黄芪总皂苷对IEC-6细胞增殖及相关蛋白表达的影响
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摘要
目的观察黄芪总皂苷对小肠上皮细胞(IEC-6)迁移和增殖的影响,并从多胺及其调控的细胞增殖相关蛋白(转录因子c-Myc、Rho-GTP酶RhoA和细胞周期依赖激酶Cdk2)表达角度研究其作用机制。方法从黄芪中提取黄芪总皂苷(皂苷含量88.8%)作为受试药,高效液相色谱仪蒸发光检测(HPLC-ELSD)获得受试药色谱图;移液器吸头划痕法检测细胞迁移率;MTT法检测细胞增殖;Western Blot法检测相关蛋白表达。结果黄芪总皂苷(25、50、100 mg·L~(-1))对细胞迁移无明显影响(与空白组比较,P>0.05)。黄芪总皂苷(50 mg·L~(-1)或100 mg·L~(-1))有促进细胞增殖作用(与空白组比较,P <0.05),且能逆转二氟甲基鸟氨酸(DFMO,多胺合成抑制剂)所致的细胞增殖抑制;可提高c-Myc、RhoA和Cdk2蛋白表达,逆转DFMO所致的c-Myc、RhoA和Cdk2蛋白表达抑制。结论黄芪总皂苷可促进IEC-6细胞增殖,其作用机制与影响多胺调控的增殖相关蛋白表达有关。
Objective This study aims to evaluate the effects of total saponins from Astragalus membranaceus on cellmigration and proliferation in intestinal epithelial cells(IEC-6),and to elucidate the potential mechanism throughpolyamines-mediated proliferation related proteins(transcription factor c-Myc,Rho GTPase RhoA and cell cycle-dependent kinase Cdk2). Methods The total saponins(88.8% purity) extracted from Astragalus membranaceus wasused as an administrated drug. High performance liquid chromatography with evaporative light detection(HPLC-ELSD) was performed to obtain the chromatogram of the administrated drug. Pipette tip scratch method was used todetect cell migration. MTT assay was carried out to determine cell proliferation. Western Blot analysis was used toevaluate the expression of proliferation related proteins. Results Treatment with total saponins from Astragalus membranaceus(25,50 and 100 mg·L~(-1)) had no effect on cell migration(P > 0.05,compared with blank group).However,total saponins from Astragalus membranaceus(50 mg·L~(-1)or 100 mg·L~(-1)) significantly promoted cellproliferation(P < 0.05,compared with the blank group),and reversed the inhibition of cell proliferation inducedby Difluoromethylornithine(DFMO, an inhibitor of polyamine synthesis). Total saponins from Astragalus membranaceus could increase the expression of c-Myc,RhoA and Cdk2 protein,and reverse the inhibition of c-Myc, RhoA and Cdk2 protein expression induced by DFMO. Conclusion Total saponins from Astragalus membranaceus promotes cell proliferation in IEC-6 partly through regulating the expression of polyamines-mediatedproliferation related proteins.
Objective This study aims to evaluate the effects of total saponins from Astragalus membranaceus on cellmigration and proliferation in intestinal epithelial cells(IEC-6),and to elucidate the potential mechanism throughpolyamines-mediated proliferation related proteins(transcription factor c-Myc,Rho GTPase RhoA and cell cycle-dependent kinase Cdk2). Methods The total saponins(88.8% purity) extracted from Astragalus membranaceus wasused as an administrated drug. High performance liquid chromatography with evaporative light detection(HPLC-ELSD) was performed to obtain the chromatogram of the administrated drug. Pipette tip scratch method was used todetect cell migration. MTT assay was carried out to determine cell proliferation. Western Blot analysis was used toevaluate the expression of proliferation related proteins. Results Treatment with total saponins from Astragalus membranaceus(25,50 and 100 mg·L~(-1)) had no effect on cell migration(P > 0.05,compared with blank group).However,total saponins from Astragalus membranaceus(50 mg·L~(-1)or 100 mg·L~(-1)) significantly promoted cellproliferation(P < 0.05,compared with the blank group),and reversed the inhibition of cell proliferation inducedby Difluoromethylornithine(DFMO, an inhibitor of polyamine synthesis). Total saponins from Astragalus membranaceus could increase the expression of c-Myc,RhoA and Cdk2 protein,and reverse the inhibition of c-Myc, RhoA and Cdk2 protein expression induced by DFMO. Conclusion Total saponins from Astragalus membranaceus promotes cell proliferation in IEC-6 partly through regulating the expression of polyamines-mediatedproliferation related proteins.
引文
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[9]涂小华,李茹柳,邓娇,等.四君子汤多糖对小肠上皮细胞迁移多胺信号通路钙调控的影响[J].中华中医药杂志,2016,31(5):1665-1673.
[10]王洲,李茹柳,徐颂芬,等.白术糖复合物对IEC-6细胞分化及绒毛蛋白表达的影响[J].中药材,2010,33(6):938-944.
[11]伍婷婷,李茹柳,曾丹,等.白术多糖调控钙离子以促进细胞迁移及E-钙黏蛋白表达的研究[J].中药新药与临床药理,2017,28(2):145-150.
[12]年立全.益气健脾中药提取物对大鼠应激性溃疡及多胺影响的研究[D].广州:广州中医药大学,2013.
[13]郭雪红.黄芪注射液的药理作用及临床应用研究进展[J].中国药房,2015,26(21):3018-3019.
[14]孙雪莲.黄芪苷对胃黏膜损伤的保护作用研究[D].广州:广州中医药大学,2011:35-40.
[15]孙雪莲,黄可儿,匡忠生,等.黄芪皂苷类不同有效组分对大鼠急性胃黏膜损伤的保护作用研究[J].中华中医药杂志,2011,26(5):1181-1185.
[16]时玉霞.四君子汤多糖对IEC-6细胞迁移Ca2+调节相关指标的影响[D].广州:广州中医药大学,2018.
[17]时玉霞,李茹柳,邓娇,等.实时细胞分析法建立小肠上皮细胞生长实验模型[J].中国药理学通报,2018,34(2):290-294.
[18]国家药典委员会.中华人民共和国药典(一部)[M].北京:中国医药科技出版社,2015:302-303.
[19]曾丹,李茹柳,伍婷婷,等.黄芪多糖对IEC-6细胞迁移多胺信号通路相关指标的影响[J].中华中医药杂志,2017,32(8):3447-3452.
[20]LAN L,LI L,RAO J N,et al.Polyamine-modulated expression of c-myc plays a critical role in stimulation of normal intestinal epithelial cell proliferation[J].Am J Physiol Cell Physiol,2005,288:C89-C99.
[21]RAY R M,MCCORMACK S A,JOHNSON L R.Polyamine depletion arrests growth of IEC-6 and Caco-2 cells by different mechanisms[J].Am J Physiol Gastrointest Liver Physiol,2001,281:G37-G43.
[22]GUO H Z,RAY R M,JOHNSON L R.RhoA stimulates IEC-6cell proliferation by increasing polyamine-dependent Cdk2 activity[J].Am J Physiol Gastrointest Liver Physiol,2003,285:G704-G713.
[23]曾丹.黄芪多糖通过多胺和钙离子调节IEC-6细胞迁移的研究[D].广州:广州中医药大学,2017.
[2]WANG J Y.Cellular signaling in rapid intestinal epithelial restitution:implication of polyamines and K+channels[J].Acta Physiologica Sinica,2003,55(4):365-337.
[3]胡灿,李茹柳,莫全毅,等.白术和黄芪不同提取部位对小肠上皮细胞增殖的影响[J].中药新药与临床药理,2010,21(2):156-160.
[4]胡灿,李茹柳,王静,等.黄芪和白术提取物对IEC-6细胞迁移的影响[J].中药新药与临床药理,2011,22(1):60-65.
[5]李茹柳,陶玉珠,温鹏,等.甘草黄酮提取物对小肠上皮细胞IEC-6迁移及多胺的影响[J].中草药,2013,44(19):2722-2726.
[6]李茹柳,曾丹,温鹏,等.党参黄酮提取部位对小肠上皮细胞迁移及多胺的影响[J].中药新药与临床药理,2014,25(5):523-526.
[7]宋厚盼,李茹柳,王一寓,等.白术提取物对IEC-6细胞迁移过程多胺信号通路钙离子调控的影响[J].中华中医药杂志,2014,29(5):1361-1367.
[8]李茹柳,陶玉珠,曾丹,等.党参、甘草糖提取物对小肠上皮细胞迁移多胺信号通路的影响[J].中国药理学通报,2016,32(2):245-251.
[9]涂小华,李茹柳,邓娇,等.四君子汤多糖对小肠上皮细胞迁移多胺信号通路钙调控的影响[J].中华中医药杂志,2016,31(5):1665-1673.
[10]王洲,李茹柳,徐颂芬,等.白术糖复合物对IEC-6细胞分化及绒毛蛋白表达的影响[J].中药材,2010,33(6):938-944.
[11]伍婷婷,李茹柳,曾丹,等.白术多糖调控钙离子以促进细胞迁移及E-钙黏蛋白表达的研究[J].中药新药与临床药理,2017,28(2):145-150.
[12]年立全.益气健脾中药提取物对大鼠应激性溃疡及多胺影响的研究[D].广州:广州中医药大学,2013.
[13]郭雪红.黄芪注射液的药理作用及临床应用研究进展[J].中国药房,2015,26(21):3018-3019.
[14]孙雪莲.黄芪苷对胃黏膜损伤的保护作用研究[D].广州:广州中医药大学,2011:35-40.
[15]孙雪莲,黄可儿,匡忠生,等.黄芪皂苷类不同有效组分对大鼠急性胃黏膜损伤的保护作用研究[J].中华中医药杂志,2011,26(5):1181-1185.
[16]时玉霞.四君子汤多糖对IEC-6细胞迁移Ca2+调节相关指标的影响[D].广州:广州中医药大学,2018.
[17]时玉霞,李茹柳,邓娇,等.实时细胞分析法建立小肠上皮细胞生长实验模型[J].中国药理学通报,2018,34(2):290-294.
[18]国家药典委员会.中华人民共和国药典(一部)[M].北京:中国医药科技出版社,2015:302-303.
[19]曾丹,李茹柳,伍婷婷,等.黄芪多糖对IEC-6细胞迁移多胺信号通路相关指标的影响[J].中华中医药杂志,2017,32(8):3447-3452.
[20]LAN L,LI L,RAO J N,et al.Polyamine-modulated expression of c-myc plays a critical role in stimulation of normal intestinal epithelial cell proliferation[J].Am J Physiol Cell Physiol,2005,288:C89-C99.
[21]RAY R M,MCCORMACK S A,JOHNSON L R.Polyamine depletion arrests growth of IEC-6 and Caco-2 cells by different mechanisms[J].Am J Physiol Gastrointest Liver Physiol,2001,281:G37-G43.
[22]GUO H Z,RAY R M,JOHNSON L R.RhoA stimulates IEC-6cell proliferation by increasing polyamine-dependent Cdk2 activity[J].Am J Physiol Gastrointest Liver Physiol,2003,285:G704-G713.
[23]曾丹.黄芪多糖通过多胺和钙离子调节IEC-6细胞迁移的研究[D].广州:广州中医药大学,2017.