食品与发酵工业 >

2024 , Vol. 50 >Issue 8: 17 - 23

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.035243

研究报告

佛手多糖对1-甲基-4-苯基-吡啶离子诱导人神经母细胞瘤(SH-SY5Y)细胞损伤的保护作用研究

  • 陈进炫 ,
  • 龚舒 ,
  • 刘天开 ,
  • 龚记熠 ,
  • 乙引 ,
  • 刘文华
展开
  • 1(贵州师范大学 生命科学学院,贵州 贵阳,550025)
    2(肇庆学院 生命科学学院,广东 肇庆,526061)
第一作者:硕士研究生(乙引教授和刘文华教授为共同通信作者,E-mail:100236417@qq.com;wenhualiu@hotmail.com)

收稿日期: 2023-02-24

  修回日期: 2023-03-27

  网络出版日期: 2024-06-11

基金资助

国家自然科学基金(31271124);广东省教育厅创新团队项目(2015KCXTD032);广东省普通高校青年创新人才项目(2018KQNCX291)

收起

Protective effect of bergamot polysaccharide on SH-SY5Y cells injured by 1-methyl-4-phenyl-pyridine ion

  • CHEN Jinxuan ,
  • GONG Shu ,
  • LIU Tiankai ,
  • GONG Jiyi ,
  • YI Yin ,
  • LIU Wenhua
Expand
  • 1(School of Life Science, Guizhou Normal University, Guiyang 550025, China)
    2(School of Life Science, Zhaoqing University, Zhaoqing 526061, China)

Received date: 2023-02-24

  Revised date: 2023-03-27

  Online published: 2024-06-11

Fold

摘要

该文探究佛手多糖对1-甲基-4-苯基-吡啶离子(1-methyl-4-phenyl-pyridine ion, MPP+)诱导人神经母细胞瘤(SH-SY5Y)细胞损伤的保护作用及其机制。佛手多糖经大孔吸附树脂AB-8进行纯化。体外培养SH-SY5Y细胞,构建帕金森病(Parkinson's disease, PD)细胞模型,实验分为对照组、MPP+模型组、佛手多糖组。采用噻唑蓝(methye thiazdye telrazlium, MTT)法检测细胞存活率,Hoechst33258染色法观察细胞形态,2',7'-二氯荧光黄双乙酸盐荧光探针检测细胞活性氧(reactive oxygen species, ROS)水平,JC-1荧光探针法检测线粒体膜电位,蛋白免疫印迹(Western blot)检测磷酸化蛋白激酶B(phosphorylated protein kinase B, p-Akt)、蛋白激酶B(protein kinase B, Akt)、磷酸化细胞外调节蛋白激酶(phosphorylated extracellular regulated protein kinases1/2, p-ERK1/2)和细胞色素c(cytochrome c, Cyt-c)蛋白表达水平。结果表明,佛手多糖的得率4.86%,纯度为44.46%,经过AB-8纯化后,纯度提高到60.81%;与对照组相比,模型组细胞的存活率显著降低,Hoechst33258染色下可见细胞破碎,细胞核皱缩,细胞内ROS显著增加,线粒体膜电位显著降低。与模型组相比,佛手多糖组的细胞存活率显著增加,细胞形态明显得到改善,ROS水平下降,线粒体膜电位升高。Western blot结果显示,佛手多糖能抑制MPP+引起的p-Akt和p-ERK1/2的降低,以及Cyt-c的上升。综上,佛手多糖对MPP+诱导SH-SY5Y细胞损伤具有保护作用,其机制可能是通过调节线粒体ROS的产生和Cyt-c的释放,进而维持线粒体稳态,激活Akt信号通路和ERK信号通路,抑制细胞的凋亡,从而起到保护作用。研究结果可为缓解帕金森病的发生发展提供理论依据,同时也能更好地开发和利用佛手资源。

关键词： 佛手多糖; 提取纯化; MPP+; SH-SY5Y细胞; 保护作用

本文引用格式

陈进炫 , 龚舒 , 刘天开 , 龚记熠 , 乙引 , 刘文华 . 佛手多糖对1-甲基-4-苯基-吡啶离子诱导人神经母细胞瘤(SH-SY5Y)细胞损伤的保护作用研究[J]. 食品与发酵工业, 2024 , 50(8) : 17 -23 . DOI: 10.13995/j.cnki.11-1802/ts.035243

Abstract

This study aimed to explore the protective effect and mechanism of bergamot polysaccharide on damaged SH-SY5Y cells induced by 1-methyl-4-phenyl-pyridine ion (MPP+).The bergamot polysaccharide was extracted and purified with AB-8 macroporous resin.SH-SY5Y cells were cultured in vitro to construct a Parkinson's disease (PD) cell model.The experiment was divided into a control group, the MPP+ model group, and the bergamot polysaccharide group.The cell viability was detected with the methye thiazdye telrazlium (MTT) method, and the cell morphology was observed by the Hoechst33258 staining method.The reactive oxygen species (ROS) level was detected by the DCFH-DA fluorescence probe, and mitochondrial membrane potential was measured by the JC-1 fluorescence probe method.The protein expression levels of phosphorylated protein kinase B (p-Akt), protein kinase B (Akt), phosphorylated extracellular regulated protein kinases1/2 (p-ERK1/2), and cytochrome c (Cyt-c) were detected by Western blot.Results showed that the yield of bergamot polysaccharide was 4.86% and the purity was 44.46%.After purification by AB-8, the purity of the polysaccharide was increased to 60.81%.Compared with the control group, the cell viability of the MPP+-treated group was significantly reduced, the cells were broken and the nucleus was shrunk after Hoechst33258 staining.Meanwhile, the intracellular ROS was significantly increased, and the mitochondrial membrane potential was significantly decreased.Compared with the model group, the cell viability of the bergamot polysaccharide group was significantly increased, the cell morphology was significantly improved, the enhancement of intracellular ROS was inhibited, and the mitochondrial membrane potential was increased.The results of the Western blot showed that bergamot polysaccharide could inhibit the decrease of p-Akt and p-ERK1/2 as well as the increase of Cyt-c caused by MPP+.Based on these data, bergamot polysaccharide had a protective effect on SH-SY5Y cells against the injury induced by MPP+, which might be correlated with antioxidant activity as well as ERK and Akt activation pathway.The research results could provide a theoretical basis for alleviating the occurrence and development of Parkinson's disease, and be also beneficial for the development and utilization of bergamot resources.

Key words： bergamot polysaccharide; extraction and purification; 1-methyl-4-phenyl-pyridine ion (MPP+); SH-SY5Y cells; protective effect

参考文献

[1] 岳玲, 程轩轩, 唐晓敏, 等.佛手的传统应用[J].中国实验方剂学杂志, 2019, 25(4):206-211.
YUE L, CHENG X X, TANG X M, et al.Traditional application of citri sarcodactylis fructus[J].Chinese Journal of Experimental Traditional Medical Formulae, 2019, 25(4):206-211.
[2] 彭宝, 文瑶, 于荣敏, 等.佛手多糖提取、结构表征及生物活性研究进展[J].食品与药品, 2018, 20(3):236-241.
PENG B, WEN Y, YU R M, et al.Advances in extraction, structure characterization and bioactivities of Citrus medica polysaccharide[J].Food and Drug, 2018, 20(3):236-241.
[3] 曹诣斌, 朱海玲, 王晓艳.不同产地佛手水溶性多糖的分离纯化及初步分析[J].浙江师范大学学报(自然科学版), 2008, 31(2):190-194.
CAO Y B, ZHU H L, WANG X Y.Preliminary analysis on purification of the water-soluble polysaccharides in bergamots from different areas[J].Journal of Zhejiang Normal University (Natural Sciences), 2008, 31(2):190-194.
[4] 李小凤, 蔡春, 程荷凤, 等.广佛手多糖的分离纯化与相关成分的气相色谱分析[J].广东医学院学报, 2005(3):240-241;259.
LI X F, CAI C, CHENG H F, et al.Isolation and purification of polysaccharide from fructus of Citri medica and analysis of its chemical composition by gas chromatography[J].Journal of Guangdong Medical College, 2005(3):240-241;259.
[5] 王淑惠, 杨玉洁, 周爱梅, 等.两种方法提取佛手渣多糖及其对巨噬细胞RAW264.7免疫调节活性的研究[J].食品工业科技, 2020, 41(15):179-187.
WANG S H, YANG Y J, ZHOU A M, et al.Study on the polysaccharide extracted from bergamot (Citrus medica L.var.sarcodactylis) residue by two methods and its immunomodulatory function in RAW264.7 cells[J].Science and Technology of Food Industry, 2020, 41(15):179-187.
[6] 邹胜. 佛手多糖的分离纯化及抗氧化活性研究[D].重庆:重庆大学, 2015.
ZOU S.Extraction and separation of polysaccharides from bergamot and their antioxidant activities in vitro[D].Chongqing:Chongqing University, 2015.
[7] 黄玲, 邝枣园. 佛手多糖对小鼠移植性肝肿瘤HAC22的抑制作用. 江西中医学院学报, 2000, 12(1):41-47.
HUANG L, KUANG Z Y. Inhibitory effect of bergamot polysaccharide on transplanted liver tumor HAC22 in mice. Journal of Jiangxi College of Traditional Chinese Medicine, 2000, 12(1):41-47.
[8] 袁惠莉, 汪璇, 张丽娟, 等.中药在防治帕金森病中的作用及研究进展[J].中国药理学通报, 2010, 26(7):850-854.
YUAN H L, WANG X, ZHANG L J, et al.Mechanism and research progress of Chinese traditional medicine in the prevention and treatment of Parkinson's disease[J].Chinese Pharmacological Bulletin, 2010, 26(7):850-854.
[9] 辛陈琦. 帕金森病发病机制与治疗研究进展[J].医学研究生学报, 2019, 32(6)646-651.
XIN C Q.The mechanism underlying pathogenesis of Parkinson’s disease and the progress in its therapeutics[J].Journal of Medical Postgraduates, 2019, 32(6)646-651.
[9] 辛陈琦, 张承武, 李林.帕金森病发病机制与治疗研究进展[J].医学研究生学报, 2019, 32(6):646-651.
XIN C Q, ZHANG C W, LI L.The mechanism underlying pathogenesis of Parkinson's disease and the progress in its therapeutics[J].Journal of Medical Postgraduates, 2019, 32(6):646-651.
[10] 庞文渊, 翟利杰, 刘依琳, 等.全球帕金森病综合治疗指南的分析[J].中国临床药理学杂志, 2022, 38(21):2638-2643.
PANG W Y, ZHAI L J, LIU Y L, et al.Analysis of global guidelines for comprehensive treatment of Parkinson's disease[J].The Chinese Journal of Clinical Pharmacology, 2022, 38(21):2638-2643.
[11] 苏燕, 周亚莉, 田琳琳, 等.中药活性成分防治帕金森病的研究进展[J].神经解剖学杂志, 2020, 36(1):111-115.
SU Y, ZHOU Y L, TIAN L L, et al.Research progress on prevention and treatment of Parkinson's disease with active components of traditional Chinese medicine[J].Chinese Journal of Neuroanatomy, 2020, 36(1):111-115.
[12] 陈浩, 张皓洁, 师亮, 等.枸杞多糖对帕金森病小鼠的抗氧化作用和神经保护效应[J].中国神经精神疾病杂志, 2018, 44(10):613-618.
CHEN H, ZHANG H J, SHI L, et al.Antioxidative and Neuroprotective effects of Lycium barbarum polysaccharide on Parkinson's disease mice[J].Chinese Journal of Nervous and Mental Diseases, 2018, 44(10):613-618.
[13] 李鹏, 杜园园, 文杰, 等.枸杞多糖通过激活PI3K/Akt通路调节MPP+诱导的帕金森病模型细胞凋亡[J].中国药物与临床, 2020, 20(1):28-30.
LI P, DU Y Y, WEN J, et al.Lycium barbarum polysaccharide regulates MPP+-induced apoptosis in Parkinson's disease model by activating PI3K/Akt pathway[J].Chinese Remedies & Clinics, 2020, 20(1):28-30.
[14] 尹帅领, 王海波, 杨硕.肉苁蓉多糖通过激活Wnt/β-catenin信号通路对6-HODA致帕金森病大鼠的神经保护作用[J].中西医结合心脑血管病杂志, 2020, 18(8):1227-1230.
YIN S L, WANG H B, YANG S.Neuroprotective effects of Cistanche deserticola polysaccharide on Parkinson's rats induced by 6-HODA caused by activating the Wnt/β-catenin signaling pathway[J].Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease, 2020, 18(8):1227-1230.
[15] 章斌, 李远志, 陈宇, 等.复合酶法提取广佛手多糖的工艺研究[J].安徽农业科学, 2010, 38(15):7833-7835;7873.
ZHANG B, LI Y Z, CHEN Y, et al.Extraction of polysaccharides from bergamot by compound enzymolysis method[J].Journal of Anhui Agricultural Sciences, 2010, 38(15):7833-7835;7873.
[16] 杨波, 韩凤波, 杨波.D301和LSA-700B大孔吸附树脂分离纯化玉竹多糖[J].食品研究与开发, 2014, 35(24):109-112.
YANG B, HAN F B, YANG B.Isolation and purification of polysaccharide from Polygonatum odoratum by D301 and LSA-700B macroporous adsorption resins[J].Food Research and Development, 2014, 35(24):109-112.
[17] 王琴, 蒋林, 张爵玉.广佛手多糖分离提取的工艺优化[J].食品工业科技, 2008, 29(4):199-202.
WANG Q, JIANG L, ZHANG J Y.Optimized technique of extraction of Bergamot polysaccharide[J].Science and Technology of Food Industry, 2008, 29(4):199-202.
[18] ZAWADA W M, BANNINGER G P, THORNTON J, et al.Generation of reactive oxygen species in 1-methyl-4-phenylpyridinium (MPP+) treated dopaminergic neurons occurs as an NADPH oxidase-dependent two-wave cascade[J].Journal of Neuroinflammation, 2011, 8:129.
[19] REDZA-DUTORDOIR M, AVERILL-BATES D A.Activation of apoptosis signalling pathways by reactive oxygen species[J].Biochimica et Biophysica Acta, 2016, 1863(12):2977-2992.
[20] LIN E, CAVANAUGH J E, LEAK R K, et al.Rapid activation of ERK by 6-hydroxydopamine promotes survival of dopaminergic cells[J].Journal of Neuroscience Research, 2008, 86(1):108-117.
[21] ALBERT-GASCÓ H, ROS-BERNAL F, CASTILLO-GÓMEZ E, et al.MAP/ERK signaling in developing cognitive and emotional function and its effect on pathological and neurodegenerative processes[J].International Journal of Molecular Sciences, 2020, 21(12):4471.
[22] SINGH J, SHARMA K, FROST E E, et al.Role of PDGF-A-activated ERK signaling mediated FAK-paxillin interaction in oligodendrocyte progenitor cell migration[J].Journal of Molecular Neuroscience, 2019, 67(4):564-573.
[23] CAO Q, QIN L Y, HUANG F, et al.Amentoflavone protects dopaminergic neurons in MPTP-induced Parkinson’s disease model mice through PI3K/Akt and ERK signaling pathways[J].Toxicology and Applied Pharmacology, 2017, 319:80-90.
[24] 袁倩倩, 赵海洲, 马延红, 等.芦丁对MPP+诱导的SH-SY5Y细胞损伤的保护作用[J].中国实验方剂学杂志, 2018, 24(16):109-114.
YUAN Q Q, ZHAO H Z, MA Y H, et al.Protective effect of rutin on SH-SY5Y cells injured by MPP+[J].Chinese Journal of Experimental Traditional Medical Formulae, 2018, 24(16):109-114.
[25] MANNING B D, TOKER A.AKT/PKB signaling:Navigating the network[J].Cell, 2017, 169(3):381-405.
[26] SONG G, OUYANG G L, BAO S D.The activation of Akt/PKB signaling pathway and cell survival[J].Journal of Cellular and Molecular Medicine, 2005, 9(1):59-71.
[27] CARPENTER R L, SIRKISOON S, ZHU D Q, et al.Combined inhibition of AKT and HSF1 suppresses breast cancer stem cells and tumor growth[J].Oncotarget, 2017, 8(43):73947-73963.
Options
文章导航

/

〈 〉

技术支持：北京玛格泰克科技发展有限公司