摘要
目的揭示丙烯酰胺(Acrylamide,ACR)原型及其主要代谢产物环氧丙酰胺(Glycidmide,GA)对神经突触前的损伤效应。方法构建pWSLV-07-Cyp2e1过表达质粒及未插入CYP2E1基因片段的pWSLV-07空载质粒,构建CYP2E1过表达HepG2 E47细胞株与低表达HepG2 C34细胞株,与人神经母细胞瘤细胞SH-SY5Y分别建立HepG2 E47/SH-SY5Y和HepG2 C34/SH-SY5Y体外共培养系统,以梯度浓度ACR(0、50、100、150、200、250和500μg/ml)分别染毒HepG2 E47/SH-SY5Y和HepG2 C34/SH-SY5Y共培养细胞24、48和72 h,CCK-8法检测各组SH-SY5Y细胞存活率以确定最终染毒浓度和时间。HPLC法检测SH-SY5Y细胞内ACR和GA含量,电子显微镜观察突触前微结构变化和线粒体损伤,Western blot检测SH-SY5Y中损伤关键蛋白SynapsinⅠ、GAP-43和SNAREs复合体蛋白(SNAP-25、Syntaxin 1A及VAMP-2)的表达。结果根据CCK-8结果确认ACR染毒浓度梯度为0、50、100和150μg/ml,染毒时间为24 h。随染毒浓度升高,与对照比较,两组SH-SY5Y细胞中ACR和GA的含量升高(P<0.05)、突触微结构异常,线粒体出现损伤性变化、Ca^(2+)-ATPase和Na^(+)-K^(+)-ATPase活性下降(P<0.05)、SynapsinⅠ和GAP-43蛋白表达下调(P<0.05),上述指标在HepG2 E47/SH-SY5Y组变化明显高于HepG2 C34/SH-SY5Y组;而SNAREs复合体各蛋白表达上调(P<0.05),且HepG2 E47/SH-SY5Y组上调程度明显高于HepG2 C34/SH-SY5Y组,差异均有统计学意义。结论在本共培养体系内,一定剂量时间的ACR作用后,ACR代谢产物GA在ACR所致突触前损伤中发挥重要作用,为ACR神经毒作用的全面解析提供一定的线索和依据。
Objective To explore the toxic effect of acrylamide(ACR)prototype and its main metabolite glycidamide(GA)on presynaptic damage.Methods The pWSLV-07-Cyp2e1 over-expression plasmid was constructed and the vector pWSLV-07 without the CYP2E1 gene fragment was inserted as an empty plasmid control,CYP2E1 over-expression HepG2 E47 cells and low expression HepG2 C34 cells were constructed and administered with ACR at different concentrations(0,50,100,150,200,250 and 500μg/ml)for 24,48 and 72hours.Relative survival rate of SH-SY5Y cells were tested by CCK-8 method.The contents of ACR and GA in SH-SY5Y cells were detected by HPLC method.The damage of presynaptic microstructure and mitochondria was observed by electron microscope.The expressions of synapsinⅠ,GAP-43 and SNAREs(SNAP-25,syntaxin1a and VAMP-2)in SH-SY5Y cells were measured by Western blot.Results Based on the result of CCK-8,gradient concentrations of ACR was set as 0,50,100 and 150μg/ml.The final exposure time was 24 hours.Contents of ACR and GA in SH-SY5Y cells were significantly increased(P<0.05),the synaptic microstructure and mitochondria damages were observed,the activities of Ca^(2+)-ATPase and Na^(+)-K^(+)-ATPase were decreased(P<0.05),and the protein expressions of synapsinⅠand GAP-43 were down regulated(P<0.05).The protein expression of SNAREs was up-regulated(P<0.05).The above indexes in HepG2E47/SH-SY5Y group were significantly higher than those in HepG2 C34/SH-SY5Y group.Conclusion In this co-culture system,after ACR administration for certain dosages and times.GA,the metabolite of ACR played an important role in ACR induced presynaptic damage,which provided certain clues and basis for the comprehensive analysis of the neurotoxic effect of ACR.
作者
付豪
王海华
陈宵
刘黎
张意
吕佳琦
李雨露
杨海涛
肖经纬
李斌
FU Hao;WANG Hai-hua;CHEN Xiao;LIU Li;ZHANG Yi;LV Jia-qi;LI Yu-lu;YANG Hai-tao;XIAO Jing-wei;LI Bin(National Institute of Occupational Health and Poison Control,Chinese Center for Disease Control and Prevention,Key Laboratory of Chemical Pollution and Health Safety,Chinese Center for Disease Control and Prevention,Beijing 100050,China)
出处
《毒理学杂志》
CAS
CSCD
2021年第3期207-214,共8页
Journal of Toxicology
基金
国家自然科学基金面上项目(81773474)
国家重点研发计划(2017YFF0211201)。
