利拉鲁肽调节Sirtuin表达对高糖诱导的人内皮集落形成细胞生物学行为的影响
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摘要
目的探索利拉鲁肽是否通过调节Sirtuin影响高糖诱导的人内皮集落形成细胞(ECFC)的生物学行为。方法采用密度梯度离心法从外周血中分离获取单个核细胞,用EBM-2诱导培养出ECFC,随机分为正糖组(5.5 mmol/L D-葡萄糖)、渗透压组(5.5mmol/LD-葡萄糖+24.5 mmol/L甘露醇)、高糖组(30 mmol/LD-葡萄糖),均处理6d。随后采用EdU实验、Transwell实验、成管实验、β-半乳糖苷酶实验分别测定ECFC的增殖、迁移、成管及衰老情况,采用免疫印迹法测定Sirtuins(Sirtuin1–7)、血管内皮生长因子(VEGF)、血管生成素的表达水平。研究利拉鲁肽对ECFC的影响时,将细胞分为4组,除上述正糖组与高糖组外,还设有正糖利拉鲁肽干预组(5.5 mmol/L D-葡萄糖+100 nmol/L利拉鲁肽)、高糖利拉鲁肽干预组(30 mmol/L D-葡萄糖+100 nmol/L利拉鲁肽),均处理6d,观察4组细胞的生物学行为变化并测定VEGF和血管生成素的表达水平。结果 EdU实验显示高糖组的细胞增殖率明显低于正糖组[(30.16±12.36)%vs.(88.00±13.77)%],Transwell实验显示高糖组的细胞迁移能力明显低于正糖组(25.11±6.05vs.64.89±10.73),成管实验显示高糖组细胞成管能力明显低于正糖组(27.50±3.90 vs. 69.61±5.48),β-半乳糖苷酶染色实验显示高糖组细胞衰老率明显高于正糖组[(87.63±9.63)%vs.(71.35±7.58)%],差异均有统计学意义(P<0.05)。高糖情况下,Sirtuins家族表达普遍降低,VEGF和血管生成素表达均显著下降(P<0.05),Sirtuin1的表达随利拉鲁肽浓度的升高而升高。予以适宜浓度的利拉鲁肽干预后,VEGF和血管生成素的表达均明显增加(P<0.05)。EdU实验显示高糖利拉鲁肽干预组的细胞增殖率明显高于正糖组[(54.09±27.29)%vs.(29.01±7.56)%],Transwell实验显示高糖利拉鲁肽干预组的细胞迁移能力明显高于高糖组(32.25±4.99 vs. 21.75±3.10),成管实验显示高糖利拉鲁肽干预组的细胞成管能力明显高于高糖组(69.61±8.11vs.39.85±5.78),β-半乳糖苷酶染色实验显示高糖利拉鲁肽干预组的细胞衰老率明显低于高糖组[(52.06±7.94)%vs.(69.03±1.57)%],差异均有统计学意义(P<0.05)。结论高糖可降低ECFC的增殖、迁移和成管能力,增加细胞衰老比例,降低Sirtuins、VEGF和血管生成素的表达;利拉鲁肽可逆转高糖导致的ECFC生物学行为改变,同时上调Sirtuin1、VEGF及血管生成素的表达。
Objective To explore whether liraglutide could affect high glucose induced-biological behavior of human endothelial colony forming cell(ECFC) by regulating sirtuin. Methods Mononuclear cells in human peripheral blood were isolated by density gradient centrifugation. ECFCs were induced and cultured with EBM-2 complete medium. The cells were randomly divided into three groups: normal glucose group(NG, 5.5 mmol/L D-glucose for 6 days), high glucose group(HG, 30 mmol/L D-glucose for6 days), and osmotic pressure group(OSM, 5.5 mmol/L D-glucose+24.5 mmol/L mannitol for 6 days). The proliferation, migration,tube formation and senescence of ECFCs in the three groups were tested by EdU, Transwell, tube formation and SA-β-gal experiments respectively. The protein expression levels of Sirtuins(Sirtuin1-7), vascular endothelial growth factor(VEGF) and angiogenin weremeasured by Western blotting. Except NG and HG groups mentioned above, NG + liraglutide intervention group(5.5 mmol/L D-glucose+100 nmol/L liraglutide) and HG+liraglutide intervention group(30 mmol/L D-glucose+100 nmol/L liraglutide) were added in order to observe the effects of liraglutide on ECFC biological behavior and the expression of VEGF and angiogenin. The intervention time in all groups is 6 days. Results EdU, Transwell and tube formation experiments showed that the proliferation rate, migration ability and tube formation ability of ECFCs were lower in HG group [(30.16±12.36)%, 25.11±6.05, 27.50±3.90, respectively] than in NG group [(88.00±13.77)%, 64.89±10.73, 69.61±5.48, respectively], while the SA-β-gal experiment showed the senescence rate of ECFCs was higher in HG group than in NG group [(87.63±9.63)% vs.(71.35±7.58)%], all with statistical significance(P<0.05).Under high glucose conditions, the expression of Sirtuins family was generally reduced, and the expressions of VEGF and angiogenin significantly decreased(P<0.05). The expression of Sirtuin1 showed an upward trend with the increase of liraglutide concentration. After intervention with appropriate concentration of liraglutide, the expressions of VEGF and angiogenin significantly increased(P<0.05). EdU experiment showed the proliferation rate of ECFCs was higher in HG+liraglutide intervention group than in NG group [(54.09±27.29)%vs.(29.01±7.56)%], the Transwell and tube formation experiments showed that the migration ability and tube formation ability of ECFCs were higher in HG+liraglutide intervention group(32.25±4.99, 69.61±8.11) than in HG group(21.75±3.10, 39.85±5.78),and the SA-β-gal experiment showed the senescence rate of ECFCs was lower in HG+liraglutide intervention group than in HG group[(52.06±7.94)% vs.(69.03±1.57)%], all with statistical significance(P<0.05). Conclusions High glucose may accelerate ECFCs senescence while decrease the ability of proliferation, migration and tube formation of ECFCs, accompanied by down-regulation of Sirtuins, VEGF and angiogenin. Liraglutide may reverse the changes of ECFCs biological behavior induced by high glucose and upregulate the expression of Sirtuin1, VEGF and angiogenin.
Objective To explore whether liraglutide could affect high glucose induced-biological behavior of human endothelial colony forming cell(ECFC) by regulating sirtuin. Methods Mononuclear cells in human peripheral blood were isolated by density gradient centrifugation. ECFCs were induced and cultured with EBM-2 complete medium. The cells were randomly divided into three groups: normal glucose group(NG, 5.5 mmol/L D-glucose for 6 days), high glucose group(HG, 30 mmol/L D-glucose for6 days), and osmotic pressure group(OSM, 5.5 mmol/L D-glucose+24.5 mmol/L mannitol for 6 days). The proliferation, migration,tube formation and senescence of ECFCs in the three groups were tested by EdU, Transwell, tube formation and SA-β-gal experiments respectively. The protein expression levels of Sirtuins(Sirtuin1-7), vascular endothelial growth factor(VEGF) and angiogenin weremeasured by Western blotting. Except NG and HG groups mentioned above, NG + liraglutide intervention group(5.5 mmol/L D-glucose+100 nmol/L liraglutide) and HG+liraglutide intervention group(30 mmol/L D-glucose+100 nmol/L liraglutide) were added in order to observe the effects of liraglutide on ECFC biological behavior and the expression of VEGF and angiogenin. The intervention time in all groups is 6 days. Results EdU, Transwell and tube formation experiments showed that the proliferation rate, migration ability and tube formation ability of ECFCs were lower in HG group [(30.16±12.36)%, 25.11±6.05, 27.50±3.90, respectively] than in NG group [(88.00±13.77)%, 64.89±10.73, 69.61±5.48, respectively], while the SA-β-gal experiment showed the senescence rate of ECFCs was higher in HG group than in NG group [(87.63±9.63)% vs.(71.35±7.58)%], all with statistical significance(P<0.05).Under high glucose conditions, the expression of Sirtuins family was generally reduced, and the expressions of VEGF and angiogenin significantly decreased(P<0.05). The expression of Sirtuin1 showed an upward trend with the increase of liraglutide concentration. After intervention with appropriate concentration of liraglutide, the expressions of VEGF and angiogenin significantly increased(P<0.05). EdU experiment showed the proliferation rate of ECFCs was higher in HG+liraglutide intervention group than in NG group [(54.09±27.29)%vs.(29.01±7.56)%], the Transwell and tube formation experiments showed that the migration ability and tube formation ability of ECFCs were higher in HG+liraglutide intervention group(32.25±4.99, 69.61±8.11) than in HG group(21.75±3.10, 39.85±5.78),and the SA-β-gal experiment showed the senescence rate of ECFCs was lower in HG+liraglutide intervention group than in HG group[(52.06±7.94)% vs.(69.03±1.57)%], all with statistical significance(P<0.05). Conclusions High glucose may accelerate ECFCs senescence while decrease the ability of proliferation, migration and tube formation of ECFCs, accompanied by down-regulation of Sirtuins, VEGF and angiogenin. Liraglutide may reverse the changes of ECFCs biological behavior induced by high glucose and upregulate the expression of Sirtuin1, VEGF and angiogenin.
引文
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[12]Matsui M,Uemura S,Takeda Y,et al.Placental growth factor as a predictor of cardiovascular events in patients with CKD from the NARA-CKD study[J].J Am Soc Nephrol,2015,26(11):2871-2881.
[13]Chuan FN,Tang K,Jiang P,et al.Reliability and validity of the perfusion,extent,depth,infection and sensation(PEDIS)classification system and score in patients with diabetic foot ulcer[J].PLoS One,2015,10(4):e0124739.
[14]Thijssen DH,Vos JB,Verseyden C,et al.Haematopoietic stem cells and endothelial progenitor cells in healthy men:effect of aging and training[J].Aging Cell,2006,5(6):495-503.
[15]Wang QL,Guo SJ.Sirtuins function as the modulators in agingrelated diseases in common or respectively[J].Chin Med J,2015,128(12):1671-1678.
[16]Balestrieri ML,Rizzo MR,Barbieri M,et al.Sirtuin 6 expression and inflammatory activity in diabetic atherosclerotic plaques:effects of incretin treatment[J].Diabetes,2015,64(4):1395-1406.
[17]Inoue T,Inoguchi T,Sonoda N,et al.GLP-1 analog liraglutide protects against cardiac steatosis,oxidative stress and apoptosis in streptozotocin-induced diabetic rats[J].Atherosclerosis,2015,240(1):250-259.
[18]Ke J,Wei R,Yu F,et al.Liraglutide restores angiogenesis in palmitate-impaired human endothelial cells through PI3K/AktFoxo1-GTPCH1 pathway[J].Peptides,2016,86:95-101.
[19]Oeseburg H,de Boer RA,Buikema H,et al.Glucagon-like peptide 1 prevents reactive oxygen species-induced endothelial cell senescence through the activation of protein kinase A[J].Arterioscler Thromb Vasc Biol,2010,30(7):1407-1414.
[20]Langlois A,Mura C,Bietiger W,et al.In vitro and in vivo investigation of the angiogenic effects of liraglutide during islet transplantation[J].PLoS One,2016,11(3):e0147068.
[21]Chai WD,Fu Z,Aylor KW,et al.L i raglutide prevent s microvascular insulin resistance and preserves muscle capillary density in high-fat diet-fed rats[J].Am J Physiol Endocrinol Metab,2016,311(3):E640-E648.
[2]Sieveking DP,Buckle A,Celermajer DS,et al.Strikingly different angiogenic properties of endothelial progenitor cell subpopulations:insights from a novel human angiogenesis assay[J].J Am Coll Cardiol,2008,51(6):660-668.
[3]Xu B,Li HL,Liu DP,et al.Promotion of exosomes derived from bone-marrow endothelial progenitor cells in repairing traumatic cutaneous deficiency in rats[J].J Jilin Univ(Med Ed),2017,43(4):672-678+857-858.[徐兵,李海乐,刘丹平,等.骨髓源内皮祖细胞分泌的外泌体对大鼠创伤性皮肤缺损修复的促进作用[J].吉林大学学报(医学版),2017,43(4):672-678,857-858.]
[4]Tepper OM,Galiano RD,Capla JM,et al.Human endothelial progenitor cells from typeⅡdiabetics exhibit impaired proliferation,adhesion,and incorporation into vascular structures[J].Circulation,2002,106(22):2781-2786.
[5]Yuan Q,Hu CP,Gong ZC,et al.Accelerated onset of senescence of endothelial progenitor cells in patients with type 2 diabetes mellitus:role of dimethylarginine dimethylaminohydrolase 2and asymmetric dimethylarginine[J].Biochem Biophys Res Commun,2015,458(4):869-876.
[6]Marso SP,Daniels GH,Brown-Frandsen K,et al.Liraglutide and cardiovascular outcomes in type 2 diabetes[J].N Engl J Med,2016,375(4):311-322.
[7]Dhatariya K,Bain SC,Buse JB,et al.The impact of liraglutide on diabetes-related foot ulceration and associated complications in patients with type 2 diabetes at high risk for cardiovascular events:results from the LEADER trial[J].Diabetes Care,2018,41(10):2229-2235.
[8]Gaspari T,Liu HB,Welungoda I,et al.A GLP-1 receptor agonist liraglutide inhibits endothelial cell dysfunction and vascular adhesion molecule expression in an ApoE-/-mouse model[J].Diab Vasc Dis Res,2011,8(2):117-124.
[9]Chen YX,Zhang XJ,He JN,et al.Delayed administration of the glucagon-like peptide 1 analog liraglutide promoting angiogenesis after focal cerebral ischemia in mice[J].J Stroke Cerebrovasc Dis,2018,27(5):1318-1325.
[10]Tang K,Zhang M,Zhou B.Hyperglycemia metabolic memory effect in late endothelial progenitor cells from human peripheral blood[J].J Chongqing Med Univ,2018,43(6):751-756.[唐康,张敏,周波.人外周血晚期内皮祖细胞中的高糖记忆效应[J].重庆医科大学学报,2018,43(6):751-756.]
[11]Chen YH,Lin SJ,Lin FY,et al.High glucose impairs early and late endothelial progenitor cells by modifying nitric oxiderelated but not oxidative stress-mediated mechanisms[J].Diabetes,2007,56(6):1559-1568.
[12]Matsui M,Uemura S,Takeda Y,et al.Placental growth factor as a predictor of cardiovascular events in patients with CKD from the NARA-CKD study[J].J Am Soc Nephrol,2015,26(11):2871-2881.
[13]Chuan FN,Tang K,Jiang P,et al.Reliability and validity of the perfusion,extent,depth,infection and sensation(PEDIS)classification system and score in patients with diabetic foot ulcer[J].PLoS One,2015,10(4):e0124739.
[14]Thijssen DH,Vos JB,Verseyden C,et al.Haematopoietic stem cells and endothelial progenitor cells in healthy men:effect of aging and training[J].Aging Cell,2006,5(6):495-503.
[15]Wang QL,Guo SJ.Sirtuins function as the modulators in agingrelated diseases in common or respectively[J].Chin Med J,2015,128(12):1671-1678.
[16]Balestrieri ML,Rizzo MR,Barbieri M,et al.Sirtuin 6 expression and inflammatory activity in diabetic atherosclerotic plaques:effects of incretin treatment[J].Diabetes,2015,64(4):1395-1406.
[17]Inoue T,Inoguchi T,Sonoda N,et al.GLP-1 analog liraglutide protects against cardiac steatosis,oxidative stress and apoptosis in streptozotocin-induced diabetic rats[J].Atherosclerosis,2015,240(1):250-259.
[18]Ke J,Wei R,Yu F,et al.Liraglutide restores angiogenesis in palmitate-impaired human endothelial cells through PI3K/AktFoxo1-GTPCH1 pathway[J].Peptides,2016,86:95-101.
[19]Oeseburg H,de Boer RA,Buikema H,et al.Glucagon-like peptide 1 prevents reactive oxygen species-induced endothelial cell senescence through the activation of protein kinase A[J].Arterioscler Thromb Vasc Biol,2010,30(7):1407-1414.
[20]Langlois A,Mura C,Bietiger W,et al.In vitro and in vivo investigation of the angiogenic effects of liraglutide during islet transplantation[J].PLoS One,2016,11(3):e0147068.
[21]Chai WD,Fu Z,Aylor KW,et al.L i raglutide prevent s microvascular insulin resistance and preserves muscle capillary density in high-fat diet-fed rats[J].Am J Physiol Endocrinol Metab,2016,311(3):E640-E648.