内吗啡肽对糖基化终末产物条件下血管内皮细胞功能的影响
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摘要
目的：探讨内吗啡肽(EMs)对糖基化终末产物(AGEs)培养条件下人脐静脉内皮细胞(HUVECs)合成和分泌血管活性物质的影响。
方法：从新鲜脐带中分离培养鉴定HUVECs,运用体外制备的糖基化修饰的牛血清白蛋白(AGEs-BSA)干预HUVECs,并进一步用不同浓度EMs(10μmol／ml,1μmol／ml,0.1μmol／ml,10nmol／ml)与AGEs-BSA共同干预HUVECs,同时设只加牛血清白蛋白(BSA)的HUVECs作阴性对照组；四甲基偶氮唑蓝(MTT)比色法检测HUVEC存活率,优化EMs的最佳作用浓度；硝酸盐还原酶法检测一氧化氮(NO)的浓度,分光光度法检测内皮型、诱导型一氧化氮合酶(eNOS、iNOS)的浓度,酶联免疫吸附(ELISA)法检测内皮素1(ET-1)的浓度,逆转录聚合酶链式反应(RT-PCR)检测eNOS、ET-1 mRNA的表达,免疫荧光法检测p38丝裂原活化蛋白激酶(p38MAPK)的表达。
结果：AGEs-BSA可使HUVECs活力降低,并呈现时间依赖性,与正常对照组比较差异有统计学意义(P<0.01),时间依赖地刺激HUVECs分泌iNOS、NO、ET-1及上调ET-1 mRNA的表达,抑制eNOS分泌及下调eNOS mRNA的表达(P<0.05),并激活p38MAPK,使其核内表达的荧光值增高；EMs各浓度预处理组均可使AGEs-BSA作用下HUVECs存活率增加,NO分泌减少(P<0.05),iNOS、ET-1产生及ET-1 mRNA表达降低(P<0.05),eNOS分泌及eNOS mRNA表达增加(P<0.05),并呈时间依赖性和浓度依赖性；EMs可使AGEs-BSA作用下p38MAPK核内表达的荧光值降低。
结论：AGEs-BSA可诱导人脐静脉内皮细胞活性下降及分泌内皮活性因子NO、eNOS、iNOS、ET-1受损；EMs对AGEs-BSA所致人脐静脉内皮细胞功能损伤有保护作用,并呈时间依赖性和浓度依赖性；EMs可抑制AGEs-BSA激活的p38MAPK的核内表达。
Objective:To investigate effects of EMs on synthesis and secretion of vasoactive substances by human umbilical vein endothelial cells (HUVECs) in the condition of advanced glycation end products (AGEs).
Methods:HUVECs were isolated from freshly collected umbilical cords, stimulated with AGEs-bovine serum albumin (AGEs-BSA), bovine serum albumin (BSA) or both AGEs-BSA and EMs, respectively. Then, HUVEC survival rate was calculated by MTT assay, the levels of NO, endothelial nitric oxide synthase and inducible nitric oxide synthase (eNOS, iNOS) were detected by colorimetric analysis, contents of endothelin-1 (ET-1) were detected by ELISA. Furthermore, expression of eNOS, ET-1 mRNA was measured by reverse transcription polymerase chain reaction (RT-PCR). In addition, expression of p38 mitogen-activated protein kinase (p38MAPK) was detected by immunofluorescence assay.
Results:Cell viability was time dependently decreased by exposed to AGEs-BSA (P＜0.01), AGEs-BSA can time dependently increase NO, iNOS, ET-1 concentration and up regulate ET-1 mRNA expression, decrease eNOS secretion and down regulate eNOS mRNA expression (P<0.05), and elevate the fluorescence intensity of p38MAPK in nucelus. While cell viability, secretion of NO、iNOS、ET-1, mRNA expression of ET-1 was significantly decreased after incubation with EMs compared to that with AGEs-BSA (P<0.05), secretion and mRNA expression of eNOS had opposite changes (P<0.05), which in a time and concentration dependent manner. EMs also decreaed the fluorescence intensity of p38MAPK in nucelus compared to AGEs-BSA group.
Conclusion:It was demonstrated that AGEs-BSA decreased the cell viability of HUVECs, and impaired the function of secreting endothelial active factors such as NO、eNOS、iNOS、ET-1. EMs has a certain protection effect on AGEs-BSA-induced injury in HUVECs as time and concentration dependent manner, and a inhibitory effect on the expression of p38MAPK in the nucelus activated by AGEs-BSA.
方法：从新鲜脐带中分离培养鉴定HUVECs,运用体外制备的糖基化修饰的牛血清白蛋白(AGEs-BSA)干预HUVECs,并进一步用不同浓度EMs(10μmol／ml,1μmol／ml,0.1μmol／ml,10nmol／ml)与AGEs-BSA共同干预HUVECs,同时设只加牛血清白蛋白(BSA)的HUVECs作阴性对照组；四甲基偶氮唑蓝(MTT)比色法检测HUVEC存活率,优化EMs的最佳作用浓度；硝酸盐还原酶法检测一氧化氮(NO)的浓度,分光光度法检测内皮型、诱导型一氧化氮合酶(eNOS、iNOS)的浓度,酶联免疫吸附(ELISA)法检测内皮素1(ET-1)的浓度,逆转录聚合酶链式反应(RT-PCR)检测eNOS、ET-1 mRNA的表达,免疫荧光法检测p38丝裂原活化蛋白激酶(p38MAPK)的表达。
结果：AGEs-BSA可使HUVECs活力降低,并呈现时间依赖性,与正常对照组比较差异有统计学意义(P<0.01),时间依赖地刺激HUVECs分泌iNOS、NO、ET-1及上调ET-1 mRNA的表达,抑制eNOS分泌及下调eNOS mRNA的表达(P<0.05),并激活p38MAPK,使其核内表达的荧光值增高；EMs各浓度预处理组均可使AGEs-BSA作用下HUVECs存活率增加,NO分泌减少(P<0.05),iNOS、ET-1产生及ET-1 mRNA表达降低(P<0.05),eNOS分泌及eNOS mRNA表达增加(P<0.05),并呈时间依赖性和浓度依赖性；EMs可使AGEs-BSA作用下p38MAPK核内表达的荧光值降低。
结论：AGEs-BSA可诱导人脐静脉内皮细胞活性下降及分泌内皮活性因子NO、eNOS、iNOS、ET-1受损；EMs对AGEs-BSA所致人脐静脉内皮细胞功能损伤有保护作用,并呈时间依赖性和浓度依赖性；EMs可抑制AGEs-BSA激活的p38MAPK的核内表达。
Objective:To investigate effects of EMs on synthesis and secretion of vasoactive substances by human umbilical vein endothelial cells (HUVECs) in the condition of advanced glycation end products (AGEs).
Methods:HUVECs were isolated from freshly collected umbilical cords, stimulated with AGEs-bovine serum albumin (AGEs-BSA), bovine serum albumin (BSA) or both AGEs-BSA and EMs, respectively. Then, HUVEC survival rate was calculated by MTT assay, the levels of NO, endothelial nitric oxide synthase and inducible nitric oxide synthase (eNOS, iNOS) were detected by colorimetric analysis, contents of endothelin-1 (ET-1) were detected by ELISA. Furthermore, expression of eNOS, ET-1 mRNA was measured by reverse transcription polymerase chain reaction (RT-PCR). In addition, expression of p38 mitogen-activated protein kinase (p38MAPK) was detected by immunofluorescence assay.
Results:Cell viability was time dependently decreased by exposed to AGEs-BSA (P＜0.01), AGEs-BSA can time dependently increase NO, iNOS, ET-1 concentration and up regulate ET-1 mRNA expression, decrease eNOS secretion and down regulate eNOS mRNA expression (P<0.05), and elevate the fluorescence intensity of p38MAPK in nucelus. While cell viability, secretion of NO、iNOS、ET-1, mRNA expression of ET-1 was significantly decreased after incubation with EMs compared to that with AGEs-BSA (P<0.05), secretion and mRNA expression of eNOS had opposite changes (P<0.05), which in a time and concentration dependent manner. EMs also decreaed the fluorescence intensity of p38MAPK in nucelus compared to AGEs-BSA group.
Conclusion:It was demonstrated that AGEs-BSA decreased the cell viability of HUVECs, and impaired the function of secreting endothelial active factors such as NO、eNOS、iNOS、ET-1. EMs has a certain protection effect on AGEs-BSA-induced injury in HUVECs as time and concentration dependent manner, and a inhibitory effect on the expression of p38MAPK in the nucelus activated by AGEs-BSA.
引文
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4. Nakagami T. DECODA Study Group. Hyperglycaemia and mortality from all causes and from cardiovascular disease in five populations of Asian origin [J]. Diabetologia,2003,47:385-394.
5. Lawes CM, Parag V, Bennett DA, et al. Blood glucose and risk of cardiovascular disease in the Asia Pacific region [J]. Diabetes care,2004,27: 2836-2842.
6. Fong DS, Aiello LP, Ferris FL 3rd, Klein R. Diabetic retinopathy [J]. Diabetes Care,2004,27(10):2540-2553.
7. Kempen JH, OColmain BJ, Leske MC, et al. Eye Diseases Prevalence Research Group. The prevalence of diabetic retinopathy among adults in the United States [J]. Arch Ophthalmol,2004,122:552-563.
8. 中华医学会糖尿病学分会慢性并发症调查组.1991～2000年全国住院糖尿病患者慢性并发症及相关大血管病变回顾性分析[J].中国医学科学院学报,2002,24：447-451.
9. Janero DR. Nitric Oxide (NO)-related pharmaceuticals:con-temporary approaches to therapeutical NO modulation [J]. Free Radieal Biology Medicine,2000,28(10):1495-1506.
10. De Artinano AA, Gonzalez VL. Endothelial dysfunctio and hypertensive vasoconstriction [J]. Pharmacological Research,1999,40:113-124.
11. Hantman HR, Bollage W. The effects of arotinoids on rat mammary carcinogenesis [J]. Cancer Chemother Pharmacol,1985,15(2):141-143.
12. Conger JD. Endothelial regulation of vascular tone [J]. HosPital Praetiee, 1994,29(10):117-122.
13. Jovanovie SV, Boone CW, Steenken S, et al. How curcumin works preferentially with water soluble antioxidants [J]. J.Am.Chem.Soc,2001, 7(13):2829-2833.
14. Brownlee M, Vlassara H. Advanced protein glycosylation in diabetes and aging [J]. Annu Rev Med,1995,46:223-234.
15.孙子林,刘乃丰,孙桂菊.糖尿病小鼠血清糖基化终末产物水平增高[J].中国糖尿病杂志,2000,8：315-320.
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21. Brewster JL, De Valoir T, Dwyer NC, et al. An osmosensing signal transduction pathway in yeast [J]. Science,1993,259:1760.
22.韩济生,万有.“内吗啡肽”的发现是阿片肽研究的一次突破[J].生理科学进展,1997,28：237-239.
23. Horvath,G. Endomorphin-1 and endomorphin-2:pharmacology of the selective endogenous μ-opioiod receptor agonists [J]. Pharmacol Ther,2000, 294:159-162.
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25. Champion HC, Zadina JE, Kastin AJ, Kadowitz PJ. Endomorphin 1 and 2 have vasodepressor activity in the anesthetized mouse [J]. Peptides,1998, 19:925-929.
26. Czapla MA, Champion HC, Zadina JE, Kastin AJ, Hackler L., Ge L.J, et al, Endomorphin 1 and 2, the endogenous mu-opioid agonists, decrease systemic arterial pressure in the rat [J]. Life Sci,1998,62:PL175-179.
27. Jing Liu, Ye Yu, Ying-zhe Fan, Hui Chang, Hong-mei Liu, Yun Cui, et al. Cardiovascular effects of endomorphins in alloxan-induced diabetic rats [J]. Peptides,2005,26:607-614.
28. Akil H, Watson SJ, Yongg E, et al. Endogenous opioids:biology and function [J]. Annu Rev Neurosci,1984,7:223-255.
29. Champion HC, Bivalacqua TJ, Friedman DE, Zadina JE, Kastin AJ, Kadowitz PJ. Nitric oxide release mediates vasodilator responses to endomorphin 1 but not nociceptin/OFQ in the hindquarters vascular bed of the rat [J]. Peptides,1995,19:1595-1602.
30. Champion HC, Bivalacqua TJ, Zadina JE, et al. Role of nitric oxide in mediating vasodilator responses to opioid peptide in the rat [J]. Clin and Exp pharmacology and phsiology,2002,29:229-232.
31. McCarthy AD, Etcheverry SB, Bruzzone L, Cortizo AM. Effects of advanced glycation end-products on the proliferation and differentiation of osteoblast-like cells [J]. Mol Cell Biochem,1997,170:43-51.
32. Jaffe EA, Nachman RL, Becker CG, et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria [J]. J Clin Invest,1973,52(11):2745-2756.
33. Maclag T, Hoover GM, Stemermam MB, et al. Serial propagation of human endothelial cells in vitro [J]. J Cell Biol,1981, (91):420-422.
34.鄂征.组织培养和分子细胞学技术[M].北京：北京出版社,2001,125-126.
35.郑超,文格波,曹仁贤,等.糖基化终末产物对人脐静脉内皮细胞凋亡及其受体表达的影响[J].中华内分泌代谢杂志,2003,19：115-118.
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2. 叶任高,陆再英,谢毅,等.内科学[M].北京：人民卫生出版社,2004,794-795.
3. Kamaua AM, Grady D, Barrett-Connor E. Explaining the sex difference in coronary heart disease mortality among patients with type 2 diabetes mellitus:a meta-analysis [J]. Arch Intern Med,2002,162:1737-1745.
4. Nakagami T. DECODA Study Group. Hyperglycaemia and mortality from all causes and from cardiovascular disease in five populations of Asian origin [J]. Diabetologia,2003,47:385-394.
5. Lawes CM, Parag V, Bennett DA, et al. Blood glucose and risk of cardiovascular disease in the Asia Pacific region [J]. Diabetes care,2004,27: 2836-2842.
6. Fong DS, Aiello LP, Ferris FL 3rd, Klein R. Diabetic retinopathy [J]. Diabetes Care,2004,27(10):2540-2553.
7. Kempen JH, OColmain BJ, Leske MC, et al. Eye Diseases Prevalence Research Group. The prevalence of diabetic retinopathy among adults in the United States [J]. Arch Ophthalmol,2004,122:552-563.
8. 中华医学会糖尿病学分会慢性并发症调查组.1991～2000年全国住院糖尿病患者慢性并发症及相关大血管病变回顾性分析[J].中国医学科学院学报,2002,24：447-451.
9. Janero DR. Nitric Oxide (NO)-related pharmaceuticals:con-temporary approaches to therapeutical NO modulation [J]. Free Radieal Biology Medicine,2000,28(10):1495-1506.
10. De Artinano AA, Gonzalez VL. Endothelial dysfunctio and hypertensive vasoconstriction [J]. Pharmacological Research,1999,40:113-124.
11. Hantman HR, Bollage W. The effects of arotinoids on rat mammary carcinogenesis [J]. Cancer Chemother Pharmacol,1985,15(2):141-143.
12. Conger JD. Endothelial regulation of vascular tone [J]. HosPital Praetiee, 1994,29(10):117-122.
13. Jovanovie SV, Boone CW, Steenken S, et al. How curcumin works preferentially with water soluble antioxidants [J]. J.Am.Chem.Soc,2001, 7(13):2829-2833.
14. Brownlee M, Vlassara H. Advanced protein glycosylation in diabetes and aging [J]. Annu Rev Med,1995,46:223-234.
15.孙子林,刘乃丰,孙桂菊.糖尿病小鼠血清糖基化终末产物水平增高[J].中国糖尿病杂志,2000,8：315-320.
16. Valassara H, Fuh H, Makita Z, et al. Exogenous advanced glycosylation end products induce complex vascular dysfunction in normal animals:a model for diabetic and aging complications [J]. Proc Natl Acad Sci USA,1992,89: 12043-12047.
17. Shifang Yan, Ravichandran Ramasamy, Yoshifumi Naka, et al. Glycation, Inflammation, and RAGE:A Scaffold for the Macrovascular Complications of Diabetes and Beyond [J]. Circ Res,2003,93:1159-1169.
18. Cohen RA. Dysfunction of vascular endothelium in diabetes mellitus [J]. Circulation,1993,87(supplⅤ):v67.
19. Farkas J, Menzel EJ. Proteins lose their nitric oxide stabilizing function after Advaneed glycosylation [J]. Bioehlm Biophys Acta,1995,1245:305-310.
20. Raingeaud J, Whitmarsh AJ, Barrett T, et al. MKK3 and MKK6 regulated gene expression in mediated by the p38 mitogen-activated protein kinase signal transduction pathway [J]. Mol Cell Biol,1996,16(3):1247.
21. Brewster JL, De Valoir T, Dwyer NC, et al. An osmosensing signal transduction pathway in yeast [J]. Science,1993,259:1760.
22.韩济生,万有.“内吗啡肽”的发现是阿片肽研究的一次突破[J].生理科学进展,1997,28：237-239.
23. Horvath,G. Endomorphin-1 and endomorphin-2:pharmacology of the selective endogenous μ-opioiod receptor agonists [J]. Pharmacol Ther,2000, 294:159-162.
24. Champion HC, Bivalacqua TJ, Lambert DG, Me Williams SM, Zadina JE, Kastin AJ, Kadowitz PJ. Endomorphinland 2,the endogenous mu-opioid agonists, produce biphasic changes in systemic arterial pressure in the cat [J]. Life Sci,1998,63:PL131-PL136.
25. Champion HC, Zadina JE, Kastin AJ, Kadowitz PJ. Endomorphin 1 and 2 have vasodepressor activity in the anesthetized mouse [J]. Peptides,1998, 19:925-929.
26. Czapla MA, Champion HC, Zadina JE, Kastin AJ, Hackler L., Ge L.J, et al, Endomorphin 1 and 2, the endogenous mu-opioid agonists, decrease systemic arterial pressure in the rat [J]. Life Sci,1998,62:PL175-179.
27. Jing Liu, Ye Yu, Ying-zhe Fan, Hui Chang, Hong-mei Liu, Yun Cui, et al. Cardiovascular effects of endomorphins in alloxan-induced diabetic rats [J]. Peptides,2005,26:607-614.
28. Akil H, Watson SJ, Yongg E, et al. Endogenous opioids:biology and function [J]. Annu Rev Neurosci,1984,7:223-255.
29. Champion HC, Bivalacqua TJ, Friedman DE, Zadina JE, Kastin AJ, Kadowitz PJ. Nitric oxide release mediates vasodilator responses to endomorphin 1 but not nociceptin/OFQ in the hindquarters vascular bed of the rat [J]. Peptides,1995,19:1595-1602.
30. Champion HC, Bivalacqua TJ, Zadina JE, et al. Role of nitric oxide in mediating vasodilator responses to opioid peptide in the rat [J]. Clin and Exp pharmacology and phsiology,2002,29:229-232.
31. McCarthy AD, Etcheverry SB, Bruzzone L, Cortizo AM. Effects of advanced glycation end-products on the proliferation and differentiation of osteoblast-like cells [J]. Mol Cell Biochem,1997,170:43-51.
32. Jaffe EA, Nachman RL, Becker CG, et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria [J]. J Clin Invest,1973,52(11):2745-2756.
33. Maclag T, Hoover GM, Stemermam MB, et al. Serial propagation of human endothelial cells in vitro [J]. J Cell Biol,1981, (91):420-422.
34.鄂征.组织培养和分子细胞学技术[M].北京：北京出版社,2001,125-126.
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