从G蛋白偶联受体119表达探讨清热降浊方调控GLP-1分泌的分子机制
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摘要
一、清热降浊方及黄连素对IEC-6细胞株增殖和凋亡的影响及其对IEC-6细胞分泌GLP-1的调控作用
目的：观察清热降浊方及黄连素对IEC-6细胞增殖和凋亡的影响以及对IEC-6细胞释放GLP-1的影响,探讨清热降浊方及黄连素对IEC-6细胞释放GLP-1的影响是否与GPR119激活有关。
方法：采用MTT比色法计算清热降浊方及黄连素干预后的IEC-6细胞存活率,观察清热降浊方对IEC-6细胞增殖的影响；采用Annexin V-FITC和PI双标流式术检测清热降浊方及黄连素对IEC-6细胞凋亡的影响。应用放射免疫法,检测细胞上清液中GLP-1、cAMP含量,观察清热降浊方及黄连素对IEC-6细胞分泌GLP-1的影响及可能的作用途径。
结果：利用MTT法检测后得到的OD值,计算IEC-6细胞存活率,清热降浊方干预24h后,与正常胎牛血清培养相比,低浓度中药复方组细胞存活率显著增加(P<0.01),其中2.5%浓度含药血清组细胞存活率最高(162.42%),故选用2.5%浓度含药血清作为工作浓度。利用WST-1法检测后得到的OD值,计算IEC-6细胞存活率,黄连素干预24h后,与正常胎牛血清培养相比,低浓度黄连素组细胞存活率显著增加,其中1μg/mL黄连素组细胞存活率最高(220.17%),故选用1μg/mL黄连素浓度作为工作浓度。通过流式细胞术检测细胞凋亡率的结果显示,清热降浊方及黄连素干预24h后,不同葡萄糖状态下细胞的凋亡率均明显下降。相比高糖(正常培养,加胎牛血清)组,高糖加含药血清组细胞凋亡率下降了7.92%,高糖加黄连素组细胞凋亡率下降了6.23%；相比低糖(加胎牛血清)组,低糖加含药血清组细胞凋亡率下降了10.92%,低糖加黄连素组细胞凋亡率下降了8.51%；相比超高糖(加胎牛血清)组,超高糖加黄连素组细胞凋亡率下降了9.3%。放免法检测结果显示,高糖加含药血清组GLP-1含量为2.167±0.167pmol/g,高糖加黄连素组为1.870±0.098pmol/g,均明显高于高糖加胎牛血清组的GLP-1含量1.687±0.196pmol/g (P<0.01);低糖加含药血清组GLP-1含量1.942±0.119pmol/g,低糖加黄连素组为1.565±0.020pmol/g,均明显高于低糖加胎牛血清组的GLP-1含量1.441±0.102pmol/g (P<0.01);超高糖加黄连素组GLP-1含量为2.286±0.105pmol/g,明显高于超高糖加胎牛血清组的GLP-1含量1.856±0.015(P<0.01)。高糖加含药血清组cAMP含量为1.820±0.128pmol/mg,高糖加黄连素组为1.683±0.063pmol/mg,均明显高于高糖加胎牛血清组的cAMP含量1.539±0.065pmol/mg (P<0.01)；低糖加含药血清组cAMP含量1.457±0.057pmol/mg,低糖加黄连素组为1.428±0.053pmol/mg,均明显高于低糖加胎牛血清组的cAMP含量1.249±0.046pmol/mg (P<0.01)；超高糖加黄连素组cAMP含量为1.799±0.045pmol/g,明显高于超高糖加胎牛血清组的GLP-1含量1.655±0.069(P<0.01)。
结论：清热降浊方能够促进IEC-6细胞增殖,减少细胞凋亡；促进IEC-6细胞分泌GLP-1,且其促GLP-1分泌作用,与GPR119-cAMP-GLP-1通路激活有关。黄连素在促进IEC-6细胞增殖、减少凋亡,促进GLP-1分泌方面的作用与清热降浊方一致。
二、清热降浊方及黄连素对IEC-6细胞GLP-1和GPR119蛋白及基因表达的影响
目的：从基因、蛋白水平探讨清热降浊方及黄连素促进IEC-6细胞分泌GLP-1作用是否与促进GPR119表达有关。
方法：应用RT-PCR技术检测IEC-6细胞表面GLP-1和GPR119mRNA的表达,根据检测的Ct值结果,计算GLP-1和GPR119mRNA相对表达量；应用Western blot技术检测IEC-6细胞表面GLP-1和GPR119蛋白表达,通过计算相对灰度值,计算GLP-1和GPR119蛋白相对含量。
结果：RT-PCR法检测GLP-1和GPR119mRNA表达,以正常培养(10%胎牛血清)组为基准样品,计算2-ΔΔCt值,比较相对表达量。高糖加复方含药血清组GLP-1mRNA相对表达量为4.90359±0.273842,高糖加黄连素组GLP-1mRNA相对表达量为4.75654±0.244486,超高糖加黄连素组GLP-1mRNA相对表达量为4.95339±0.216146,超高糖加胎牛血清组GLP-1mRNA相对表达量为4.47110±0.199938,低糖加复方含药血清组GLP-1mRNA相对表达量为1.04591±0.163524,低糖加黄连素组GLP-1mRNA相对表达量为0.94792±0.085629,低糖加胎牛血清组GLP-1mRNA相对表达量为0.54016±0.041613。高糖加复方含药血清组GPR119mRNA相对表达量为5.00823±0.294559,高糖加黄连素组GPR119mRNA相对表达量为4.75283±0.307996,超高糖加黄连素组GPR119mRNA相对表达量为4.91934±0.284853,超高糖加胎牛血清组GPR119mRNA相对表达量为4.35557±0.237960,低糖加复方含药血清组GPR119mRNA相对表达量为1.00210±0.107958,低糖加黄连素组GPR119mRNA相对表达量为0.95795±0.075175,低糖加胎牛血清组GPR119mRNA相对表达量为0.44108±0.034275。高糖加复方含药血清组、高糖加黄连素组GLP-1mRNA及GPR119mRNA相对表达量显著高于正常高糖培养组(P<0.01)；低糖加复方含药血清组、低糖加黄连素组GLP-1mRNA及GPR119mRNA相对表达量显著高于低糖加胎牛血清组(P<0.01)；超高糖加黄连素组GLP-1mRNA和GPR119mRNA相对表达量显著高于超高糖加胎牛血清组(P<0.01)。Western blot法检测GLP-1和GPR119蛋白表达,计算目的蛋白与β-actin灰度值之比,结果为,高糖加复方含药血清组GLP-1蛋白相对含量为1.66333±0.074976,高糖加黄连素组GLP-1蛋白相对含量为1.26583±0.025262,高糖加胎牛血清组GLP-1蛋白相对含量为0.90643±0.025290,低糖加复方含药血清组GLP-1蛋白相对含量为0.80650±0.024825,低糖加黄连素组GLP-1蛋白相对含量为0.79833±0.025201,低糖加胎牛血清组GLP-1蛋白相对含量为0.49217±0.043582,超高糖加黄连素组GLP-1蛋白相对含量为1.63833±0.052504,超高糖加胎牛血清组GLP-1蛋白相对含量为1.24667±0.035387。高糖加复方含药血清组GPR119蛋白相对含量为1.59017±0.056912,高糖加黄连素组GPR119蛋白相对含量为1.37133±0.035478,高糖加胎牛血清组GPR119蛋白相对含量为0.77183±0.025725,低糖加复方含药血清组GPR119蛋白相对含量为0.67033±0.040820,低糖加黄连素组GPR119蛋白相对含量为0.66643±0.022541,低糖加胎牛血清组GPR119蛋白相对含量为0.45283±0.024879,超高糖加黄连素组GPR119蛋白相对含量为1.60883±0.057881,超高糖加胎牛血清组GPR119蛋白相对含量为0.98058±0.062956。高糖加复方含药血清组、高糖加黄连素组GLP和GPR119蛋白相对含量显著高于正常高糖培养(高糖加胎牛血清)组(P<0.01)；低糖加复方含药血清组、低糖加黄连素组GLP和GPR119蛋白相对含量显著高于低糖加胎牛血清组(P<0.01)；超高糖加黄连素组GLP和GPR119蛋白相对含量显著高于超高糖加胎牛血清组(P<0.01)。
结论：清热降浊方和黄连素能够促进IEC-6细胞表面GLP-1的表达,其促进表达的机制与清热降浊方及黄连素对细胞表面GPR119表达的上调作用有关。
Part1The effection of Qingrejiangzhuo Decotion and berberine on the proliferation and apoptosis of IEC-6cells,and the effects of Qingrejiangzhuo Decotion and berberine on GLP-1and cAMP secretion of IEC-6cells
Objective:To observe the effection of Qingrejiangzhuo Decotion and berberine on the proliferation and apoptosis of IEC-6cells,and the effects of Qingrejiangzhuo Decotion and berberine on GLP-1secretion of IEC-6cells,and whether the effects is related to the activation of GPR119.
Methods:The proliferation of IEC-6cells was observed by MTT assay.The apoptosis of IEC-6cells was observed through by flow cytometry.The content of GLP-1,GPR119and cAMP was determined by radioimmunoassay.
Results:The survival rate of IEC-6cells was significantly raised after intervention with Qingrejiangzhuo Decotion,comparing with normal culture group. The survival rate of IEC-6cells was highest after intervention with2.5%medicated serum,with a survival rate of162.42%.So2.5%medicated serum was chosed as the working concentration. The survival rate of IEC-6cells was significantly raised after intervention with berberine,comparing with normal culture group. The survival rate of IEC-6cells was highest after intervention with1μg/mLberberine,with a survival rate of220.17%.So1μg/mLberberine solution was chosed as the working concentration.The apotosis rate of IEC-6cells decreased significantly after Qingrejiangzhuo Decotion and berberine intervention.Compared with the normal culture group,the apoptosis rate of DMEM high glucose medium containing medicated serum group decreased by7.92%,the apoptosis rate of DMEM high glucose medium containing berberine group decreased by6.23%.Compared with the DMEM low glucose medium containing fetal bovine serum group,the apoptosis rate of DMEM low glucose medium containing medicated serum group decreased by10.92%,the apoptosis rate of DMEM low glucose medium containing berberine group decreased by8.51%.Compared with the DMEM higer glucose medium containing fetal bovine serum group,the apoptosis rate of DMEM higher glucose medium containing berberine group decreased by9.3%.The content of GLP-1of the DMEM high glucose medium containing medicated serum group was2.167±0.167pmol/g, the DMEM high glucose medium containing berberine group1.870±0.098pmol/g,both significantly higher than the normal culture group(1.687±0.196pmol/g,P<0.01).The content of GLP-1of the DMEM low glucose medium containing medicated serum group was1.942±0.119pmol/g,the DMEM low glucose medium containing berberine group1.565±0.020pmol/g,both significantly higher than the DMEM low glucose medium containing fetal bovine serum group (1.441±0.102pmol/g,P<0.01).The content of GLP-1of the DMEM higher glucose medium containing berberine group2.286±0.105pmol/g, significantly higher than the DMEM higher glucose medium containing fetal bovine serum group (1.856±0.015,P<0.01).The content of cAMP of the DMEM high glucose medium containing medicated serum group was1.820±0.128pmol/mg,the DMEM high glucose medium containing berberine group1.683±0.063pmol/mg,both significantly higher than the normal culture group (1.539±0.065pmol/mg,P<0.01).The content of cAMP of the DMEM low glucose medium containing medicated serum group was1.457±0.057pmol/mg,the DMEM low glucose medium containing berberine group1.428±0.053pmol/mg,both significantly higher than the DMEM low glucose medium containing fetal bovine serum group (1.249±0.046pmol/mg.P<0.01).The content of cAMP of the DMEM higher glucose medium containing berberine group1.799±0.045pmol/g,significantly higher than the DMEM higher glucose medium containing fetal bovine serum group (1.655±0.069,P<0.01)
Conclusion:Qingrejiangzhuo decotion could promote the proliferation of IEC-6cells,reduce the apoptosis of IEC-6cells,and promote IEC-6cells to secrete GLP-1.The promotion effects could be attributed to the activation of GPR119-cAMP-GLP-1pathway.The berberine showed the same effects with Qingrejiangzhuo decotion.
Part2The effection of Qingrejiangzhuo Decotion and berberine on the expression of protein and mRNA of GLP-1and GPR119in IEC-6cells
Objective:To observe the promotion effects of Qingrejiangzhuo decotion and berberine on GLP-1secretion whether can be attributed to the promotion effects on the expression of GPPR119.
Methods:The relative expression of mRNA of GLP-1and GPR119were determined by RT-PCR,calculating2(-Delta Delta C[T]).The relative expression of proteins of GLP-1and GPR119were determined by Western blot.
Results:The relative expression of GLP-1mRNA of the DMEM high glucose medium containing medicated serum group was4.90359±0.273842,the relative expression of GLP-1mRNA of the DMEM high glucose medium containing berberine group was4.75654±0.244486,the relative expression of GLP-1mRNA of the DMEM higher glucose medium containing berberine group was4.95339±0.216146,the relative expression of GLP-1mRNA of the DMEM higher glucose medium containing fetal bovine serum group was4.47110±0.199938,the relative expression of GLP-1mRNA of the DMEM low glucose medium containing medicated serum group was1.04591±0.163524,the relative expression of GLP-1mRNA of the DMEM low glucose medium containing berberine group was0.94792±0.085629,and the relative expression of GLP-1mRNA of the DMEM low glucose medium containing fetal bovine serum group was0.54016±0.041613.The relative expressions of GLP-1mRNA of the DMEM high glucose medium containing medicated serum group and the DMEM high glucose medium containing berberine group were significantly higher than the normal culture group (P<0.01).The relative expressions of GLP-1mRNA of the DMEM low glucose medium containing medicated serum group and the DMEM low glucose medium containing berberine group were significantly higher than the DMEM low glucose medium containing fetal bovine serum group (P<0.01).The relative expression of GLP-1mRNA of the DMEM higher glucose medium containing berberine group was higher than the DMEM higher glucose medium containing fetal bovine serum group (P<0.01).The relative expression of GPR119mRNA of the DMEM high glucose medium containing medicated serum group was5.00823±0.294559,the relative expression of GPR119mRNA of the DMEM high glucose medium containing berberine group was4.75283±0.307996,the relative expression of GPR119mRNA of the DMEM higher glucose medium containing berberine group was4.91934±0.284853,the relative expression of GPR119mRNA of the DMEM higher glucose medium containing fetal bovine serum group was4.35557±0.237960,the relative expression of GPR119mRNA of the DMEM low glucose medium containing medicated serum group was1.00210±0.107958,the relative expression of GPR119mRNA of the DMEM low glucose medium containing berberine group was0.95795±0.075175,and the relative expression of GPR119mRNA of the DMEM low glucose medium containing fetal bovine serum group was0.44108±0.034275.The relative expressions of GPR119mRNA of the DMEM high glucose medium containing medicated serum group and the DMEM high glucose medium containing berberine group were significantly higher than the normal culture group (P<0.01).The relative expressions of GPR119mRNA of the DMEM low glucose medium containing medicated serum group and the DMEM low glucose medium containing berberine group were significantly higher than the DMEM low glucose medium containing fetal bovine serum group(P<0.01).The relative expression of GPR119mRNA of the DMEM higher glucose medium containing berberine group was higher than the DMEM higher glucose medium containing fetal bovine serum group (P<0.01).The relative expression of protein of GLP-1was1.66333±0.074976of the DMEM high glucose medium containing medicated serum group,the relative expression of protein of GLP-1was1.26583±0.025262of the DMEM high glucose medium containing berberine group,the relative expression of protein of GLP-1was0.90643±0.025290of the DMEM high glucose medium containing fetal bovine serum group,the relative expression of protein of GLP-1was1.63833±0.052504of the DMEM higher glucose medium containing berberine group,the relative expression of protein of GLP-1was1.24667±0.035387of the DMEM higher glucose medium containing fetal bovine serum group,the relative expression of protein of GLP-1was0.80650±0.024825of the DMEM low glucose medium containing medicated serum group,the relative expression of protein of GLP-1was0.79833±0.025201of the DMEM low glucose medium containing berberine group,and the relative expression of protein of GLP-1was0.49217±0.043582of the DMEM low glucose medium containing fetal bovine serum group.The protein relative expressions of GLP-1of the DMEM high glucose medium containing medicated serum group and the DMEM high glucose medium containing berberine group were significantly higher than the normal culture group (P<0.01). The protein relative expressions of GLP-1of the DMEM low glucose medium containing medicated serum group and the DMEM low glucose medium containing berberine group were significantly higher than the DMEM low glucose medium containing fetal bovine serum group (P<0.01).The protein relative expression of GLP-1of the DMEM higher glucose medium containing berberine group was higher than the DMEM higher glucose medium containing fetal bovine serum group (P<0.01). The relative expression of protein of GPR119was1.59017±0.056912of the DMEM high glucose medium containing medicated serum group,the relative expression of protein of GPR119was1.37133±0.035478of the DMEM high glucose medium containing berberine group,the relative expression of protein of GPR119was0.77183±0.025725of the DMEM high glucose medium containing fetal bovine serum group,the relative expression of protein of GPR119was1.60883±0.057881of the DMEM higher glucose medium containing berberine group,the relative expression of protein of GPR119was0.98058±0.062956of the DMEM higher glucose medium containing fetal bovine serum group,the relative expression of protein of GPR119was0.67033±0.040820of the DMEM low glucose medium containing medicated serum group,the relative expression of protein of GPR119was0.66643±0.022541of the DMEM low glucose medium containing berberine group,and the relative expression of protein of GPR119was0.45283±0.024879of the DMEM low glucose medium containing fetal bovine serum group.The protein relative expressions of GPR119of the DMEM high glucose medium containing medicated serum group and the DMEM high glucose medium containing berberine group were significantly higher than the normal culture group(P<0.01). The protein relative expressions of GPR119of the DMEM low glucose medium containing medicated serum group and the DMEM low glucose medium containing berberine group were significantly higher than the DMEM low glucose medium containing fetal bovine serum group (P<0.01). The protein relative expression of GPR119of the DMEM higher glucose medium containing berberine group was higher than the DMEM higher glucose medium containing fetal bovine serum group (P<0.01)
Conclusion:Qingrejiangzhuo decotion and berberine can promote the expression of GLP-1in IEC-6cells,and the mechanism of promotion can be attributed to the up-regulation of GPR119expression.
目的：观察清热降浊方及黄连素对IEC-6细胞增殖和凋亡的影响以及对IEC-6细胞释放GLP-1的影响,探讨清热降浊方及黄连素对IEC-6细胞释放GLP-1的影响是否与GPR119激活有关。
方法：采用MTT比色法计算清热降浊方及黄连素干预后的IEC-6细胞存活率,观察清热降浊方对IEC-6细胞增殖的影响；采用Annexin V-FITC和PI双标流式术检测清热降浊方及黄连素对IEC-6细胞凋亡的影响。应用放射免疫法,检测细胞上清液中GLP-1、cAMP含量,观察清热降浊方及黄连素对IEC-6细胞分泌GLP-1的影响及可能的作用途径。
结果：利用MTT法检测后得到的OD值,计算IEC-6细胞存活率,清热降浊方干预24h后,与正常胎牛血清培养相比,低浓度中药复方组细胞存活率显著增加(P<0.01),其中2.5%浓度含药血清组细胞存活率最高(162.42%),故选用2.5%浓度含药血清作为工作浓度。利用WST-1法检测后得到的OD值,计算IEC-6细胞存活率,黄连素干预24h后,与正常胎牛血清培养相比,低浓度黄连素组细胞存活率显著增加,其中1μg/mL黄连素组细胞存活率最高(220.17%),故选用1μg/mL黄连素浓度作为工作浓度。通过流式细胞术检测细胞凋亡率的结果显示,清热降浊方及黄连素干预24h后,不同葡萄糖状态下细胞的凋亡率均明显下降。相比高糖(正常培养,加胎牛血清)组,高糖加含药血清组细胞凋亡率下降了7.92%,高糖加黄连素组细胞凋亡率下降了6.23%；相比低糖(加胎牛血清)组,低糖加含药血清组细胞凋亡率下降了10.92%,低糖加黄连素组细胞凋亡率下降了8.51%；相比超高糖(加胎牛血清)组,超高糖加黄连素组细胞凋亡率下降了9.3%。放免法检测结果显示,高糖加含药血清组GLP-1含量为2.167±0.167pmol/g,高糖加黄连素组为1.870±0.098pmol/g,均明显高于高糖加胎牛血清组的GLP-1含量1.687±0.196pmol/g (P<0.01);低糖加含药血清组GLP-1含量1.942±0.119pmol/g,低糖加黄连素组为1.565±0.020pmol/g,均明显高于低糖加胎牛血清组的GLP-1含量1.441±0.102pmol/g (P<0.01);超高糖加黄连素组GLP-1含量为2.286±0.105pmol/g,明显高于超高糖加胎牛血清组的GLP-1含量1.856±0.015(P<0.01)。高糖加含药血清组cAMP含量为1.820±0.128pmol/mg,高糖加黄连素组为1.683±0.063pmol/mg,均明显高于高糖加胎牛血清组的cAMP含量1.539±0.065pmol/mg (P<0.01)；低糖加含药血清组cAMP含量1.457±0.057pmol/mg,低糖加黄连素组为1.428±0.053pmol/mg,均明显高于低糖加胎牛血清组的cAMP含量1.249±0.046pmol/mg (P<0.01)；超高糖加黄连素组cAMP含量为1.799±0.045pmol/g,明显高于超高糖加胎牛血清组的GLP-1含量1.655±0.069(P<0.01)。
结论：清热降浊方能够促进IEC-6细胞增殖,减少细胞凋亡；促进IEC-6细胞分泌GLP-1,且其促GLP-1分泌作用,与GPR119-cAMP-GLP-1通路激活有关。黄连素在促进IEC-6细胞增殖、减少凋亡,促进GLP-1分泌方面的作用与清热降浊方一致。
二、清热降浊方及黄连素对IEC-6细胞GLP-1和GPR119蛋白及基因表达的影响
目的：从基因、蛋白水平探讨清热降浊方及黄连素促进IEC-6细胞分泌GLP-1作用是否与促进GPR119表达有关。
方法：应用RT-PCR技术检测IEC-6细胞表面GLP-1和GPR119mRNA的表达,根据检测的Ct值结果,计算GLP-1和GPR119mRNA相对表达量；应用Western blot技术检测IEC-6细胞表面GLP-1和GPR119蛋白表达,通过计算相对灰度值,计算GLP-1和GPR119蛋白相对含量。
结果：RT-PCR法检测GLP-1和GPR119mRNA表达,以正常培养(10%胎牛血清)组为基准样品,计算2-ΔΔCt值,比较相对表达量。高糖加复方含药血清组GLP-1mRNA相对表达量为4.90359±0.273842,高糖加黄连素组GLP-1mRNA相对表达量为4.75654±0.244486,超高糖加黄连素组GLP-1mRNA相对表达量为4.95339±0.216146,超高糖加胎牛血清组GLP-1mRNA相对表达量为4.47110±0.199938,低糖加复方含药血清组GLP-1mRNA相对表达量为1.04591±0.163524,低糖加黄连素组GLP-1mRNA相对表达量为0.94792±0.085629,低糖加胎牛血清组GLP-1mRNA相对表达量为0.54016±0.041613。高糖加复方含药血清组GPR119mRNA相对表达量为5.00823±0.294559,高糖加黄连素组GPR119mRNA相对表达量为4.75283±0.307996,超高糖加黄连素组GPR119mRNA相对表达量为4.91934±0.284853,超高糖加胎牛血清组GPR119mRNA相对表达量为4.35557±0.237960,低糖加复方含药血清组GPR119mRNA相对表达量为1.00210±0.107958,低糖加黄连素组GPR119mRNA相对表达量为0.95795±0.075175,低糖加胎牛血清组GPR119mRNA相对表达量为0.44108±0.034275。高糖加复方含药血清组、高糖加黄连素组GLP-1mRNA及GPR119mRNA相对表达量显著高于正常高糖培养组(P<0.01)；低糖加复方含药血清组、低糖加黄连素组GLP-1mRNA及GPR119mRNA相对表达量显著高于低糖加胎牛血清组(P<0.01)；超高糖加黄连素组GLP-1mRNA和GPR119mRNA相对表达量显著高于超高糖加胎牛血清组(P<0.01)。Western blot法检测GLP-1和GPR119蛋白表达,计算目的蛋白与β-actin灰度值之比,结果为,高糖加复方含药血清组GLP-1蛋白相对含量为1.66333±0.074976,高糖加黄连素组GLP-1蛋白相对含量为1.26583±0.025262,高糖加胎牛血清组GLP-1蛋白相对含量为0.90643±0.025290,低糖加复方含药血清组GLP-1蛋白相对含量为0.80650±0.024825,低糖加黄连素组GLP-1蛋白相对含量为0.79833±0.025201,低糖加胎牛血清组GLP-1蛋白相对含量为0.49217±0.043582,超高糖加黄连素组GLP-1蛋白相对含量为1.63833±0.052504,超高糖加胎牛血清组GLP-1蛋白相对含量为1.24667±0.035387。高糖加复方含药血清组GPR119蛋白相对含量为1.59017±0.056912,高糖加黄连素组GPR119蛋白相对含量为1.37133±0.035478,高糖加胎牛血清组GPR119蛋白相对含量为0.77183±0.025725,低糖加复方含药血清组GPR119蛋白相对含量为0.67033±0.040820,低糖加黄连素组GPR119蛋白相对含量为0.66643±0.022541,低糖加胎牛血清组GPR119蛋白相对含量为0.45283±0.024879,超高糖加黄连素组GPR119蛋白相对含量为1.60883±0.057881,超高糖加胎牛血清组GPR119蛋白相对含量为0.98058±0.062956。高糖加复方含药血清组、高糖加黄连素组GLP和GPR119蛋白相对含量显著高于正常高糖培养(高糖加胎牛血清)组(P<0.01)；低糖加复方含药血清组、低糖加黄连素组GLP和GPR119蛋白相对含量显著高于低糖加胎牛血清组(P<0.01)；超高糖加黄连素组GLP和GPR119蛋白相对含量显著高于超高糖加胎牛血清组(P<0.01)。
结论：清热降浊方和黄连素能够促进IEC-6细胞表面GLP-1的表达,其促进表达的机制与清热降浊方及黄连素对细胞表面GPR119表达的上调作用有关。
Part1The effection of Qingrejiangzhuo Decotion and berberine on the proliferation and apoptosis of IEC-6cells,and the effects of Qingrejiangzhuo Decotion and berberine on GLP-1and cAMP secretion of IEC-6cells
Objective:To observe the effection of Qingrejiangzhuo Decotion and berberine on the proliferation and apoptosis of IEC-6cells,and the effects of Qingrejiangzhuo Decotion and berberine on GLP-1secretion of IEC-6cells,and whether the effects is related to the activation of GPR119.
Methods:The proliferation of IEC-6cells was observed by MTT assay.The apoptosis of IEC-6cells was observed through by flow cytometry.The content of GLP-1,GPR119and cAMP was determined by radioimmunoassay.
Results:The survival rate of IEC-6cells was significantly raised after intervention with Qingrejiangzhuo Decotion,comparing with normal culture group. The survival rate of IEC-6cells was highest after intervention with2.5%medicated serum,with a survival rate of162.42%.So2.5%medicated serum was chosed as the working concentration. The survival rate of IEC-6cells was significantly raised after intervention with berberine,comparing with normal culture group. The survival rate of IEC-6cells was highest after intervention with1μg/mLberberine,with a survival rate of220.17%.So1μg/mLberberine solution was chosed as the working concentration.The apotosis rate of IEC-6cells decreased significantly after Qingrejiangzhuo Decotion and berberine intervention.Compared with the normal culture group,the apoptosis rate of DMEM high glucose medium containing medicated serum group decreased by7.92%,the apoptosis rate of DMEM high glucose medium containing berberine group decreased by6.23%.Compared with the DMEM low glucose medium containing fetal bovine serum group,the apoptosis rate of DMEM low glucose medium containing medicated serum group decreased by10.92%,the apoptosis rate of DMEM low glucose medium containing berberine group decreased by8.51%.Compared with the DMEM higer glucose medium containing fetal bovine serum group,the apoptosis rate of DMEM higher glucose medium containing berberine group decreased by9.3%.The content of GLP-1of the DMEM high glucose medium containing medicated serum group was2.167±0.167pmol/g, the DMEM high glucose medium containing berberine group1.870±0.098pmol/g,both significantly higher than the normal culture group(1.687±0.196pmol/g,P<0.01).The content of GLP-1of the DMEM low glucose medium containing medicated serum group was1.942±0.119pmol/g,the DMEM low glucose medium containing berberine group1.565±0.020pmol/g,both significantly higher than the DMEM low glucose medium containing fetal bovine serum group (1.441±0.102pmol/g,P<0.01).The content of GLP-1of the DMEM higher glucose medium containing berberine group2.286±0.105pmol/g, significantly higher than the DMEM higher glucose medium containing fetal bovine serum group (1.856±0.015,P<0.01).The content of cAMP of the DMEM high glucose medium containing medicated serum group was1.820±0.128pmol/mg,the DMEM high glucose medium containing berberine group1.683±0.063pmol/mg,both significantly higher than the normal culture group (1.539±0.065pmol/mg,P<0.01).The content of cAMP of the DMEM low glucose medium containing medicated serum group was1.457±0.057pmol/mg,the DMEM low glucose medium containing berberine group1.428±0.053pmol/mg,both significantly higher than the DMEM low glucose medium containing fetal bovine serum group (1.249±0.046pmol/mg.P<0.01).The content of cAMP of the DMEM higher glucose medium containing berberine group1.799±0.045pmol/g,significantly higher than the DMEM higher glucose medium containing fetal bovine serum group (1.655±0.069,P<0.01)
Conclusion:Qingrejiangzhuo decotion could promote the proliferation of IEC-6cells,reduce the apoptosis of IEC-6cells,and promote IEC-6cells to secrete GLP-1.The promotion effects could be attributed to the activation of GPR119-cAMP-GLP-1pathway.The berberine showed the same effects with Qingrejiangzhuo decotion.
Part2The effection of Qingrejiangzhuo Decotion and berberine on the expression of protein and mRNA of GLP-1and GPR119in IEC-6cells
Objective:To observe the promotion effects of Qingrejiangzhuo decotion and berberine on GLP-1secretion whether can be attributed to the promotion effects on the expression of GPPR119.
Methods:The relative expression of mRNA of GLP-1and GPR119were determined by RT-PCR,calculating2(-Delta Delta C[T]).The relative expression of proteins of GLP-1and GPR119were determined by Western blot.
Results:The relative expression of GLP-1mRNA of the DMEM high glucose medium containing medicated serum group was4.90359±0.273842,the relative expression of GLP-1mRNA of the DMEM high glucose medium containing berberine group was4.75654±0.244486,the relative expression of GLP-1mRNA of the DMEM higher glucose medium containing berberine group was4.95339±0.216146,the relative expression of GLP-1mRNA of the DMEM higher glucose medium containing fetal bovine serum group was4.47110±0.199938,the relative expression of GLP-1mRNA of the DMEM low glucose medium containing medicated serum group was1.04591±0.163524,the relative expression of GLP-1mRNA of the DMEM low glucose medium containing berberine group was0.94792±0.085629,and the relative expression of GLP-1mRNA of the DMEM low glucose medium containing fetal bovine serum group was0.54016±0.041613.The relative expressions of GLP-1mRNA of the DMEM high glucose medium containing medicated serum group and the DMEM high glucose medium containing berberine group were significantly higher than the normal culture group (P<0.01).The relative expressions of GLP-1mRNA of the DMEM low glucose medium containing medicated serum group and the DMEM low glucose medium containing berberine group were significantly higher than the DMEM low glucose medium containing fetal bovine serum group (P<0.01).The relative expression of GLP-1mRNA of the DMEM higher glucose medium containing berberine group was higher than the DMEM higher glucose medium containing fetal bovine serum group (P<0.01).The relative expression of GPR119mRNA of the DMEM high glucose medium containing medicated serum group was5.00823±0.294559,the relative expression of GPR119mRNA of the DMEM high glucose medium containing berberine group was4.75283±0.307996,the relative expression of GPR119mRNA of the DMEM higher glucose medium containing berberine group was4.91934±0.284853,the relative expression of GPR119mRNA of the DMEM higher glucose medium containing fetal bovine serum group was4.35557±0.237960,the relative expression of GPR119mRNA of the DMEM low glucose medium containing medicated serum group was1.00210±0.107958,the relative expression of GPR119mRNA of the DMEM low glucose medium containing berberine group was0.95795±0.075175,and the relative expression of GPR119mRNA of the DMEM low glucose medium containing fetal bovine serum group was0.44108±0.034275.The relative expressions of GPR119mRNA of the DMEM high glucose medium containing medicated serum group and the DMEM high glucose medium containing berberine group were significantly higher than the normal culture group (P<0.01).The relative expressions of GPR119mRNA of the DMEM low glucose medium containing medicated serum group and the DMEM low glucose medium containing berberine group were significantly higher than the DMEM low glucose medium containing fetal bovine serum group(P<0.01).The relative expression of GPR119mRNA of the DMEM higher glucose medium containing berberine group was higher than the DMEM higher glucose medium containing fetal bovine serum group (P<0.01).The relative expression of protein of GLP-1was1.66333±0.074976of the DMEM high glucose medium containing medicated serum group,the relative expression of protein of GLP-1was1.26583±0.025262of the DMEM high glucose medium containing berberine group,the relative expression of protein of GLP-1was0.90643±0.025290of the DMEM high glucose medium containing fetal bovine serum group,the relative expression of protein of GLP-1was1.63833±0.052504of the DMEM higher glucose medium containing berberine group,the relative expression of protein of GLP-1was1.24667±0.035387of the DMEM higher glucose medium containing fetal bovine serum group,the relative expression of protein of GLP-1was0.80650±0.024825of the DMEM low glucose medium containing medicated serum group,the relative expression of protein of GLP-1was0.79833±0.025201of the DMEM low glucose medium containing berberine group,and the relative expression of protein of GLP-1was0.49217±0.043582of the DMEM low glucose medium containing fetal bovine serum group.The protein relative expressions of GLP-1of the DMEM high glucose medium containing medicated serum group and the DMEM high glucose medium containing berberine group were significantly higher than the normal culture group (P<0.01). The protein relative expressions of GLP-1of the DMEM low glucose medium containing medicated serum group and the DMEM low glucose medium containing berberine group were significantly higher than the DMEM low glucose medium containing fetal bovine serum group (P<0.01).The protein relative expression of GLP-1of the DMEM higher glucose medium containing berberine group was higher than the DMEM higher glucose medium containing fetal bovine serum group (P<0.01). The relative expression of protein of GPR119was1.59017±0.056912of the DMEM high glucose medium containing medicated serum group,the relative expression of protein of GPR119was1.37133±0.035478of the DMEM high glucose medium containing berberine group,the relative expression of protein of GPR119was0.77183±0.025725of the DMEM high glucose medium containing fetal bovine serum group,the relative expression of protein of GPR119was1.60883±0.057881of the DMEM higher glucose medium containing berberine group,the relative expression of protein of GPR119was0.98058±0.062956of the DMEM higher glucose medium containing fetal bovine serum group,the relative expression of protein of GPR119was0.67033±0.040820of the DMEM low glucose medium containing medicated serum group,the relative expression of protein of GPR119was0.66643±0.022541of the DMEM low glucose medium containing berberine group,and the relative expression of protein of GPR119was0.45283±0.024879of the DMEM low glucose medium containing fetal bovine serum group.The protein relative expressions of GPR119of the DMEM high glucose medium containing medicated serum group and the DMEM high glucose medium containing berberine group were significantly higher than the normal culture group(P<0.01). The protein relative expressions of GPR119of the DMEM low glucose medium containing medicated serum group and the DMEM low glucose medium containing berberine group were significantly higher than the DMEM low glucose medium containing fetal bovine serum group (P<0.01). The protein relative expression of GPR119of the DMEM higher glucose medium containing berberine group was higher than the DMEM higher glucose medium containing fetal bovine serum group (P<0.01)
Conclusion:Qingrejiangzhuo decotion and berberine can promote the expression of GLP-1in IEC-6cells,and the mechanism of promotion can be attributed to the up-regulation of GPR119expression.
引文
[1]Zimmet P,et al.Global and societal implications of the diabetes epidemic. [J]Nature, 2001,414:782-787.
[2]Wenying Yang,M.D.,Juming,Lu,M.D.,et,al.Prevalence of Diabetes among Men and Women in China.[J]The New England Journal of Medicine,2010,362:1090-101.
[3]UK prospective Diabetes study(UKPD) Group.Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes(UKPDS 34).[J]Lancet,1998,353(9131):854-865.
[4]Wright AD,Cull CA,Macleod KM,et,al.Hypoglycemia in type 2 diabetic patients randomized to and maintained on monotherapy with diet,sulfonylurea,metformin,or insulin for 6 years from diagnosis:UKPDS73.[J]J Diabetes Complications,2006,20(6):395-401.
[5]B T Srinivasan,J Jarvis,K Khunti,M J Davies.Recent advances in the management of type 2diabetes mellitus:a review.[J]Postgrad Med J,2008;84:524-531.
[6]Al-Sabah S, Donnelly D.A model for receptor peptide binding at the glucagon-like peptide 1(GLP-1) receptor through the analysis of truncated ligands and receptors. [J]Br J Pharmacol,2003,140(2):339-346.
[7]Vilsboll T,Zdravkovic M,Le-Thi T,et al.Liraglutide,a long-acting human GLP-1 analog, given as mono therapy significantly improves glycemic control and lowers body weight without risk of hypoglycemia in patients with type 2 diabetes mellitus. [J]Diabetes Care,2007,30(6):1608-1610.
[8]Nauck MA,El-Ouaghlidi A,Gabrys B,et al.Secretion of incretin hormones (GIP and GLP-1) and incretin effect after oral glucose in first-degree relatives of patients with type 2 diabetes.[J]Regulatory Peptides,2004,122(3):209-217.
[9]Kim W,Egan JM.The role of incretins in glucose homeostasis and diabetes treatment.[J]Pharmacol Rev,2008,60(4):470-512.
[10]Arulmozhi DK,Portha B.GLP-1 based therapy for type 2 diabetes.[J]Eur J Pharm Sci,2006,28(1/2):96-108.
[11]Holst JJ.The physiology of glucagon-like peptide-1.[J]Phsio Rev,2007,87(4): 1409-1439.
[12]Edwin T. Parlevliet, Judith E. de Leeuw van Weenen, Johannes A. Romijn,et,al.GLP-1 treatment reduces endogenous insulin resistance via activation of central GLP-1 receptors in mice fed a high-fat diet.[J]Am J Physiol Endocrinol Metab,2010,299:E318-E324.
[13]Meier JJ,Nauck MA,Kranz D,et,al.Secretion,degradation,and elimination of glucagon-like peptide land gastric inhibitory polypeptide in patients with chronic renal insufficiency and healthy control subjects.[J]Dabetes,2004,53(3):654-662.
[14]Klonoff DC, Buse JB, Nielsen LL,et al.Exenatide effects on diabetes, obesity, cardiovascular risk factors and hepatic biomarkers in patients with type 2 diabetes treated for at least 3 years.[J]Curr Med Res Opin,2008,24(1):275-286.
[15]Davies MJ,Donnelly R,Barnett AH,et,al.Exenatide compared with long-acting insulin to achieve glycamic control with minimal weight gain in patients with type 2 diabetes:results of the Helping Evaluate Exenatide in patients with diabetes compared with Long-Acting insulin(HEELA)study.[J]Diabetes Obes Metab,2009,11(12):1153-1162.
[16]Garber A,Heny R,Ratner R,et,al.Liraglutide versus glimepiride monotherapy for ty-pe 2 diabetes(LEAD-3Mono):a randomized,52-week,phase Ⅲ,double-blind, paralleltreatment tri-al. [J]Lancet,2009,373(9662):473-481.
[17]Bosi E,Camisasca RP,Collober C,et al.Effects of vildagliptin on glucose control over 24weeks in patients with type 2 diabetes inadequately controlled with metformin.[J]Diabetes Care,2007,30(4):890-895.
[18]Chia CW,Egan JM.Incretin-based therapies in type 2 diabetes mellitus.[J]J Clin Endocrinol,2008,93(10):3703-3716.
[19]Fonseca V,Schweizer A,Albrecht D,et al. Addition of vildagliptin to insulin improves glycaemic control in type 2 diabetes.[J]Diabetology,2007,50(6):1148-1155.
[20]Scott R, Wu M, Sanchez M,et al.Efficacy and tolerability of the dipepfidyl peptidase-4 inhibitor sitagliptin as monotherapy over 12 weeks in patients with type 2 diabetes.[J]Int J Clin Pract,2007,61(1):171-180.
[21]Jones RM,et al.GPR119 agonists for the treatment of type 2 diabetes. [J]Expert Opin Ther Pat,2009,19:1339-1359.
[22]Soga T,Ohishi T,Matsui T,et al.Lysophosphatidylcholine enhances glucose-dependent insulin secrention via an orphan G-protein-coupled receptor.[J]Biochem Biophy Res Commun, 2005,326 (4):744-751.
[23]Overton HA,Babbs AJ,Doel SM,et al.Deorphanization of a G protein-coupled receptor for oleoylethanolamide and its use in the discovery of small-molecule hypophagic agents.[J]Cell Metab,2006,3(3):167-175.
[24]Chu ZL,Carroll C,Alfonso J,et al.A role for intestinal endocrine cell-expressed G protein-coupled receptor 119 in glycemic control by enhancing glucagon-like peptide-1 and glucose-dependent insulinotropic peptide release.[J]Endocrinology,2008,149(5):2038-2047.
[25]Siddiqui NI,et al.Incretin mimetics and DPP-4 inhibitors:new approach to treatment of type 2 diabetes mellitus.[J]Mymensingh Med J,2009,18:113-124.
[26]Grace Flock,Dianne Holland,Yutaka Seino.GPR119 Regulates Murine Glucose Homeostasis Through Incretin Receptor-Dependent and Independent Mechanisms. [J] Endocrinology,2011,152(2):374-383.
[27]McIntosh CH,et al.Glucose-dependent insulinotropic polypeptide(gastric inhibitory polypeptide; GIP).[J]Vitam Horm,2009,80:409-471.
[28]Chu ZL,et al.A role for beta-cell-expressed G protein-coupled receptor 119 in gly-cemic control by enhancing glucose-dependent insulin release. [J]Endocrinology,2007, 148(6):2601-2609.
[29]Chu ZL,et al.Noleoyldopamine enhances glucose homeostasis through the activation of GPR119.[J]Mol Endocrinol,2009,24:161-170.
[30]Madiraju SR,et al.G protein-coupled receptors and insulin secretion:119 and counting.[J]Endocrinology,2007,148:2598-2600.
[31]Ansarullah,Yan Lu,Martha Holstein,et al.Stimulating b-Cell Regeneration by Combining a GPR119 Agonist with a DPP-IV Inhibitor.[J]PLOS ONS,2013,8(1):e53345.
[32]Bonini JA,Borowsky BE,Adham N,et al.Methods of identifying compounds that bind to SNORF25 receptors.[J]US patent,2002,6,468:756-B1.
[33]Griffin G.Methods for identification of modulators of OSGPR116 activity.[J]US patent 2006,7,083:933-B 1.
[34]Overton HA,et al.GPR119,a novel G protein-coupled receptor target for the treatment of type 2 diabetes and obesity.[J]Br J Pharmacol,2008,153(Suppl 1):S76-81.
[35]Soga T,Ohishi T,Matsui T,Saito T,Matsumoto M,Takasaki J et al. Lysophosphatidylc-holine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled recept-or. [J]Biochem Biophys Res Commun,2005,326:744-751.
[36]Jones RM,Semple G,Fioravanti B,Pereira G,Calderon I,et al.1,2,3-trisubstituted aryl and heteroaryl derivatives as modulators of metabolism and the prophylaxis and treatment of disorders related thereto such as diabetes and hyperglycaemia.[J]International Patent Publication,2004,WO 2004/065380.
[37]Semple G,Fioravanti B,Pepeira RP,et al.Discovery of the first potent and orally eff-icacious agonist of the orphan G-protein coupled receptor119.[J]J Med Chem,2008,51(17): 5172-5174.
[38]Fyfe M,Babbs AJ,Bertram LS,et al.Synthesis,SAR,and in viov efficacy of novel GPR119 agonist with a 4-[3-(4-methanesulfinylphenoxy) propyl]-1-Boc-piperidine core.[C] American Chemical Society National Meeting,Boston,MA,USA,2007,234:MEDI 062.
[39]Dhayal S,Morgan NG.The significance of GPR119 agonists as a future treatment for type 2 diabetes.[J]Drug News Perspect,2010,23(7):418-424.
[40]Mascitti V,Stevens BD,Choi C,et al.Design and evaluation of a 2-(2,3,6-trifluorope-nyl) acetamide derivative as an agonist of the GPR119 receptor.[J]Bioorg Med Chem,2011, 21(9):1306-1309.
[41]Chu ZL,Carrol C,Chen RP,et al.N-oleoyldopamine enhances glucose homeostasis through the activation of GPR119.[J]Mol Endocrinol,2010,24(1):161-170.
[42]Lina M.Lauffer,Roman Iakoubov,Patricia L.Brubaker.GPR119 is essential for Oleoylethanolamide-Induced glucagon-like peptide-1 secretion from the intestinal enteroendocrine L-Cell.[J]Diabetes,2009,58(5):1058-1066.
[43]Katrine B. Hansen, Mette M. Rosenkilde, Filip K. Knop,et al.2-Oleoyl Glycerol Is a GPR119 Agonist and Signals GLP-1 Release in Humans.[J]J Clin Endocrinol Metab,2011, 96(9):E1409-E1417.
[44]Kogure R,Toyama K,Hiyamuta S,et al.5-Hydroxy-eicosapentaenoic acid is an endogenous GPR119 agonist and enhances glucose-dependent insulin secretion.[J]Biochem Biophys Res Commun,2011,416(1-2):58-63.
[45]Gao J,Tian L,Weng G,O'Brien TD,et al.Stimulating β-cell replication and improving islet graft function by AR231453,A GPR119 agonist.[J]Transplant Proc,2011,43(9):3217-20.
[46]Katz LB,Gambale JJ,Rothenberg PL,et al.Pharmacokinetics, pharmacodynamics, safety, and tolerability of JNJ-38431055,a novel GPR119 receptor agonist and potential antidiabetes agent,in healthy male subjects.[J]Clin Pharmacol Ther,2011,90(5):685-692.
[47]Katz LB,Gambale JJ,Rothenberg PL,et al.Effects of JNJ-38431055,a novel GPR119 receptor agonist,in randomized,double-blind,placebo-controlled studies in subjects with type 2 diabetes.[J]Diabetes Obes Metab,2012,14(8):709-716.
[48]Oshima H,Yoshida S,Ohishi T,et al.Novel GPR119 agonist AS1669058 potentiates insulin secretion from rat islets and has potent anti-diabetic effects in ICR and diabetic db/db mice.[J]Life Sci,2013,92(2):167-73.
[49]Yoshida S,Ohishi T,Matsui T,et al.Identification of a novel GPR119 agonist, AS 1269574, with in vitro and in vivo glucosestimulated insulin secretion. [J]Biochem Biophys Res Commun,2010,400(3):437-441.
[50]Yoshida S,Ohishi T,Matsui T,et al.Novel GPR119 agonist AS1535907 contributes to first-phase insulin secretion in rat perfused pancreas and diabetic db/db mice.[J]Biochem Biophys Res Commun,2010,402(2):280-285.
[51]Yoshida S,Ohishi T,Matsui T,et al.Discovery and biological evaluation of novel 4-amino-2-phenylpyrimidine derivatives as potent and orally active GPR119 agonists. [J] Bioorg Med Chem Lett,2010,20(17):5235-5246.
[52]Yoshida S,Ohishi T,Matsui T, et al.AS1907417,a novel GPR119 agonist, as an insulinotropic and β-cell preservative agent for the treatment of type 2 diabetes. [J]Bioorg Med Chem Lett,2010,40(17):745-751.
[53]HILL D.Metabolex announces positive results from phasela clinical trial of MBX-2982[EB/OL].[2011-10-12].http://www.metabolex.com/news/Nov122008.html.
[54]Roberts B,Gregoire FM,Karpf DB,et al.MBX-2982,a novel oral GPR119 agnoist for the treatment of type 2 diabetes:results of single & multiple dose studies. [C] American Diabetes Association-69th Meeting,New Orleans;2009.
[55]Wilson MEJohnson J.Oxymethylene aryl compounds and usesthereof:WO, 2009123992.[P]2009-10-08,[2012-10-09].
[56]Unmesh Shah.GPRl 19 agonists:a promising new approach for the treatment of type 2 diabetes and related metabolic disorders.[J]Curr Opin Drug Discov Dev,2009,12(4): 519-532.
[57]Jones RM,Leonard JN,Buzard DJ,et al.GPR119 agonists for the treatment of type 2 diabetes.[J]Expert Opin Ther Pat,2009,19,(10):1339-1359.
[58]Katamreddy SR,Carpenter AJ,Ammala CE,et al.Discovery of 6,7-dihydro-5H-pyrrolo [2,3-a] pyrimidines as orally available G protein-coupled receptor 119 agonists.[J]J Med Che-m, 2012,55(24):10972-10994.
[59]Negoro K,Yonetoku Y,Moritomo A,et al.Synthesis and structure-activity relationship of fused-pyrimidine derivatives as a series of novel GPR119 agonists.[J]Bioorg Med Chem, 2012,20(21):6442-6451.
[60]Negoro K,Yonetoku Y,Misawa-Mukai H,et al.Discovery and biological evaluation of novel 4-amino-2-phenylpyrimidine derivatives as potent and orally active GPR119 agonists.[J] Bioorg Med Chem,2012,20(17):5235-5246.
[61]Negoro K,Yonetoku Y,Maruyama T,et al. Synthesis and structure-activity relationship of 4-amino-2-phenylpyrimidine derivatives as a series of novel GPR119 agonists.[J]Bioorg Med Chem,2012,20(7):2369-2375.
[62]Xia Y,Chackalamannil S,Greenlee WJ,et al.Discovery of a nortropanol derivative as a potent and orally active GPR119 agonist for type 2 diabetes. [J]Bioorg Med Chem,2011, 21(11):3290-3296.
[63]Scott JS,Birch AM,Brocklehurst KJ,et al.Use of small-molecule crystal structures to address solubility in a novel series of G protein coupled receptor 119 agonists:optimization of a lead and in vivo evaluation.[J]J Med Chem,2012,55(11):5361-5379.
[64]Carpenter AJ,Fang J.Chemical compounds and uses:WO,2010014593.[P] 2010-02-04,[2012-10-09].
[1]高钧,卢守四,张蕾等.人参皂苷Re促进胰高血糖素样肽-1分泌的研究.[J]中国药物与临床,2011,11(12)：1383-1385.
[2]周丽嫦,徐艳燕,陈伟标等.大黄素通过激活PPARδ促进胰高血糖素样肽1分泌的研究.[J]中国中医药现代远程教育,2011,9(21)：142-144.
[3]李艳君,欧叶涛,李晓涛等刺五加叶皂苷对2型糖尿病大鼠GLP-1和血糖分泌的影响.[J]解剖科学进展,2003,9(3)：238-239.
[4]杨扬,张义栋,姜吉文.刺五加叶皂苷对GLP-1分泌影响的实验研究[J].福建中医药,2004,35(3)：38-41.
[5]吕秋菊,曹淑强,蒲强红.黄连素对糖尿病大鼠分泌GLP-1的影响.[J]内江科技,2010(12)：64.
[6]俞蕴莉,刘晓东.小檗碱促进胰高血糖素样肽-1分泌及机制研究.[C]第九届全国药物和化学异物代谢学术会议论文集,2009：281-282.
[7]蔡春容.金钗石斛总生物碱对糖尿病动物模型的影响.[D]贵州,遵义医学院,2011.
[8]苗雷.传统中药中二肽基肽酶Ⅳ抑制剂的筛选及体内药效学的初步研究.[D]无锡,江南大学,2008.
[9]陶枫,朱蕴华,姚政等.健脾清化方对糖尿病模型大鼠GLP-1表达的影响.[C]首届国际中西医结合内分泌代谢病学术大会暨糖尿病论坛论文集,2008：664-668.
[10]陶枫,姚政,陆灏,等.健胰方影响2型糖尿病患者肠促胰岛素GLP-1表达的临床观察.[J]中药应用,2007,18(12)：934-936.
[11]Yu YL,Lu SS,Yu S,et al.Huang-lian-jie-du-decoction modulates glucagon-like peptide-1 secretion in diabetic rats.[J]J Ethnopharmacol,2009,124(3):444-449.
[12]余文珍,施红,郑燕芳.石斛合剂对衰老糖尿病大鼠血清GLP-1水平的影响.[J]海南医学院学报,2006,12(3)：196-199.
[13]李艳君,武艳丽,云长海等.消渴汤方剂对2型糖尿病大鼠GLP-1分泌的影响.[J]解剖科学进展,2005,11(1)：43-44,48.
[14]赵振爱.中药与糖尿病胰岛素抵抗.[J]中国误诊学杂志,2007,7(15)：3528-3529.
[1]Francesco R.Is Type 2 Diabetes an Operable Intestinal Disease?[J]Diabetes Care, 2008,31 (S2):S290-S296.
[2]Xiaokang W,Chaofeng M,Lei Han,et,al.Molecular Characterisation of the Faecal Microbiota in Patients with Type II Diabetes. [J]Curr Microbiol,2010,61:69-78.
[3]Shigeru Yoshida,Yoshitaka Kajimoto,Tetsuyuki Yasuda,et al.PDX-1 Induces Differentiation of Intestinal Epithelioid IEC-6 Into Insulin-Producing Cells. [J]Diabetes,2002, 51:2505-2513.
[4]Hideto Kojima,Takaaki Nakamura,Yukihiro Fujita,et,al.Combined Expression of Pancreatic DuodenalHomeobox 1 and Islet Factor 1 Induces Immature Enterocytes to Produce Insulin.[J]Diabetes,2002,51:1398-1408.
[5]袁名泽.从GLP-1探讨清热降浊方有效部位提取物对自发糖尿病大鼠的降糖机制.[D]北京中医药大学,2012.
[6]Jens Juul Holst.The Physiology of Glucagon-like Peptide 1.[J]Physiol Rev,2007,87: 1409-1439.
[7]Min Jin Moon,Sumi Park,Dong-Kyu Kim,et al. Structural and molecular conservation of glucagon-like peptide-1 and its receptor confers selective ligand-receptor interaction. [J] Frontiers in Endocrinology,2012,3:1-9.
[8]Tsujihata Y,Ito Y,Suzuki M,Harada A,Negoro N,Yasuma T,Momose Y,Takeuchi K.TAK-875,an orally available G protein-coupled receptor 40/free fatty acid receptor 1 agonist,enhances glucose-dependent insulin secretion and improves both postprandial and fasting hyperglycemia in type 2 diabetic rats.[J]J Pharmacol Exp Therapeutics,2011, 339(1):228-237.
[9]Jian Luo,Gayathri Swaminath,Sean P.Brown,Jane Zhang,Qi Guo,Michael Chen,Kathy Nguyen,Thanhvien Tran,Lynn Miao,Paul J,Dransfield,Marc Vimolratana, Jonathan B.Houze,Simon Wong,Maria Toteva,Bei Shan,Frank Li,Run Zhang. A Potent Class of GPR40 Full Agonists Engages the EnteroInsular Axis to Promote Glucose Control in Rodents.[J]PLOS ONE,2012,7(1):e46300.
[10]Fujiwara K,Maekawa F,Yada T.Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet beta-cells:mediation by PLC and L-type Ca2+channel and link to insulin release.[J]Am J Physiol Endocrinol Metab,2005,289(4):E670-E677.
[11]Liou A P,Lu XP,Sei Y,Zhao XL,Pechhold S,Carrero RJ,Raybould HE,Wank S.The G-protein-coupled receptors GPR40 directly meditates long-chain fatty acid-induced secretion of cholecystokinin.[J]Gastroenterology,2011,140(3):903-912.
[12]Gotoh C,Hong YH,Roh SG,et al.The regulation of adipogenesis through GPR120.[J] Biochem Biophys Res Commun,2007,354(2):591-597.
[13]Toshiki Tanaka,Susumu Katsuma,Tetsuya Adachi,et al.Free fatty acids induce cholecystokinin secretion through GPR120.[J]Nauyn-Schmiedeberg_s Arch Pharmacol,2008, 377:523-527.
[14]Toshiki Tanaka,Takeaki Yano,Tetsuya Adachi,et al.Cloning and characterization of the rat free fatty acid receptor GPR120:in vivo effect of the natural ligand on GLP-1 secretion and proliferation of pancreatic β cells. [J]Nauyn-Schmiedeberg_s Arch Pharmacol,2008, 377:515-522.
[15]Hyeung-Jin Jang,Zaza Kokrashvili,Michael J.Theodorakis,et,al.Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1.[J]PNAS,2007, 104(38):15069-15074.
[16]Jones RM.GPR119 agonists for the treatment of type 2 diabetes. [J]Expert Opin Ther Pat,2009,19:1339-1359.
[17]Shah U.GPR119 agonists:a promising new approach for the treatment of type 2 di-abetes and related metabolic disorders.[J]Curr Opin Drug Discov Devel,2009,12:519-532.
[18]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C[T])method.[J]Methods,2001,25(4):402-408.
[19]仝小林,毕桂芝,甄仲等.2518例肥胖2型糖尿病中医证型分类研究.[J]世界中西医结合杂志,2008,3(1)：26-28.
[20]Mayer-Davis E.Low-fat diets for diabetes prevention.[J]Diabetes Care,2001,24: 613-614.
[21]仝小林,姬航宇,李敏等.脾瘅新论.[J]中华中医药杂志,2009,24(8)：988-991.
[22]Buchwald H,Avidor Y,Braunwald E,et al.Bariatric surgery:a systematic review and meta-analysis.[J] JAMA,2004,292(14):1724-1737.
[23]Nadja Larsen, Finn K. Vogensen,Frans W. J. van den Berg,et al.Gut Microbiota in Human Adults with Type 2 Diabetes Differs from Non-Diabetic Adults. [J]PLOS ONE,2010, 5(2):e9085.
[24]Ley,R.E.,Tumbaugh,P.J.,Klein,S.&Gordon,J.I.Microbial ecology:human gut microbes associated with obesity. [J]Nature,2006,444:1022-1023.
[25]连凤梅,仝小林,白煜等.中药降糖复方与二甲双胍对照治疗2型糖尿病的临床研究.[J]中国临床药理学杂志,2008,24(6)：501-504.
[26]GAO Z,LENG S,LU F,et al.Effect of berberine on expression of hepatocyte nuclear factor-4alpha in rats with fructose-induced insulin resistance. [J]J Huazhong Univ Sci Tec-hnolog Med Sci,2008,28(3):261-265.
[27]YIN J,GAO Z,LIU D,et al.Berberine improves glucose metabolism through inducti-on of glycolysis.[J]Am J Physiol Endocrinol Metab,2008,294(1):E148-E156.
[28]KO BS,CHOI SB,PARK SK,et al.Insulin sensitizing and insulinotropic action of berberine from Cortidis rhizome.[J]Biol Pharm Bull,2005,28(8):1431-1437.
[29]LU SS,YU YL,ZHU HJ,et al.Berberine promotes glucagon-like peptide-l(7-36) ami-de secretion in streptozotocin-induced diabetic rats.[J]J Endocrinol,2009,200(2):159-165.
[30]HUANG C,ZHANG Y,GONG Z,et al.Berberine inhibits 3T3-L1 adipocyte differentiation through the PPAR-gamma pathway. [J]Biochem Biophys Res Commun,2006, 348(2):571-578.
[31]KO WH,YAO XQ,LAU CW,et al.Vasorelaxant and antiproliferative effects of berberine. [J]Eur J Pharmacol,2000,399(2-3):187-196.
[2]Wenying Yang,M.D.,Juming,Lu,M.D.,et,al.Prevalence of Diabetes among Men and Women in China.[J]The New England Journal of Medicine,2010,362:1090-101.
[3]UK prospective Diabetes study(UKPD) Group.Effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes(UKPDS 34).[J]Lancet,1998,353(9131):854-865.
[4]Wright AD,Cull CA,Macleod KM,et,al.Hypoglycemia in type 2 diabetic patients randomized to and maintained on monotherapy with diet,sulfonylurea,metformin,or insulin for 6 years from diagnosis:UKPDS73.[J]J Diabetes Complications,2006,20(6):395-401.
[5]B T Srinivasan,J Jarvis,K Khunti,M J Davies.Recent advances in the management of type 2diabetes mellitus:a review.[J]Postgrad Med J,2008;84:524-531.
[6]Al-Sabah S, Donnelly D.A model for receptor peptide binding at the glucagon-like peptide 1(GLP-1) receptor through the analysis of truncated ligands and receptors. [J]Br J Pharmacol,2003,140(2):339-346.
[7]Vilsboll T,Zdravkovic M,Le-Thi T,et al.Liraglutide,a long-acting human GLP-1 analog, given as mono therapy significantly improves glycemic control and lowers body weight without risk of hypoglycemia in patients with type 2 diabetes mellitus. [J]Diabetes Care,2007,30(6):1608-1610.
[8]Nauck MA,El-Ouaghlidi A,Gabrys B,et al.Secretion of incretin hormones (GIP and GLP-1) and incretin effect after oral glucose in first-degree relatives of patients with type 2 diabetes.[J]Regulatory Peptides,2004,122(3):209-217.
[9]Kim W,Egan JM.The role of incretins in glucose homeostasis and diabetes treatment.[J]Pharmacol Rev,2008,60(4):470-512.
[10]Arulmozhi DK,Portha B.GLP-1 based therapy for type 2 diabetes.[J]Eur J Pharm Sci,2006,28(1/2):96-108.
[11]Holst JJ.The physiology of glucagon-like peptide-1.[J]Phsio Rev,2007,87(4): 1409-1439.
[12]Edwin T. Parlevliet, Judith E. de Leeuw van Weenen, Johannes A. Romijn,et,al.GLP-1 treatment reduces endogenous insulin resistance via activation of central GLP-1 receptors in mice fed a high-fat diet.[J]Am J Physiol Endocrinol Metab,2010,299:E318-E324.
[13]Meier JJ,Nauck MA,Kranz D,et,al.Secretion,degradation,and elimination of glucagon-like peptide land gastric inhibitory polypeptide in patients with chronic renal insufficiency and healthy control subjects.[J]Dabetes,2004,53(3):654-662.
[14]Klonoff DC, Buse JB, Nielsen LL,et al.Exenatide effects on diabetes, obesity, cardiovascular risk factors and hepatic biomarkers in patients with type 2 diabetes treated for at least 3 years.[J]Curr Med Res Opin,2008,24(1):275-286.
[15]Davies MJ,Donnelly R,Barnett AH,et,al.Exenatide compared with long-acting insulin to achieve glycamic control with minimal weight gain in patients with type 2 diabetes:results of the Helping Evaluate Exenatide in patients with diabetes compared with Long-Acting insulin(HEELA)study.[J]Diabetes Obes Metab,2009,11(12):1153-1162.
[16]Garber A,Heny R,Ratner R,et,al.Liraglutide versus glimepiride monotherapy for ty-pe 2 diabetes(LEAD-3Mono):a randomized,52-week,phase Ⅲ,double-blind, paralleltreatment tri-al. [J]Lancet,2009,373(9662):473-481.
[17]Bosi E,Camisasca RP,Collober C,et al.Effects of vildagliptin on glucose control over 24weeks in patients with type 2 diabetes inadequately controlled with metformin.[J]Diabetes Care,2007,30(4):890-895.
[18]Chia CW,Egan JM.Incretin-based therapies in type 2 diabetes mellitus.[J]J Clin Endocrinol,2008,93(10):3703-3716.
[19]Fonseca V,Schweizer A,Albrecht D,et al. Addition of vildagliptin to insulin improves glycaemic control in type 2 diabetes.[J]Diabetology,2007,50(6):1148-1155.
[20]Scott R, Wu M, Sanchez M,et al.Efficacy and tolerability of the dipepfidyl peptidase-4 inhibitor sitagliptin as monotherapy over 12 weeks in patients with type 2 diabetes.[J]Int J Clin Pract,2007,61(1):171-180.
[21]Jones RM,et al.GPR119 agonists for the treatment of type 2 diabetes. [J]Expert Opin Ther Pat,2009,19:1339-1359.
[22]Soga T,Ohishi T,Matsui T,et al.Lysophosphatidylcholine enhances glucose-dependent insulin secrention via an orphan G-protein-coupled receptor.[J]Biochem Biophy Res Commun, 2005,326 (4):744-751.
[23]Overton HA,Babbs AJ,Doel SM,et al.Deorphanization of a G protein-coupled receptor for oleoylethanolamide and its use in the discovery of small-molecule hypophagic agents.[J]Cell Metab,2006,3(3):167-175.
[24]Chu ZL,Carroll C,Alfonso J,et al.A role for intestinal endocrine cell-expressed G protein-coupled receptor 119 in glycemic control by enhancing glucagon-like peptide-1 and glucose-dependent insulinotropic peptide release.[J]Endocrinology,2008,149(5):2038-2047.
[25]Siddiqui NI,et al.Incretin mimetics and DPP-4 inhibitors:new approach to treatment of type 2 diabetes mellitus.[J]Mymensingh Med J,2009,18:113-124.
[26]Grace Flock,Dianne Holland,Yutaka Seino.GPR119 Regulates Murine Glucose Homeostasis Through Incretin Receptor-Dependent and Independent Mechanisms. [J] Endocrinology,2011,152(2):374-383.
[27]McIntosh CH,et al.Glucose-dependent insulinotropic polypeptide(gastric inhibitory polypeptide; GIP).[J]Vitam Horm,2009,80:409-471.
[28]Chu ZL,et al.A role for beta-cell-expressed G protein-coupled receptor 119 in gly-cemic control by enhancing glucose-dependent insulin release. [J]Endocrinology,2007, 148(6):2601-2609.
[29]Chu ZL,et al.Noleoyldopamine enhances glucose homeostasis through the activation of GPR119.[J]Mol Endocrinol,2009,24:161-170.
[30]Madiraju SR,et al.G protein-coupled receptors and insulin secretion:119 and counting.[J]Endocrinology,2007,148:2598-2600.
[31]Ansarullah,Yan Lu,Martha Holstein,et al.Stimulating b-Cell Regeneration by Combining a GPR119 Agonist with a DPP-IV Inhibitor.[J]PLOS ONS,2013,8(1):e53345.
[32]Bonini JA,Borowsky BE,Adham N,et al.Methods of identifying compounds that bind to SNORF25 receptors.[J]US patent,2002,6,468:756-B1.
[33]Griffin G.Methods for identification of modulators of OSGPR116 activity.[J]US patent 2006,7,083:933-B 1.
[34]Overton HA,et al.GPR119,a novel G protein-coupled receptor target for the treatment of type 2 diabetes and obesity.[J]Br J Pharmacol,2008,153(Suppl 1):S76-81.
[35]Soga T,Ohishi T,Matsui T,Saito T,Matsumoto M,Takasaki J et al. Lysophosphatidylc-holine enhances glucose-dependent insulin secretion via an orphan G-protein-coupled recept-or. [J]Biochem Biophys Res Commun,2005,326:744-751.
[36]Jones RM,Semple G,Fioravanti B,Pereira G,Calderon I,et al.1,2,3-trisubstituted aryl and heteroaryl derivatives as modulators of metabolism and the prophylaxis and treatment of disorders related thereto such as diabetes and hyperglycaemia.[J]International Patent Publication,2004,WO 2004/065380.
[37]Semple G,Fioravanti B,Pepeira RP,et al.Discovery of the first potent and orally eff-icacious agonist of the orphan G-protein coupled receptor119.[J]J Med Chem,2008,51(17): 5172-5174.
[38]Fyfe M,Babbs AJ,Bertram LS,et al.Synthesis,SAR,and in viov efficacy of novel GPR119 agonist with a 4-[3-(4-methanesulfinylphenoxy) propyl]-1-Boc-piperidine core.[C] American Chemical Society National Meeting,Boston,MA,USA,2007,234:MEDI 062.
[39]Dhayal S,Morgan NG.The significance of GPR119 agonists as a future treatment for type 2 diabetes.[J]Drug News Perspect,2010,23(7):418-424.
[40]Mascitti V,Stevens BD,Choi C,et al.Design and evaluation of a 2-(2,3,6-trifluorope-nyl) acetamide derivative as an agonist of the GPR119 receptor.[J]Bioorg Med Chem,2011, 21(9):1306-1309.
[41]Chu ZL,Carrol C,Chen RP,et al.N-oleoyldopamine enhances glucose homeostasis through the activation of GPR119.[J]Mol Endocrinol,2010,24(1):161-170.
[42]Lina M.Lauffer,Roman Iakoubov,Patricia L.Brubaker.GPR119 is essential for Oleoylethanolamide-Induced glucagon-like peptide-1 secretion from the intestinal enteroendocrine L-Cell.[J]Diabetes,2009,58(5):1058-1066.
[43]Katrine B. Hansen, Mette M. Rosenkilde, Filip K. Knop,et al.2-Oleoyl Glycerol Is a GPR119 Agonist and Signals GLP-1 Release in Humans.[J]J Clin Endocrinol Metab,2011, 96(9):E1409-E1417.
[44]Kogure R,Toyama K,Hiyamuta S,et al.5-Hydroxy-eicosapentaenoic acid is an endogenous GPR119 agonist and enhances glucose-dependent insulin secretion.[J]Biochem Biophys Res Commun,2011,416(1-2):58-63.
[45]Gao J,Tian L,Weng G,O'Brien TD,et al.Stimulating β-cell replication and improving islet graft function by AR231453,A GPR119 agonist.[J]Transplant Proc,2011,43(9):3217-20.
[46]Katz LB,Gambale JJ,Rothenberg PL,et al.Pharmacokinetics, pharmacodynamics, safety, and tolerability of JNJ-38431055,a novel GPR119 receptor agonist and potential antidiabetes agent,in healthy male subjects.[J]Clin Pharmacol Ther,2011,90(5):685-692.
[47]Katz LB,Gambale JJ,Rothenberg PL,et al.Effects of JNJ-38431055,a novel GPR119 receptor agonist,in randomized,double-blind,placebo-controlled studies in subjects with type 2 diabetes.[J]Diabetes Obes Metab,2012,14(8):709-716.
[48]Oshima H,Yoshida S,Ohishi T,et al.Novel GPR119 agonist AS1669058 potentiates insulin secretion from rat islets and has potent anti-diabetic effects in ICR and diabetic db/db mice.[J]Life Sci,2013,92(2):167-73.
[49]Yoshida S,Ohishi T,Matsui T,et al.Identification of a novel GPR119 agonist, AS 1269574, with in vitro and in vivo glucosestimulated insulin secretion. [J]Biochem Biophys Res Commun,2010,400(3):437-441.
[50]Yoshida S,Ohishi T,Matsui T,et al.Novel GPR119 agonist AS1535907 contributes to first-phase insulin secretion in rat perfused pancreas and diabetic db/db mice.[J]Biochem Biophys Res Commun,2010,402(2):280-285.
[51]Yoshida S,Ohishi T,Matsui T,et al.Discovery and biological evaluation of novel 4-amino-2-phenylpyrimidine derivatives as potent and orally active GPR119 agonists. [J] Bioorg Med Chem Lett,2010,20(17):5235-5246.
[52]Yoshida S,Ohishi T,Matsui T, et al.AS1907417,a novel GPR119 agonist, as an insulinotropic and β-cell preservative agent for the treatment of type 2 diabetes. [J]Bioorg Med Chem Lett,2010,40(17):745-751.
[53]HILL D.Metabolex announces positive results from phasela clinical trial of MBX-2982[EB/OL].[2011-10-12].http://www.metabolex.com/news/Nov122008.html.
[54]Roberts B,Gregoire FM,Karpf DB,et al.MBX-2982,a novel oral GPR119 agnoist for the treatment of type 2 diabetes:results of single & multiple dose studies. [C] American Diabetes Association-69th Meeting,New Orleans;2009.
[55]Wilson MEJohnson J.Oxymethylene aryl compounds and usesthereof:WO, 2009123992.[P]2009-10-08,[2012-10-09].
[56]Unmesh Shah.GPRl 19 agonists:a promising new approach for the treatment of type 2 diabetes and related metabolic disorders.[J]Curr Opin Drug Discov Dev,2009,12(4): 519-532.
[57]Jones RM,Leonard JN,Buzard DJ,et al.GPR119 agonists for the treatment of type 2 diabetes.[J]Expert Opin Ther Pat,2009,19,(10):1339-1359.
[58]Katamreddy SR,Carpenter AJ,Ammala CE,et al.Discovery of 6,7-dihydro-5H-pyrrolo [2,3-a] pyrimidines as orally available G protein-coupled receptor 119 agonists.[J]J Med Che-m, 2012,55(24):10972-10994.
[59]Negoro K,Yonetoku Y,Moritomo A,et al.Synthesis and structure-activity relationship of fused-pyrimidine derivatives as a series of novel GPR119 agonists.[J]Bioorg Med Chem, 2012,20(21):6442-6451.
[60]Negoro K,Yonetoku Y,Misawa-Mukai H,et al.Discovery and biological evaluation of novel 4-amino-2-phenylpyrimidine derivatives as potent and orally active GPR119 agonists.[J] Bioorg Med Chem,2012,20(17):5235-5246.
[61]Negoro K,Yonetoku Y,Maruyama T,et al. Synthesis and structure-activity relationship of 4-amino-2-phenylpyrimidine derivatives as a series of novel GPR119 agonists.[J]Bioorg Med Chem,2012,20(7):2369-2375.
[62]Xia Y,Chackalamannil S,Greenlee WJ,et al.Discovery of a nortropanol derivative as a potent and orally active GPR119 agonist for type 2 diabetes. [J]Bioorg Med Chem,2011, 21(11):3290-3296.
[63]Scott JS,Birch AM,Brocklehurst KJ,et al.Use of small-molecule crystal structures to address solubility in a novel series of G protein coupled receptor 119 agonists:optimization of a lead and in vivo evaluation.[J]J Med Chem,2012,55(11):5361-5379.
[64]Carpenter AJ,Fang J.Chemical compounds and uses:WO,2010014593.[P] 2010-02-04,[2012-10-09].
[1]高钧,卢守四,张蕾等.人参皂苷Re促进胰高血糖素样肽-1分泌的研究.[J]中国药物与临床,2011,11(12)：1383-1385.
[2]周丽嫦,徐艳燕,陈伟标等.大黄素通过激活PPARδ促进胰高血糖素样肽1分泌的研究.[J]中国中医药现代远程教育,2011,9(21)：142-144.
[3]李艳君,欧叶涛,李晓涛等刺五加叶皂苷对2型糖尿病大鼠GLP-1和血糖分泌的影响.[J]解剖科学进展,2003,9(3)：238-239.
[4]杨扬,张义栋,姜吉文.刺五加叶皂苷对GLP-1分泌影响的实验研究[J].福建中医药,2004,35(3)：38-41.
[5]吕秋菊,曹淑强,蒲强红.黄连素对糖尿病大鼠分泌GLP-1的影响.[J]内江科技,2010(12)：64.
[6]俞蕴莉,刘晓东.小檗碱促进胰高血糖素样肽-1分泌及机制研究.[C]第九届全国药物和化学异物代谢学术会议论文集,2009：281-282.
[7]蔡春容.金钗石斛总生物碱对糖尿病动物模型的影响.[D]贵州,遵义医学院,2011.
[8]苗雷.传统中药中二肽基肽酶Ⅳ抑制剂的筛选及体内药效学的初步研究.[D]无锡,江南大学,2008.
[9]陶枫,朱蕴华,姚政等.健脾清化方对糖尿病模型大鼠GLP-1表达的影响.[C]首届国际中西医结合内分泌代谢病学术大会暨糖尿病论坛论文集,2008：664-668.
[10]陶枫,姚政,陆灏,等.健胰方影响2型糖尿病患者肠促胰岛素GLP-1表达的临床观察.[J]中药应用,2007,18(12)：934-936.
[11]Yu YL,Lu SS,Yu S,et al.Huang-lian-jie-du-decoction modulates glucagon-like peptide-1 secretion in diabetic rats.[J]J Ethnopharmacol,2009,124(3):444-449.
[12]余文珍,施红,郑燕芳.石斛合剂对衰老糖尿病大鼠血清GLP-1水平的影响.[J]海南医学院学报,2006,12(3)：196-199.
[13]李艳君,武艳丽,云长海等.消渴汤方剂对2型糖尿病大鼠GLP-1分泌的影响.[J]解剖科学进展,2005,11(1)：43-44,48.
[14]赵振爱.中药与糖尿病胰岛素抵抗.[J]中国误诊学杂志,2007,7(15)：3528-3529.
[1]Francesco R.Is Type 2 Diabetes an Operable Intestinal Disease?[J]Diabetes Care, 2008,31 (S2):S290-S296.
[2]Xiaokang W,Chaofeng M,Lei Han,et,al.Molecular Characterisation of the Faecal Microbiota in Patients with Type II Diabetes. [J]Curr Microbiol,2010,61:69-78.
[3]Shigeru Yoshida,Yoshitaka Kajimoto,Tetsuyuki Yasuda,et al.PDX-1 Induces Differentiation of Intestinal Epithelioid IEC-6 Into Insulin-Producing Cells. [J]Diabetes,2002, 51:2505-2513.
[4]Hideto Kojima,Takaaki Nakamura,Yukihiro Fujita,et,al.Combined Expression of Pancreatic DuodenalHomeobox 1 and Islet Factor 1 Induces Immature Enterocytes to Produce Insulin.[J]Diabetes,2002,51:1398-1408.
[5]袁名泽.从GLP-1探讨清热降浊方有效部位提取物对自发糖尿病大鼠的降糖机制.[D]北京中医药大学,2012.
[6]Jens Juul Holst.The Physiology of Glucagon-like Peptide 1.[J]Physiol Rev,2007,87: 1409-1439.
[7]Min Jin Moon,Sumi Park,Dong-Kyu Kim,et al. Structural and molecular conservation of glucagon-like peptide-1 and its receptor confers selective ligand-receptor interaction. [J] Frontiers in Endocrinology,2012,3:1-9.
[8]Tsujihata Y,Ito Y,Suzuki M,Harada A,Negoro N,Yasuma T,Momose Y,Takeuchi K.TAK-875,an orally available G protein-coupled receptor 40/free fatty acid receptor 1 agonist,enhances glucose-dependent insulin secretion and improves both postprandial and fasting hyperglycemia in type 2 diabetic rats.[J]J Pharmacol Exp Therapeutics,2011, 339(1):228-237.
[9]Jian Luo,Gayathri Swaminath,Sean P.Brown,Jane Zhang,Qi Guo,Michael Chen,Kathy Nguyen,Thanhvien Tran,Lynn Miao,Paul J,Dransfield,Marc Vimolratana, Jonathan B.Houze,Simon Wong,Maria Toteva,Bei Shan,Frank Li,Run Zhang. A Potent Class of GPR40 Full Agonists Engages the EnteroInsular Axis to Promote Glucose Control in Rodents.[J]PLOS ONE,2012,7(1):e46300.
[10]Fujiwara K,Maekawa F,Yada T.Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet beta-cells:mediation by PLC and L-type Ca2+channel and link to insulin release.[J]Am J Physiol Endocrinol Metab,2005,289(4):E670-E677.
[11]Liou A P,Lu XP,Sei Y,Zhao XL,Pechhold S,Carrero RJ,Raybould HE,Wank S.The G-protein-coupled receptors GPR40 directly meditates long-chain fatty acid-induced secretion of cholecystokinin.[J]Gastroenterology,2011,140(3):903-912.
[12]Gotoh C,Hong YH,Roh SG,et al.The regulation of adipogenesis through GPR120.[J] Biochem Biophys Res Commun,2007,354(2):591-597.
[13]Toshiki Tanaka,Susumu Katsuma,Tetsuya Adachi,et al.Free fatty acids induce cholecystokinin secretion through GPR120.[J]Nauyn-Schmiedeberg_s Arch Pharmacol,2008, 377:523-527.
[14]Toshiki Tanaka,Takeaki Yano,Tetsuya Adachi,et al.Cloning and characterization of the rat free fatty acid receptor GPR120:in vivo effect of the natural ligand on GLP-1 secretion and proliferation of pancreatic β cells. [J]Nauyn-Schmiedeberg_s Arch Pharmacol,2008, 377:515-522.
[15]Hyeung-Jin Jang,Zaza Kokrashvili,Michael J.Theodorakis,et,al.Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1.[J]PNAS,2007, 104(38):15069-15074.
[16]Jones RM.GPR119 agonists for the treatment of type 2 diabetes. [J]Expert Opin Ther Pat,2009,19:1339-1359.
[17]Shah U.GPR119 agonists:a promising new approach for the treatment of type 2 di-abetes and related metabolic disorders.[J]Curr Opin Drug Discov Devel,2009,12:519-532.
[18]Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C[T])method.[J]Methods,2001,25(4):402-408.
[19]仝小林,毕桂芝,甄仲等.2518例肥胖2型糖尿病中医证型分类研究.[J]世界中西医结合杂志,2008,3(1)：26-28.
[20]Mayer-Davis E.Low-fat diets for diabetes prevention.[J]Diabetes Care,2001,24: 613-614.
[21]仝小林,姬航宇,李敏等.脾瘅新论.[J]中华中医药杂志,2009,24(8)：988-991.
[22]Buchwald H,Avidor Y,Braunwald E,et al.Bariatric surgery:a systematic review and meta-analysis.[J] JAMA,2004,292(14):1724-1737.
[23]Nadja Larsen, Finn K. Vogensen,Frans W. J. van den Berg,et al.Gut Microbiota in Human Adults with Type 2 Diabetes Differs from Non-Diabetic Adults. [J]PLOS ONE,2010, 5(2):e9085.
[24]Ley,R.E.,Tumbaugh,P.J.,Klein,S.&Gordon,J.I.Microbial ecology:human gut microbes associated with obesity. [J]Nature,2006,444:1022-1023.
[25]连凤梅,仝小林,白煜等.中药降糖复方与二甲双胍对照治疗2型糖尿病的临床研究.[J]中国临床药理学杂志,2008,24(6)：501-504.
[26]GAO Z,LENG S,LU F,et al.Effect of berberine on expression of hepatocyte nuclear factor-4alpha in rats with fructose-induced insulin resistance. [J]J Huazhong Univ Sci Tec-hnolog Med Sci,2008,28(3):261-265.
[27]YIN J,GAO Z,LIU D,et al.Berberine improves glucose metabolism through inducti-on of glycolysis.[J]Am J Physiol Endocrinol Metab,2008,294(1):E148-E156.
[28]KO BS,CHOI SB,PARK SK,et al.Insulin sensitizing and insulinotropic action of berberine from Cortidis rhizome.[J]Biol Pharm Bull,2005,28(8):1431-1437.
[29]LU SS,YU YL,ZHU HJ,et al.Berberine promotes glucagon-like peptide-l(7-36) ami-de secretion in streptozotocin-induced diabetic rats.[J]J Endocrinol,2009,200(2):159-165.
[30]HUANG C,ZHANG Y,GONG Z,et al.Berberine inhibits 3T3-L1 adipocyte differentiation through the PPAR-gamma pathway. [J]Biochem Biophys Res Commun,2006, 348(2):571-578.
[31]KO WH,YAO XQ,LAU CW,et al.Vasorelaxant and antiproliferative effects of berberine. [J]Eur J Pharmacol,2000,399(2-3):187-196.