生与细胞间粘附分子．国外医学老年医学分册，1997，18(4)：152
14．张进虎综述．粘附分子与冠心病．心血管病学进展，1997，18(1)：38
15. Hwang SJ, Ballaantyne CM, Shrrett AR, etal Circulating adhesion molecules VCAM ICAMI andselectin in carotid atherosclerosi and incident coronary heart disease cases:the atherosclerosis risk in communities (ARIC) study Circulation11997, 96 (12): 4219
16. Gornas P, Groski A The rile of cell adhesion molecules in the pathogenesis of coronary artery disease Pol Arch Med Mewn 1996, 96 (2): 183
17. Khan BV, Harrison DG Nitric oxide regulates vascularcell adhesion molecule lgene expression and redoxsensitie events in human vascular endothlialcell Proc Natl Acad Sei USA,1996, 93 (17): 9114
18．葛璐璐，张薇，戴云，等．大蒜精油抑制白细胞介素-1a诱导的单核细胞-内皮细胞粘附及机理探讨{1}．中国中西医结合杂志，1999，19(3)：152-154
19．严鸣，龚肖崎，张亚霖，等．白细胞与内皮细胞粘附分子的调节{J}．中国病理生理杂志，1993，9(2)：272-275
20. O'Brien KD, Allen MD, Mcddonald TO, et al. Vascular cell adhesion molecule-1 is expressed in human coronary atherosclerotic plagues: implication for. The mode of pro-gression of advanced coronary atherosclerosis. J. Clin. Invest, 993,92(2):945-951
21. Li H, Crbuisky MI, Gimbrone MA Jr, et al.Inducible expression of vascular cell adhesion molecule-1 by vascular smooth muscle cells in vitro and within rabbit atheroma.Am J Pathol,1993,143:1551-1559
22. Amberger A, Maczek C, Jurgens G, et al.Co-expression of ICAM-1, VCAM-1, ELAM-1, and Hsp60 in human arterial and venous endothelial cells in response to ey-tokines and oxidized low-density lipoproteins.Cell Stress Chaperones,1997, 2:94-103
23. Crrotendorst G, Seppa, H.E.J, Kleinman, HK, and Martin, G. Attachment of smooth cells to collagen and their migration toward platelet-derived growth factor. Proc. Natl. Acad. Sci. U.S.A. 78:3669,1981
24. Waters D ,et al.Effects of monotherapy with an HMGCOA reductase inhibitor on the progression of coronary atherosclerosis as assessed by serial quantita tive arteriography, The Canadian Atheroscleros is Intervention rial,Circulation, T, 1994,89:959
25. Srtoo-Rahm A, Hultgardh-Nilsson A, Regnstrom J, et al. Native and oxidized LDL enhances production of PDGFAA and the surface exptession of PDGF receptors in cultued hum an sm oth muscle cells.Arteriosler Thromb.1992.12:1099