CGRP通过激活cAMP/PPARγ/eNOS途径改善血管内皮细胞胰岛素抵抗
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摘要
目的:探讨降钙素基因相关肽(CGRP)对人脐静脉内皮细胞胰岛素抵抗的保护作用及其机制。方法:用人脐静脉内皮细胞(HUVEC)建立胰岛素抵抗细胞模型(irHUVEC),分别用葡萄糖氧化酶法和蒽酮-硫酸法检测细胞葡萄糖消耗能力和糖原合成能力;RT-PCR和Western blot分别检测目的蛋白和基因表达。结果:33.3 mmol/L的高糖和5μmol/L的胰岛素处理24 h可以成功诱导内皮细胞产生胰岛素抵抗。与正常HUVEC相比,irHUVEC体积变大,边界模糊,形态异常;当细胞用高糖和高胰岛素分别诱导24 h和48 h时,irHUVEC的葡萄糖消耗量分别减少20%和31%,并且糖原合成分别减少了55%和64%,差异都具有显著性;同时,过氧化物酶增殖体激活受体γ(PPARγ)和还原型一氧化氮合酶(eNOS)的表达均降低。CGRP可以明显增加irHUVEC葡萄糖的消耗量约20%和糖原合成增加约70%,并能增加PPARγ和eNOS蛋白及mRNA表达;SQ22536(cAMP阻断剂)能使PPARγ和eNOS的蛋白和基因表达量明显减少。结论:CGRP可以改善内皮细胞的胰岛素抵抗,其机制可能与上调环磷酸腺苷(cAMP),增加PPARγ和eNOS表达有关。
AIM: To explore the protective effect and the mechanism of calcitonin gene-relatedpeptide(CGRP) on the insulin-resisted human umbilical vein endothelial cells(irHUVECs). METHODS: The irHUVECs was established using human umbilical veinendothelial cells(HUVECs), gucose oxidase(GOP-POD) method and anthrone-sulfuric acid method were employed to detect glucose consumption ability and glycogen synthesis ability, respectively. RT-PCR and Western blot were used to detect the mRNA and protein expressions of peroxisome proliferator-activated receptor gamma(PPARγ) and endothelial nitric oxide synthase(eNOS), respectively. RESULTS:The irHUVECs was successfully established when the HUVECs were treated with 33.3 mmol/L high glucose and 5 μmol/L insulin. Compared to the normal group of endothelial cells, the features of irHUVECs were a larger volume, fuzzy boundary and abnormal morphology, when cells induced were incubated with high glucose and high insulin in 24 h and 48 h, respectively, the glucose consumption was reduced by 20% and 31%, and the glycogen synthesis was reduced by 55% and 64%, meanwhile, the expressions of PPARγ and eNOS were decreased. Treatment of CGRP significantly increased about 20% glucose consumption and about 70% glycogen synthesis, and increased the expressions of PPARγ and eNOS, SQ22536(cAMP inhibitor) can decreased the expressions of PPARγ and eNOS. CONCLUSION: CGRP could improve the endothelial cell insulin resistance, the mechanism may be related to up-regulated cAMP, and the increased expressions of PPARγ and eNOS.
AIM: To explore the protective effect and the mechanism of calcitonin gene-relatedpeptide(CGRP) on the insulin-resisted human umbilical vein endothelial cells(irHUVECs). METHODS: The irHUVECs was established using human umbilical veinendothelial cells(HUVECs), gucose oxidase(GOP-POD) method and anthrone-sulfuric acid method were employed to detect glucose consumption ability and glycogen synthesis ability, respectively. RT-PCR and Western blot were used to detect the mRNA and protein expressions of peroxisome proliferator-activated receptor gamma(PPARγ) and endothelial nitric oxide synthase(eNOS), respectively. RESULTS:The irHUVECs was successfully established when the HUVECs were treated with 33.3 mmol/L high glucose and 5 μmol/L insulin. Compared to the normal group of endothelial cells, the features of irHUVECs were a larger volume, fuzzy boundary and abnormal morphology, when cells induced were incubated with high glucose and high insulin in 24 h and 48 h, respectively, the glucose consumption was reduced by 20% and 31%, and the glycogen synthesis was reduced by 55% and 64%, meanwhile, the expressions of PPARγ and eNOS were decreased. Treatment of CGRP significantly increased about 20% glucose consumption and about 70% glycogen synthesis, and increased the expressions of PPARγ and eNOS, SQ22536(cAMP inhibitor) can decreased the expressions of PPARγ and eNOS. CONCLUSION: CGRP could improve the endothelial cell insulin resistance, the mechanism may be related to up-regulated cAMP, and the increased expressions of PPARγ and eNOS.
引文
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[14] Shinozaki K,Ayajiki K,Kashiwagi A,et al.Malfunction of vascular control in lifestyle-related diseases:mechanisms underlying endothelial dysfunction in the insulin-resistant state[J].J Pharmacol Sci,2004;96(4):401-405.
[15] Qin XP,Ye F,Liao DF,et al.Involvement of calcitonin gene-related peptide in the depressor effects of losartan and perindopril in rats[J].Eur J Pharmacol,2003,464(1):63-67.
[16] 欧奇林,曾泗宇,骆竞妃,等.CGRP通过抑制p38MAPK/NOX4 通路保护氧化应激诱导的血管内皮细胞损伤[J].中南医学科学杂志,2015,43(4):374-378.
[17] Huang J,Stohl LL,Zhou X,et al.Calcitonin gene-related peptide inhibits chemokine production by human dermal microvascular endothelial cells[J].Brain Behav Immun,2011,25(4):787-799.
[18] Zeng SY,Yang L,Hong CL,et al.Evidence that ADAM17 mediates the protective action of CGRP against angiotensin II-Induced inflammation in vascular smooth muscle cells[J].Mediators Inflamm,2018,12;2018:2109352.doi:10.1155/2018/2109352.
[2] 刘明明,李爱玲,修瑞娟.糖尿病微血管内皮细胞功能受损机制研究进展[J].中华糖尿病杂志,2016,8(7):439-442.
[3] Russell FA,King R,Smillie SJ,et al.Calcitonin gene-related peptide:physiology and pathophysiology[J].Physiol Rev,2014,94(4):1099-142.
[4] Walker CS,Hay DL.CGRP in the trigeminovascular system:a role for CGRP,adrenomedullin and amylin receptors [J] B J Pharmacol,2013,170(7):1293-307.
[5] González M,Rojas S,Avila P,et al.Insulin reverses D-glucose-increased nitric oxide and reactive oxygen species generation in human umbilical vein endothelial cells[J].PLoS One,2015,10(4):e0122398.
[6] Takatori S,Zamami Y,Hashikawa-Hobara N,et al.Insulin resistance-induced hypertension and a role of perivascular CGRPergic nerves[J].Curr Protein Pept Sci,2013,14(4):275-281
[7] Li QY,Chen L,Yan MM,et al.Tectorigenin regulates adipogenic differentiation and adipocytokines secretion via PPARgamma and IKK/NF-kappaB signaling[J].Pharmaceul Biol,2015,1-9.
[8] 肖维民,姚尚龙,武庆平,等.人脐静脉内皮细胞胰岛素抵抗细胞模型的建立[J].中国工程组织研究,2012,16(24):4481-4485.
[9] 谌赟,戴忠,刘彦梅,等.Caveolae/caveolin-1/ERK1/2信号通路在降钙素基因相关肽抑制血管平滑肌细胞增殖中的作用[J].生物化学与生物物理进展,2013,40(5):445-453.
[10] 赵守琪,于晓光,潘洪明,等.降钙素基因相关肽对血管内皮细胞凋亡的保护作用[J].齐齐哈尔医学院学报,2006,27(2):133-134.
[11] Andreozzi F,Laratta E,Procopio C,et al.Interleukin-6 impairs the insulin signaling pathway,promoting production of nitric oxide in human umbilical vein endothelial cells[J].Mol Cellular Biol,2007,27(6):2372-2383.
[12] Polikandriotis JA,Mazzella LJ,Rupnow HL,et al.Peroxisome proliferator-activated receptor gamma ligands stimulate endothelial nitric oxide production through distinct peroxisome proliferator-activated receptor gamma-dependent mechanisms[J].Arterioscler Thromb Vasc Biol,2005,25(9):1810-1816.
[13] Merlin J,Sato M,Nowell C,et al.The PPARγ agonist rosiglitazone promotes the induction of brite adipocytes,increasing β-adrenoceptor-mediated mitochondrial function and glucose uptake[J].Cell Signal,2018,42:54-66.
[14] Shinozaki K,Ayajiki K,Kashiwagi A,et al.Malfunction of vascular control in lifestyle-related diseases:mechanisms underlying endothelial dysfunction in the insulin-resistant state[J].J Pharmacol Sci,2004;96(4):401-405.
[15] Qin XP,Ye F,Liao DF,et al.Involvement of calcitonin gene-related peptide in the depressor effects of losartan and perindopril in rats[J].Eur J Pharmacol,2003,464(1):63-67.
[16] 欧奇林,曾泗宇,骆竞妃,等.CGRP通过抑制p38MAPK/NOX4 通路保护氧化应激诱导的血管内皮细胞损伤[J].中南医学科学杂志,2015,43(4):374-378.
[17] Huang J,Stohl LL,Zhou X,et al.Calcitonin gene-related peptide inhibits chemokine production by human dermal microvascular endothelial cells[J].Brain Behav Immun,2011,25(4):787-799.
[18] Zeng SY,Yang L,Hong CL,et al.Evidence that ADAM17 mediates the protective action of CGRP against angiotensin II-Induced inflammation in vascular smooth muscle cells[J].Mediators Inflamm,2018,12;2018:2109352.doi:10.1155/2018/2109352.