人参糖肽的结构及其对Aβ_(25-35)处理PC12细胞的抗凋亡作用
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摘要
目的:研究人参糖肽的结构,探讨其对β淀粉样蛋白25-35(Aβ_(25-35))处理PC12细胞凋亡的保护作用,为开发人参抗阿尔茨海默病(AD)药物奠定理论基础。方法:采用反相高效液相色谱法(RP-HPLC)与Q Exactive Orbitrap质谱联用分析人参糖肽结构。将鼠嗜铬细胞瘤PC12细胞分为6组,加入含不同浓度(0、6.25、12.50、25.00、50.00和100.00μmol·L~(-1))Aβ_(25-35)的培养基,采用细胞计数试剂盒8 (CCK-8)法测定PC12细胞的存活率。将PC12细胞分为空白对照组、模型组和给药组,模型组加入含有50.00μmol·L~(-1)Aβ_(25-35)的培养基,给药组加入不同浓度(0.03、0.10、0.30和1.00g·L~(-1))人参糖肽和含有50.00μmol·L~(-1)Aβ_(25-35)的培养基。采用CCK-8法测定PC12细胞的存活率,采用AnnexinⅤ-FITC/PI法测定人参糖肽和Aβ_(25-35)处理后PC12细胞的凋亡率。将PC12细胞分为空白对照组、模型组和人参糖肽给药组,模型组加入含有50.00μmol·L~(-1) Aβ_(25-35)的培养基,给药组加入不同浓度(0.10和0.30g·L~(-1))人参糖肽和含有50.00μmol·L~(-1) Aβ_(25-35)的培养基,采用流式细胞术测定不同细胞周期PC12细胞的百分比。结果:人参糖肽结构分析得到肽链为NLSHYHSGSS、糖基为N1-HexNAc,肽链为SGSSSSSSSEDDGMGR、糖基为S6-HexNAc等20多个人参糖肽结构。当Aβ_(25-35)浓度为50μmol·L~(-1)时,PC12细胞存活率为(55.45±2.34)%;与空白对照组比较,不同浓度Aβ_(25-35)处理组PC12细胞存活率明显降低(P<0.01)。与空白对照组比较,模型组PC12细胞存活率明显降低(P<0.01);与模型组比较,给药组细胞存活率明显升高(P<0.05)。与空白对照组比较,模型组PC12细胞凋亡率升高(P<0.01);与模型组比较,给药组细胞凋亡率降低(P<0.05)。与空白对照组比较,模型组S期PC12细胞百分比升高(P<0.05);与模型组比较,给药组S期PC12细胞百分比降低(P<0.05)。结论:利用质谱法分析得到人参糖肽结构。人参糖肽可有效抑制Aβ_(25-35)处理PC12细胞的凋亡,具有神经细胞保护作用,其抗凋亡活性作用可能与抑制细胞S期阻滞有关。
Objective:To study the structures of ginseng glycopeptides,and to explore the protective effect on the apoptosis of PC12 cells treated with amyloid beta25-35(Aβ_(25-35)),and to lay a theoretical foundation for the development of ginseng anti-Alzheimer's disease(AD)drugs.Methods:The structures of ginseng glycopeptides were analyzed by reversed-phase high performance liquid chromatography coupled with Q Exactive Orbitrap mass spectrometry.The PC12 cells were divided into 6 groups,and treated with the medium including different concentrations(0,6.25,12.50,25.00,50.00,and 100.00μmol·L~(-1))of Aβ_(25-35),and the survival rates of PC12 cells were measured by cell counting kit-8(CCK-8)method.The PC12 cells were divided into blank control group,model group,and ginseng glycopeptides administration group.The medium including 50.00μmol·L~(-1) Aβ_(25-35) was added in model group,and different concentrations(0.03,0.10,0.30,and of 1.00 g·L~(-1))ginseng glycopeptides+50μmol·L~(-1) Aβ_(25-35) were added in administration groups.The survival rates of PC12 cells were measured by CCK-8 method.The apoptotic rates of PC12 cells after treated with glycopeptides and Aβ_(25-35) were detected by AnnexinⅤ-FITC/PI method. The PC12 cells were divided into blank control group, model group(50.00μmol·L~(-1) Aβ_(25-35))and different concentrations(0.10 and 0.30 g· L~(-1))of ginseng glycopeptides administration groups;the percentages of PC12 cells in different cell cycles were tested by flow cytometry.Results:More than 20 glycopeptide structures were obtained,such as the peptide chain was NLSHYHSGSS,the glycosyl group was N1-HexNAc,and the peptide chain was SGSSSSSSSEDDGMGR,the glycosyl group was S6-HexNAc.When the concentration of Aβ_(25-35) was 50μmol·L~(-1),the survival rate of PC12 was(55.45±2.34)%and the survival rate was significantly lower than that in blank control group(P<0.01).Compared with blank control group,the survival rate of the PC12 cells in model group was significantly decreased(P<0.01);compared with model group,the survival rates of the PC12 cells in administration groups were significantly increased(P<0.05).Compared with blank control group,the apoptotic rate of the PC12 cells in model group were increased(P<0.01);compared with model group,the apoptotic rates of the PC12 cells in administration groups were decreased(P<0.05).Compared with blank control group,the percentage of PC12 cells in S phase in model group was increased(P<0.05);compared with model group,the percentages of PC12 cells in S phase in administration groups were decreased(P<0.05).Conclusion:The structures of ginseng glycopeptides are obtained with mass spectrometry.Ginseng glycopeptides can effectively inhibit the apoptosis of PC12 cells treated with Aβ_(25-35),and they have the neuroprotective effect;the anti-apoptotic activity effect may be related to the inhibition of S phase arrest.
Objective:To study the structures of ginseng glycopeptides,and to explore the protective effect on the apoptosis of PC12 cells treated with amyloid beta25-35(Aβ_(25-35)),and to lay a theoretical foundation for the development of ginseng anti-Alzheimer's disease(AD)drugs.Methods:The structures of ginseng glycopeptides were analyzed by reversed-phase high performance liquid chromatography coupled with Q Exactive Orbitrap mass spectrometry.The PC12 cells were divided into 6 groups,and treated with the medium including different concentrations(0,6.25,12.50,25.00,50.00,and 100.00μmol·L~(-1))of Aβ_(25-35),and the survival rates of PC12 cells were measured by cell counting kit-8(CCK-8)method.The PC12 cells were divided into blank control group,model group,and ginseng glycopeptides administration group.The medium including 50.00μmol·L~(-1) Aβ_(25-35) was added in model group,and different concentrations(0.03,0.10,0.30,and of 1.00 g·L~(-1))ginseng glycopeptides+50μmol·L~(-1) Aβ_(25-35) were added in administration groups.The survival rates of PC12 cells were measured by CCK-8 method.The apoptotic rates of PC12 cells after treated with glycopeptides and Aβ_(25-35) were detected by AnnexinⅤ-FITC/PI method. The PC12 cells were divided into blank control group, model group(50.00μmol·L~(-1) Aβ_(25-35))and different concentrations(0.10 and 0.30 g· L~(-1))of ginseng glycopeptides administration groups;the percentages of PC12 cells in different cell cycles were tested by flow cytometry.Results:More than 20 glycopeptide structures were obtained,such as the peptide chain was NLSHYHSGSS,the glycosyl group was N1-HexNAc,and the peptide chain was SGSSSSSSSEDDGMGR,the glycosyl group was S6-HexNAc.When the concentration of Aβ_(25-35) was 50μmol·L~(-1),the survival rate of PC12 was(55.45±2.34)%and the survival rate was significantly lower than that in blank control group(P<0.01).Compared with blank control group,the survival rate of the PC12 cells in model group was significantly decreased(P<0.01);compared with model group,the survival rates of the PC12 cells in administration groups were significantly increased(P<0.05).Compared with blank control group,the apoptotic rate of the PC12 cells in model group were increased(P<0.01);compared with model group,the apoptotic rates of the PC12 cells in administration groups were decreased(P<0.05).Compared with blank control group,the percentage of PC12 cells in S phase in model group was increased(P<0.05);compared with model group,the percentages of PC12 cells in S phase in administration groups were decreased(P<0.05).Conclusion:The structures of ginseng glycopeptides are obtained with mass spectrometry.Ginseng glycopeptides can effectively inhibit the apoptosis of PC12 cells treated with Aβ_(25-35),and they have the neuroprotective effect;the anti-apoptotic activity effect may be related to the inhibition of S phase arrest.
引文
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[18]BRADLEYWHITMAN M A,LOVELL M A.Biomarkers of lipid peroxidation in Alzheimer disease(AD):an update[J].Arch Toxicol,2015,89(7):1035-1044.
[19]吕京南,罗浩铭,陈英红,等.人参非皂苷成分中具有治疗老年痴呆作用的活性成分筛选及其结构表征[J].时珍国医国药,2015,26(11):2561-2563.
[20]QI S,CALVI B R.Different cell cycle modifications repress apoptosis at different steps independent of developmental signaling in Drosophila[J].Mol Biol Cell,2016,27(12):1885-1897.
[21]COUNTS S E,MUFSON E J.Regulator of cell cycle(RGCC)expression during the progression of Alzheimer’s disease[J].Cell Transplant,2016,26(4):693-702.
[22]KUHLA A,LUDWIG S C,KUHLA B,et al.Advanced glycation end products are mitogenic signals and trigger cell cycle reentry of neurons in Alzheimer’s disease brain[J].Neurobiol Aging,2015,36(2):753-761.
[23]SUN S,XIE F,XU X,et al.Advanced oxidation protein products induce S-phase arrest of hepatocytes via the ROS-dependent,β-catenin-CDK2-mediated pathway[J].Redox Biol,2018,14:338-353.
[24]JI X J,HUMENIK J,YANG D,et al.PolyC-binding proteins enhance expression of the CDK2cell cycle regulatory protein via alternative splicing[J].Nucleic Acids Res,2018,46(4):2030-2044.
[25]安明,赵国君,韦新成,等.人参皂苷Rg1保护心血管和中枢神经系统的药理活性研究进展[J].中国临床药理学杂志,2012,28(1):75-77.
[26]LUO J H,WARMLANDER S K,GRASLUND A,et al.Non-chaperone proteins can inhibit aggregation and cytotoxicity of Alzheimer amyloidβpeptide[J].J Biol Chem,2014,289(40):27766-27775.
[27]MCCLEAN P L,PARTHSARATHY V,FAIVRE E,et al.The diabetes drug liraglutide prevents degenerative processes in a mouse model of Alzheimer’s disease[J].J Neurosci,2011,31(17):6587-6594.
[2]GRAHAM W V,BONITOOLIVA A,SAKMAR T P.Update on Alzheimer’s disease therapy and prevention strategies[J].Annu Rev Med,2017,68:413-430.
[3]THEOFILAS P,EHRENBERG A J,NGUY A,et al.Probing the correlation of neuronal loss,neurofibrillary tangles,and cell death markers across the Alzheimer’s disease Braak stages:aquantitative study in humans[J].Neurobiol Aging,2018,61:1-12.
[4]MINTER M R,TAYLOR J M,CRACK P J.The contribution of neuroinflammation to amyloid toxicity in Alzheimer’s disease[J].J Neurochem,2016,136(3):457-474.
[5]TAGA M,MINETT T,CLASSEY J,et al.Metaflammasome components in the human brain:a role in dementia with Alzheimer’s pathology?[J].Brain Pathol,2017,27(3):266-275.
[6]AWASTHI M,SINGH S,PANDEY V P,et al.Alzheimer’s disease:An overview of amyloid beta dependent pathogenesis and its therapeutic implications along with in silico approaches emphasizing the role of natural products[J].J Neurol Sci,2016,361:256-271.
[7]ONUKI R,BANDO Y,SUYAMA E,et al.An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer’s disease[J].EMBO J,2004,23(4):959-968.
[8]HALAWA A A,DAMBORSKY J C,SLATON G S,et al.Activation of immediate early genes by nicotine after chronic neonatal nicotine exposure in brain areas involved in stress and anxiety responses[J].Brain Res,2018,1687:32-40.
[9]MAJEED F,MALIK F Z,AHMED Z,et al.Ginseng phytochemicals as therapeutics in oncology:Recent perspectives[J].Biomed Pharmacother,2018,100:52-63.
[10]陈文学,杨铭,于德伟,等.人参糖肽对糖尿病气阴两虚证大鼠降血糖作用及其机制研究[J].中国药学杂志,2014,49(21):1903-1907.
[11]罗浩铭,王颖,陈英红,等人参糖蛋白对Aβ25~35诱导SH-SY5Y细胞凋亡的影响[J].中国老年学杂志,2016,36(24):6077-6080.
[12]王颖,白珺,宋伍,等.人参糖蛋白降低Aβ25-35诱导SH-SY5Y细胞凋亡中一氧化氮释放的保护作用[J].吉林中医药,2017(5):497-500.
[13]罗浩铭,陈英红,周婷婷,等.人参糖蛋白对小鼠学习和记忆能力的影响[J].吉林大学学报:医学版,2016,42(3):439-445.
[14]LUO H M,HU J T,WANG Y,et al.In vivo and in vitro neuroprotective effects of Panax ginseng glycoproteins[J].Int J Biol Macromol,2018,113:607-615.
[15]DEAN D N,PATE K M,MOSS M A,et al.Conformational dynamics of specific Aβoligomers govern their ability to replicate and induce neuronal apoptosis[J].Biochemistry,2016,55(15):2238-2250.
[16]ALTUNOGLU E,GUNTAS G,ERDENEN F,et al.Ischemia-modified albumin and advanced oxidation protein products as potential biomarkers of protein oxidation in Alzheimer’s disease[J].Geriatr Gerontol Int,2015,15(7):872-880.
[17]AKKAYA,YAVUZER S S,YAVUZER H,et al.DNAdamage,DNA susceptibility to oxidation and glutathione redox status in patients with Alzheimer’s disease treated with and without memantine[J].J Neurol Sci,2017,378:158-162.
[18]BRADLEYWHITMAN M A,LOVELL M A.Biomarkers of lipid peroxidation in Alzheimer disease(AD):an update[J].Arch Toxicol,2015,89(7):1035-1044.
[19]吕京南,罗浩铭,陈英红,等.人参非皂苷成分中具有治疗老年痴呆作用的活性成分筛选及其结构表征[J].时珍国医国药,2015,26(11):2561-2563.
[20]QI S,CALVI B R.Different cell cycle modifications repress apoptosis at different steps independent of developmental signaling in Drosophila[J].Mol Biol Cell,2016,27(12):1885-1897.
[21]COUNTS S E,MUFSON E J.Regulator of cell cycle(RGCC)expression during the progression of Alzheimer’s disease[J].Cell Transplant,2016,26(4):693-702.
[22]KUHLA A,LUDWIG S C,KUHLA B,et al.Advanced glycation end products are mitogenic signals and trigger cell cycle reentry of neurons in Alzheimer’s disease brain[J].Neurobiol Aging,2015,36(2):753-761.
[23]SUN S,XIE F,XU X,et al.Advanced oxidation protein products induce S-phase arrest of hepatocytes via the ROS-dependent,β-catenin-CDK2-mediated pathway[J].Redox Biol,2018,14:338-353.
[24]JI X J,HUMENIK J,YANG D,et al.PolyC-binding proteins enhance expression of the CDK2cell cycle regulatory protein via alternative splicing[J].Nucleic Acids Res,2018,46(4):2030-2044.
[25]安明,赵国君,韦新成,等.人参皂苷Rg1保护心血管和中枢神经系统的药理活性研究进展[J].中国临床药理学杂志,2012,28(1):75-77.
[26]LUO J H,WARMLANDER S K,GRASLUND A,et al.Non-chaperone proteins can inhibit aggregation and cytotoxicity of Alzheimer amyloidβpeptide[J].J Biol Chem,2014,289(40):27766-27775.
[27]MCCLEAN P L,PARTHSARATHY V,FAIVRE E,et al.The diabetes drug liraglutide prevents degenerative processes in a mouse model of Alzheimer’s disease[J].J Neurosci,2011,31(17):6587-6594.
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