抵抗素对人脐静脉内皮细胞内NO及cAMP的影响
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摘要
背景:血管内皮细胞功能紊乱是代谢综合征(metabolic syndrome, MS)的一个重要特征,而抵抗素(resistin, Re)为新近发现的脂肪细胞因子,在代谢综合征发生、发展过程中起重要的调控作用,抵抗素可能促进血管内皮细胞功能紊乱。目前已证实一氧化氮(nitric oxide, NO)及环磷腺苷(cyclic adenosine monophosphate, cAMP)在维持正常的血管内皮功能中起重要作用。
目的:本研究主要观察抵抗素对人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)内NO的分泌及cAMP水平的影响,探讨其对血管内皮功能调节的作用机制。
方法:(1)体外培养HUVECs,取4-6代细胞用于实验。培养的HUVECs接种于96孔板,生长至80%以上融合状态,将细胞分为3组:Ⅰ组为单纯培养基对照组,Ⅱ组为50ng/ml抵抗素组,Ⅲ组为20ng/ml肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)组。加入上述试剂处理12小时后,取上清液离心去除细胞碎片,硝酸还原酶法测定NO含量。
(2)体外培养HUVECs,取4-6代细胞用于实验。培养的HUVECs接种于细胞培养瓶中,生长至80%以上融合状态,分组同上。加入上述试剂处理培养6小时后,提取蛋白,ELISA法测定cAMP含量。
结果:(1)与单纯培养基对照组相比,抵抗素组及TNF-α组均能轻度抑制HUVECs内NO的分泌,但无统计学意义(P>0.05)。
(2)与单纯培养基对照组相比,经抵抗素及TNF-α培养6小时后,HUVECs内cAMP的水平均明显下降(P<0.05)。
结论:抵抗素对HUVECs内NO的分泌无明显影响,但能明显降低cAMP水平,提示抵抗素可能通过抑制cAMP合成而引起血管内皮功能障碍。
Background:Vascular endothelial cells dysfunction is a main trait of metabolic syndrome (MS), and resistin is a recently discovered adipokinine, which plays an important role in the pathophysiological development of MS. Resistin may cast adverse effects on endothelial cell dysfunction. Nitric oxide (NO) and cyclic adenosine monophosphate (cAMP) have been found to play important roles in maintenance of vascular endothelial function.
Objective: In this study we evaluated the effects of resistin on NO and cAMP levels in cultured human umbilical vein endothelial cells (HUVECs) to explore the potential mechanism of resistin on vascular endothelial function mediation.
Methods: (1) Cultured HUVECs within passage 4-6 were inoculated in 96-well plate to reach 80 percent of confluence to be divided into 3 treatment groups:Ⅰmedium only control group,Ⅱ50ng/ml resistin incubation group,Ⅲ20ng/ml tumor necrosis factor-α(TNF-α) incubation group for 12h, and the supernatant obtained from the cultured HUVECs were collected, centrifuged to remove cell debris, then measured with the method of Nitrate reductase.
(2) Cultured HUVECs within passage 4-6 were inoculated in Cell Culture Flasks to reach 80 percent of confluence to be divided into 3 treatment groups:Ⅰmedium only control group,Ⅱ50ng/ml resistin incubation group,Ⅲ20ng/ml TNF-αincubation group for 6h. Extracted and then measured the cAMP levels with Enzyme Linked Immunosorbent Assay kits (ELISA).
Result: (1) Compared with control group, neither 50ng/ml resistin nor 20ng/ml TNF-αdecreased NO levels significantly in cultured HUVECs (p>0.05).
(2) Compared with that of the control group, cAMP levels were significantly lower in resistin or TNF-αtreatment cells (p<0.05).
Conclusion: Resistin has no effect on the secretion of NO from HUVECs, but decreased the cAMP level significantly, suggesting that resistin may cause endothelial dysfunction, partly, by reducing cAMP concentration.
目的:本研究主要观察抵抗素对人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)内NO的分泌及cAMP水平的影响,探讨其对血管内皮功能调节的作用机制。
方法:(1)体外培养HUVECs,取4-6代细胞用于实验。培养的HUVECs接种于96孔板,生长至80%以上融合状态,将细胞分为3组:Ⅰ组为单纯培养基对照组,Ⅱ组为50ng/ml抵抗素组,Ⅲ组为20ng/ml肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)组。加入上述试剂处理12小时后,取上清液离心去除细胞碎片,硝酸还原酶法测定NO含量。
(2)体外培养HUVECs,取4-6代细胞用于实验。培养的HUVECs接种于细胞培养瓶中,生长至80%以上融合状态,分组同上。加入上述试剂处理培养6小时后,提取蛋白,ELISA法测定cAMP含量。
结果:(1)与单纯培养基对照组相比,抵抗素组及TNF-α组均能轻度抑制HUVECs内NO的分泌,但无统计学意义(P>0.05)。
(2)与单纯培养基对照组相比,经抵抗素及TNF-α培养6小时后,HUVECs内cAMP的水平均明显下降(P<0.05)。
结论:抵抗素对HUVECs内NO的分泌无明显影响,但能明显降低cAMP水平,提示抵抗素可能通过抑制cAMP合成而引起血管内皮功能障碍。
Background:Vascular endothelial cells dysfunction is a main trait of metabolic syndrome (MS), and resistin is a recently discovered adipokinine, which plays an important role in the pathophysiological development of MS. Resistin may cast adverse effects on endothelial cell dysfunction. Nitric oxide (NO) and cyclic adenosine monophosphate (cAMP) have been found to play important roles in maintenance of vascular endothelial function.
Objective: In this study we evaluated the effects of resistin on NO and cAMP levels in cultured human umbilical vein endothelial cells (HUVECs) to explore the potential mechanism of resistin on vascular endothelial function mediation.
Methods: (1) Cultured HUVECs within passage 4-6 were inoculated in 96-well plate to reach 80 percent of confluence to be divided into 3 treatment groups:Ⅰmedium only control group,Ⅱ50ng/ml resistin incubation group,Ⅲ20ng/ml tumor necrosis factor-α(TNF-α) incubation group for 12h, and the supernatant obtained from the cultured HUVECs were collected, centrifuged to remove cell debris, then measured with the method of Nitrate reductase.
(2) Cultured HUVECs within passage 4-6 were inoculated in Cell Culture Flasks to reach 80 percent of confluence to be divided into 3 treatment groups:Ⅰmedium only control group,Ⅱ50ng/ml resistin incubation group,Ⅲ20ng/ml TNF-αincubation group for 6h. Extracted and then measured the cAMP levels with Enzyme Linked Immunosorbent Assay kits (ELISA).
Result: (1) Compared with control group, neither 50ng/ml resistin nor 20ng/ml TNF-αdecreased NO levels significantly in cultured HUVECs (p>0.05).
(2) Compared with that of the control group, cAMP levels were significantly lower in resistin or TNF-αtreatment cells (p<0.05).
Conclusion: Resistin has no effect on the secretion of NO from HUVECs, but decreased the cAMP level significantly, suggesting that resistin may cause endothelial dysfunction, partly, by reducing cAMP concentration.
引文
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2. Ford ES, Giles WH. A comparison of the prevalence of the metabolic syndrome using two proposed definitions[J]. Diabetes Care. 2003;26(3):575-81.
3.顾东风, Reynolds K,杨文杰等.中国成年人代谢综合征的患病率[J].中华糖尿病杂志. 2005;13(3):18l-6.
4. Prabhakaran D, Anand SS. The metabolic syndrome: an emerging risk state for cardiovascular disease[J]. Vasc Med. 2004;9(1):55-68.
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7. Satoh H, Nguyen MT, et al. Adenovirus-mediated chronic "hyper-resistinemia" leads to in vivo insulin resistance in normal rats[J]. J Clin Invest. 2004;114(2):224-31.
8. Shen YH, Zhang L, Gan Y, et al. Up-regulation of PTEN (phosphatase and tensin homolog deleted on chromosome ten) mediates p38 MAPK stress signal-induced inhibition of insulin signaling. A cross-talk between stress signaling and insulin signaling inresistin-treated human endothelial cells[J]. J Biol Chem. 2006;281(12):7727-36.
9. Gonzalez MA, Selwyn AP. Endothelial function,inflammation, and prognosis in cardiovascular disease[J]. Am J Med. 2003;8(115):99-106.
10. Pinkney JH, Stehouwer CD, Coppack SW, et al. Endothelial dysfunction: cause of the insulin resistance syndrome[J]. Diabetes. 1997;2(46):S9-13.
11. Chen H, Montagnani M, Funahashi T, et al. Adiponectin stimulates production of nitric oxide in vascular endothelial cells[J]. J Biol Chem. 2003;278(45):45021-6.
12. Quehenberger P, Exner M, Sunder-Plassmann R, et al. Leptin induces endothelin-1 inendothelial cells in vitro[J]. Circ Res. 2002;90(6):711-8.
13. Li Y, Wang Y, Li Q, et al. Effect of resistin on vascular endothelium secretion dysfunction in rats[J]. Endothelium. 2007;14(4-5):207-14.
14. Verma S, Li SH, Wang CH, et al. Resistin promotes endothelial cell activation: further evidence of adipokine-endothelial interaction[J]. Circulation. 2003;108(6):736-40.
15. Curat CA, Wegner V, Sengenes C, et al. Macrophages in human visceral Adipose tissue:increased accumulation in obesity and a source of resistin and visfatin[J]. Diabetologia. 2006;49(4):744-7.
16. Eisenreich A, Boltzen U, et a1. Effects of the Cdc2-like kinase-family and DNA topoisomerase-I on the alternative splicing of eNOS in TNF-alpha-stimulated human endothelial cells[J]. Biol Chem. 2008;389(10):1333-8.
17. S. Koga S.Morris, S Ogawa, D. M. Stern, et al. TNF modulates endothelial properties by decreasing cAMP[J]. Am J Physiol. 1995;268(5 Pt 1):C1104-13.
18. Shestakova MV, Butrova SA, Sukhareva OIu. Metabolic syndrome as a precursor of diabetes mellitus type 2 and cardiovascular diseases[J]. Ter Arkh. 2007;79(10):5-8.
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21. Greenfield JR, Campbell LV. Insulin resistance and obesity[J]. Clin Dermatol. 2004; 22(4):289-95.
22. Lazar MA. How obesity causes diabetes: not a tall talel[J]. Science. 2005;307(5708):373-5.
23. Gluckman PD, Hanson MA. The developmental origins of the metabolic syndrome[J]. Trends Endocrinol Metab. 2004;15(4):183-7.
24. Guerre-Millo M. Adipose tissue secretory function:implication in metabolic and cardiovascular complications of obesity[J]. J Soc Biol. 2006;200(1):37-43.
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30. Kougias P, Chai H, Lin PH, et al. Adipocyte-derived cytokine resistin causes endothelial dysfunction of porcine coronary arteries[J]. J Vasc Surg. 2005;41(4):691-8.
31. Kawanami D, Maemura K, Takeda N, et al. Direct reciprocal effects of resistin and adiponectin on vascular endothelial cells: a new insight into adipocytokine-endothelial cell interactions[J]. Biochem Biophys Res Commun. 2004;314(2):415-9.
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1. Chen H, Montagnani M, Funahashi T, et al. Adiponectin stimulates production of nitric oxide in vascular endothelial cells[J]. J Biol Chem. 2003;278(45):45021-6.
2. Steppan CM, Bailey ST, Bhat S, et al. The hormone resistin links obesity to diabetes[J]. Nature. 2001;409(6818):307-12.
3. Ghosh S, Singh AK, Aruna B, et al. The genomic organization of mouse resistin reveals major differences from the human resistin: functional implications[J]. Gene. 2003;305(1):27-34.
4. Banerjee RR, Lazar MA. Dimerization of resistin and resistin-like molecules is determined by a single cysteine[J]. J Biol Chem. 2001;276(28):25970-3.
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6. Steppan CM, Brown EJ, Wright CM, et al. A family of tissue-specific resistin-like molecules[J]. Proc Natl Acad Sci USA. 2001;98(2):502-6.
7. Patel L, Buckels AC, Kinghorn IJ, et al. Resistin is expressed in human macrophages and directly regulated by PPAR gamma activators[J]. Biochem Biophys Res Commun. 2003;00(2):472-6.
8. Nagaev I, Smith U. Insulin resistance and type 2 diabetes are not related to resistin expression in human fat cells or skeletal muscle[J]. Biochem Biophys Res Commun. 2001;285(2):561-4.
9. Steppan CM, Wang J, Whiteman EL, et al. Activation of SOCS-3 by resistin[J]. Mol Cell Biol. 2005;25(4):1569-75.
10. de Luis DA, Gonzalez Sagrado M, Conde R, et al. Relation of resistin levels with cardiovascular risk factors and insulin resistance in non-diabetes obese patients[J]. Diabetes Res Clin Pract. 2009;84(2):174-8.
11. Cohen G, H?rl WH. Resistin as a cardiovascular and atherosclerotic risk factor and uremic toxin[J]. Semin Dial. 2009;22(4):373-7.
12. Sato N, Kobayashi K, Inoguchi T, et al. Adenovirus-mediated high expression of resistin causes dyslipidemia in mice[J]. Endocrinology. 2005;146(1):273-9.
13. Bokarewa M, Nagaev I, Dahlberg L, et al. Resistin, an adipokine with potent proinflammatory properties[J]. J Immunol. 2005;174(9):5789-95.
14. Gonzalez MA, Selwyn AP. Endothelial function, inflammation, and prognosis in cardiovascular disease[J]. Am J Med. 2003;115 Suppl 8A:99S-106S.
15. Moncada S, Higgs A. The L-arginine-nitric oxide pathway[J]. N Engl J Med. 1993;329(27):2002-12.
16. Gentile MT, Vecchione C, Marino G, et al. Resistin impairs insulin-evoked vasodilation[J]. Diabetes. 2008;57(3):577-83.
17. Kougias P, Chai H, Lin PH, et al. Adipocyte-derived cytokine resistin causes endothelial dysfunction of porcine coronary arteries[J]. J Vasc Surg. 2005;41(4):691-8.
18. Münzel T, Daiber A, Ullrich V, et al. Vascular consequences of endothelial nitric oxide synthase uncoupling for the activity and expression of the soluble guanylyl cyclase and the cGMP-dependent protein kinase[J]. Arterioscler Thromb Vasc Biol. 2005;25(8):1551-7.
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20. Verma S, Li SH, Wang CH, et al. Resistin promotes endothelial cell activation: further evidence of adipokine-endothelial interaction[J]. Circulation. 2003;108(6):736-40.
21. Jung HS, Park KH, Cho YM, et al. Resistin is secreted from macrophages in atheromas and promotes atherosclerosis[J]. Cardiovasc Res. 2006;69(1):76-85.
22. Silswal N, Singh AK, Aruna B, et al. Human resistin stimulates the pro-inflammatory cytokines TNF-alpha and IL-12 in macrophages by NF-kappa B dependent pathway[J]. Biochem Biophys Res Commun. 2005;334(4):1092-101.
23. Kawanami D, Maemura K, Takeda N, et al. Direct reciprocal effects of resistin and adiponectin on vascular endothelial cells: a new insight into adipocytokine-endothelial cell interactions[J]. Biochem Biophys Res Commun. 2004;314(2):415-9
24. Urbich C, Mallat Z, Tedgui A, et al. Upregulation of TRAF-3 by shear stress blocks CD40-mediated endothelial activation[J]. J Clin Invest. 2001;108(10):1451-8.
25. Rae C, Graham A. Human resistin promotes macrophage lipid accumulation[J]. Diabetologia. 2006;49(5):1112-4.
26. Ryoo S, Lemmon CA, Soucy KG, et al. Oxidized low-density lipoprotein-dependent endothelial arginase II activation contributes to impaired nitric oxide signaling[J]. Circ Res. 2006;99(9):951-60.
2. Ford ES, Giles WH. A comparison of the prevalence of the metabolic syndrome using two proposed definitions[J]. Diabetes Care. 2003;26(3):575-81.
3.顾东风, Reynolds K,杨文杰等.中国成年人代谢综合征的患病率[J].中华糖尿病杂志. 2005;13(3):18l-6.
4. Prabhakaran D, Anand SS. The metabolic syndrome: an emerging risk state for cardiovascular disease[J]. Vasc Med. 2004;9(1):55-68.
5. Goralski KB, Sinal CJ. Type 2 diabetes and cardiovascular disease: getting to the fat of the matter[J]. Can J Physiol Pharmacol. 2007;85(1):113-32.
6. Steppan CM, Bailey ST, Bhat S, et al. The hormone resistin links obesity to diabetes[J]. Nature. 2001;409(6818):307-12.
7. Satoh H, Nguyen MT, et al. Adenovirus-mediated chronic "hyper-resistinemia" leads to in vivo insulin resistance in normal rats[J]. J Clin Invest. 2004;114(2):224-31.
8. Shen YH, Zhang L, Gan Y, et al. Up-regulation of PTEN (phosphatase and tensin homolog deleted on chromosome ten) mediates p38 MAPK stress signal-induced inhibition of insulin signaling. A cross-talk between stress signaling and insulin signaling inresistin-treated human endothelial cells[J]. J Biol Chem. 2006;281(12):7727-36.
9. Gonzalez MA, Selwyn AP. Endothelial function,inflammation, and prognosis in cardiovascular disease[J]. Am J Med. 2003;8(115):99-106.
10. Pinkney JH, Stehouwer CD, Coppack SW, et al. Endothelial dysfunction: cause of the insulin resistance syndrome[J]. Diabetes. 1997;2(46):S9-13.
11. Chen H, Montagnani M, Funahashi T, et al. Adiponectin stimulates production of nitric oxide in vascular endothelial cells[J]. J Biol Chem. 2003;278(45):45021-6.
12. Quehenberger P, Exner M, Sunder-Plassmann R, et al. Leptin induces endothelin-1 inendothelial cells in vitro[J]. Circ Res. 2002;90(6):711-8.
13. Li Y, Wang Y, Li Q, et al. Effect of resistin on vascular endothelium secretion dysfunction in rats[J]. Endothelium. 2007;14(4-5):207-14.
14. Verma S, Li SH, Wang CH, et al. Resistin promotes endothelial cell activation: further evidence of adipokine-endothelial interaction[J]. Circulation. 2003;108(6):736-40.
15. Curat CA, Wegner V, Sengenes C, et al. Macrophages in human visceral Adipose tissue:increased accumulation in obesity and a source of resistin and visfatin[J]. Diabetologia. 2006;49(4):744-7.
16. Eisenreich A, Boltzen U, et a1. Effects of the Cdc2-like kinase-family and DNA topoisomerase-I on the alternative splicing of eNOS in TNF-alpha-stimulated human endothelial cells[J]. Biol Chem. 2008;389(10):1333-8.
17. S. Koga S.Morris, S Ogawa, D. M. Stern, et al. TNF modulates endothelial properties by decreasing cAMP[J]. Am J Physiol. 1995;268(5 Pt 1):C1104-13.
18. Shestakova MV, Butrova SA, Sukhareva OIu. Metabolic syndrome as a precursor of diabetes mellitus type 2 and cardiovascular diseases[J]. Ter Arkh. 2007;79(10):5-8.
19. Stern MP. Diabetes and cardiovascular disease: The "common soil" hypothesis[J]. Diabetes. 1995;44(4):369-74.
20. Harris NS, Winter WE. The chemical pathology of insulin resistance and the metabolic syndrome[J]. MLO Med Lab Obs. 2004;36(10):20, 22-5.
21. Greenfield JR, Campbell LV. Insulin resistance and obesity[J]. Clin Dermatol. 2004; 22(4):289-95.
22. Lazar MA. How obesity causes diabetes: not a tall talel[J]. Science. 2005;307(5708):373-5.
23. Gluckman PD, Hanson MA. The developmental origins of the metabolic syndrome[J]. Trends Endocrinol Metab. 2004;15(4):183-7.
24. Guerre-Millo M. Adipose tissue secretory function:implication in metabolic and cardiovascular complications of obesity[J]. J Soc Biol. 2006;200(1):37-43.
25. Saltiel AR, Kahn CR. Insulin signalling and the regulation of glucose and lipid metabolism[J]. Nature. 2001;414(6865):799-806.
26. Steppan CM, Wang J, Whiteman EL, et al. Activation of SOCS-3 by resistin[J]. Mol Cell Biol. 2005;25(4):1569-75.
27. de Luis DA, Gonzalez Sagrado M, Conde R, et al. Relation of resistin levels with cardiovascular risk factors and insulin resistance in non-diabetes obese patients[J]. Diabetes Res Clin Pract. 2009;84(2):174-8.
28. Burnett MS, Lee CW, Kinnaird TD, et al. The potential role of resistin in atherogenesis[J]. Atherosclerosis. 2005;182(2):241-8.
29. Sato N, Kobayashi K, Inoguchi T, et al. Adenovirus-mediated high expression of resistin causes dyslipidemia in mice[J]. Endocrinology. 2005;146(1):273-9.
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