Exendin-4高活性短肽模拟物的筛选及生物活性
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摘要
以细胞表面胰高血糖素样肽(GLP-1)受体为靶标,利用噬菌体筛选技术,通过Exendin-4竞争性洗脱筛选GLP-1受体激动剂,得到了12肽模拟物EPA.细胞增殖实验结果表明:EPA和Exendin-4均可促进胰岛β细胞增殖活性,在40～200μmol/L内,EPA比Exendin-4的活性高5%～25%;在100mmol/L高浓度葡萄糖毒性下,EPA通过抑制bax基因和上调bcl-2基因表达及抑制Caspase-3活性来抑制胰岛β细胞的凋亡;EPA是具有高活性的Exendin-4短肽模拟物,可通过bcl-2/bax…Caspase-3信号通路抑制线粒体的凋亡.
Using the technique of phage screening,we utilized the glucagon-like peptide1 receptor(GLP-1R)of RINm-5 Fcell as the target to screen GLP-1 receptor agonists through competitive elution by Exendin-4 and obtained 12-mer peptide mimics EPA.The experimental results show that EPA and Exendin-4 can promote the proliferative activity of isletβcells,and the activity of EPA is 5%—25% higher than that of Exendin-4 in the range of 40—200μmol/L.EPA inhibits the apoptosis of isletβcells by inhibiting bax gene and up-regulating bcl-2 gene expression,and inhibiting Caspase-3 activity at 100 mmol/L high concentration of glucose toxicity,EPA is a high-activity short-peptide mimics of Exendin-4,which can inhibit the mitochondrial apoptosis through bcl-2/bax…Casepase-3 signaling pathway.
Using the technique of phage screening,we utilized the glucagon-like peptide1 receptor(GLP-1R)of RINm-5 Fcell as the target to screen GLP-1 receptor agonists through competitive elution by Exendin-4 and obtained 12-mer peptide mimics EPA.The experimental results show that EPA and Exendin-4 can promote the proliferative activity of isletβcells,and the activity of EPA is 5%—25% higher than that of Exendin-4 in the range of 40—200μmol/L.EPA inhibits the apoptosis of isletβcells by inhibiting bax gene and up-regulating bcl-2 gene expression,and inhibiting Caspase-3 activity at 100 mmol/L high concentration of glucose toxicity,EPA is a high-activity short-peptide mimics of Exendin-4,which can inhibit the mitochondrial apoptosis through bcl-2/bax…Casepase-3 signaling pathway.
引文
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[12]FAN Yichao,LIU Kun,WANG Qingping,et al.Exendin-4 Protects Retinal Cells from Early Diabetes in Goto-Kakizaki Rats by Increasing the Bcl-2/Bax and Bcl-xL/Bax Ratios and Reducing Reactive Gliosis[J].Mol Vis,2014,20:1557-1568.
[13]LI Yazhou,HANSOTIA T,YUSTA B,et al.Glucagon-Like Peptide-1 Receptor Signaling Modulatesβ-Cell Apoptosis[J].J Biol Chem,2003,278(1):471-478.
[14]HAO Ming,KUANG Hongyu,FU Zheng,et al.Exenatide Prevents High-Glucose-Induced Damage of Retinal Ganglion Cells through a Mitochondrial Mechanism[J].Neurochem Int,2012,61(1):1-6.
[15]YANG Yan,TONG Yuzhen,GONG Meng,et al.Activation of PPARβ/δProtects PancreaticβCells from Palmitate-Induced Apoptosis by Upregulating the Expression of GLP-1Receptor[J].Cell Signal,2014,26(2):268-278.
[16]LI Zhen,ZHOU Zhiguang,HUANG Gan,et al.Exendin-4Protects Mitochondria from Reactive Oxygen Species Induced Apoptosis in Pancreatic Beta Cells[J].PLoS One,2013,8(10):e76172-1-e76172-6.
[17]LI Haotian,JIA Zhiqiang,LI Gang,et al.Neuroprotective Effects of Exendin-4in Rat Model of Spinal Cord Injury via Inhibiting Mitochondrial Apoptotic Pathway[J].Int J Clin Exp Pathol,2015,8(5):4837-4843.
[18]SHEU J J,CHANG Mengwei,WALLACE C G,et al.Exendin-4Protected against Critical Limb Ischemia in Obese Mice[J].Am J Transl Res,2015,7(3):445-459.
[2]CVETKOVIC'R S,PLOSKER G L.Exenatide:A Review of Its Use with Type 2 Diabetes Mellitus(as an Adjunct to Metformin and/or a Sulfonylurea)[J].Drugs,2007,67(6):935-954.
[3]CHEN Desu,LIAO Jiayu,LI Na,et al.A Nonpeptidic Agonist of Glucagon-Like Peptide 1 Receptors with Efficacy in Diabetic db/db Mice[J].Proc Natl Acad Sci U S A,2007,104(3):943-948.
[4]CHAN W C,WHITE P D.Fomc Solide Phase Peptides Synthesis[M].London:Oxford University Press,2000:765-769.
[5]THORENS B,PORRET A,BüHLER L,et al.Cloning and Functional Expression of the Human Islet GLP-1Receptor:Demonstration That Exendin-4Is an Agonist and Exendin-(9-39)an Antagonist of the Receptor[J].Diabetes,1993,42(11):1678-1682.
[6]MONTROSE-RAFIZADEH C,YANG Huan,RODGERS B D,et al.High Potency Antagonists of the Pancreatic Glucagon-Like Peptide-1Receptor[J].J Biol Chem,1997,272(34):21201-21206.
[7]MENTHIERE C S,de,CHAVANIEU A,GRASSY G,et al.Structural Requirements of the N-Terminal Region of GLP-1-[7-37]-NH2for Receptor Interaction and cAMP Production[J].Eur J Med Chem,2004,39(6):473-480.
[8]ZHANG Huinan,LIU Yunhan,GUAN Shaoyu,et al.An Orally Active Allosteric GLP-1Receptor Agonist Is Neuroprotective in Cellular and Rodent Models of Stroke[J].PLoS One,2016,11(2):e0148827-1-e0148827-16.
[9]TUOMILEHTO J,LINDSTROM J,ERIKSSON J G,et al.Prevention of Type 2Diabetes Mellitus by Changes in Lifestyle among Subjects with Impaired Glucose Tolerance[J].N Engl J Med,2001,344(18):1343-1350.
[10]ZHAO Shumei,GAO Hongli,WANG Yongliang,et al.Attenuation of High Glucose-Induced Rat Cardiomyocyte Apoptosis by Exendin-4via Intervention of HO-1/Nrf-2and the PI3K/AKT Signaling Pathway[J].Chin JPhysiol,2017,60(2):89-96.
[11]CHEN Junqin,COUTO F M,MINN A H,et al.Exenatide Inhibitsβ-Cell Apoptosis by Decreasing,Thioredoxin-Interacting Protein[J].Biochem Biophys Res Commun,2006,346(3):1067-1074.
[12]FAN Yichao,LIU Kun,WANG Qingping,et al.Exendin-4 Protects Retinal Cells from Early Diabetes in Goto-Kakizaki Rats by Increasing the Bcl-2/Bax and Bcl-xL/Bax Ratios and Reducing Reactive Gliosis[J].Mol Vis,2014,20:1557-1568.
[13]LI Yazhou,HANSOTIA T,YUSTA B,et al.Glucagon-Like Peptide-1 Receptor Signaling Modulatesβ-Cell Apoptosis[J].J Biol Chem,2003,278(1):471-478.
[14]HAO Ming,KUANG Hongyu,FU Zheng,et al.Exenatide Prevents High-Glucose-Induced Damage of Retinal Ganglion Cells through a Mitochondrial Mechanism[J].Neurochem Int,2012,61(1):1-6.
[15]YANG Yan,TONG Yuzhen,GONG Meng,et al.Activation of PPARβ/δProtects PancreaticβCells from Palmitate-Induced Apoptosis by Upregulating the Expression of GLP-1Receptor[J].Cell Signal,2014,26(2):268-278.
[16]LI Zhen,ZHOU Zhiguang,HUANG Gan,et al.Exendin-4Protects Mitochondria from Reactive Oxygen Species Induced Apoptosis in Pancreatic Beta Cells[J].PLoS One,2013,8(10):e76172-1-e76172-6.
[17]LI Haotian,JIA Zhiqiang,LI Gang,et al.Neuroprotective Effects of Exendin-4in Rat Model of Spinal Cord Injury via Inhibiting Mitochondrial Apoptotic Pathway[J].Int J Clin Exp Pathol,2015,8(5):4837-4843.
[18]SHEU J J,CHANG Mengwei,WALLACE C G,et al.Exendin-4Protected against Critical Limb Ischemia in Obese Mice[J].Am J Transl Res,2015,7(3):445-459.