D-氨基酸氧化酶在砷致仔鼠学习记忆损伤中作用的研究.pdf

454D-氨基酸氧化酶在砷致仔鼠学习记忆损伤中作用的研究沈阳医学院学报Journal of ShenyangMedical College?第2 5卷第5期2 0 2 3年9月论著牛泽华#，李金蕊，张桐”，葛莉莉，黄睿”，吴玉刚，王艳3（1.沈阳医学院公共卫生学院劳动卫生与环境卫生学专业研究生，辽宁沈阳110 0 34；2.预防医学专业本科生；3.职业卫生与职业医学教研室）摘要】目的：探讨D-氨基酸氧化酶（DAAO）在砷所致仔鼠学习记忆损伤中的作用。方法：建立仔鼠砷暴露模型（6 0mg/LNaAsO,），出生后6 周的仔鼠腹腔注射DAAO的抑制剂6-氯苯并d】异恶唑-3-醇（CBIO）（30 mg/k g）干预2 周，Real-timeRT-PCR测定DAAO、突触素（SYP）和突触后致密物95（PSD95）m RNA 表达水平，UHPLC-MS/MS法测定D-丝氨酸和L-丝氨酸水平。结果：与对照组比较，NaAsO,组仔鼠海马DAAOmRNA表达水平升高而SYP和PSD95mRNA表达水平下降（P0.05），D-丝氨酸和L-丝氨酸水平下降（P0.05)；与NaAsO,组比较，NaAsO,+CBIO组仔鼠海马DAAOmRNA表达水平下降而SYPmRNA表达水平升高（P0.05），D-丝氨酸水平升高（P0.05）。结论：砷暴露可使仔鼠海马D-丝氨酸水平降低和DAAO转录水平升高，抑制DAAO水平可使D-丝氨酸和突触相关蛋白SYP水平升高，从而可能改善砷暴露所致的仔鼠学习记忆损害。关键词砷；D-丝氨酸；D-氨基酸氧化酶；学习记忆中图分类号R994.6doi:10.16753/ki.1008-2344.2023.05.002Effects of D-amino acid oxidase on learning and memory impairment in offspring mice induced by arsenicNIU Zehua,LI Jinrui*,ZHANG Tong,GE Lili?,HUANG Rui,WU Yugang,WANG Yan(1.Postgraduate Majoring in Occupational and Environmental Health,School of Public Health,Shenyang Medical College,Shenyang 110034,China;2.Undergraduate Majoring in Preventive Medicine;3.Department of Occupational Health andOccupational Medicine)Abstract Objective:To investigate the effects of D-amino acid oxidase(DAAO)on learning and memory impairment inoffspring mice induced by arsenic.Methods:The model of offspring mice exposed to arsenite(60 mg/L NaAsO,)was established.The mice model of exposure to arsenic during pregnancy was induced using 60 mg/L NaAsO.At 6 weeks after birth,theDAAO inhibitor CBIO was given intraperitoneally for 2 weeks.The mRNA levels of DAAO,synaptophysin(SYP)andpostsynaptic density 95(PSD95)were detected by Real-time RT-PCR,the levels of D-serine and L-serine were determinedby UHPLC-MS/MS.Results:Compared with the control group,the level of DAAO mRNA increased,the levels of SYP andPSD95 mRNA decreased in the hippocampus of mice,and D-serine and L-serine levels significantly decreased in the NaAsO,group(P0.05).Compared with the NaAsO,group,the level of DAAO mRNA decreased,the level of SYP mRNAincreased significantly in the hippocampus of mice,and D-serine increased significantly in the NaAsO,+CBIO group(P0.05).Conclusions:Arsenic exposure can decrease the levels of D-serine and increase the transcription levels of DAAO in thehippocampus of offspring mice.And inhibition of DAAO can increase the levels of D-serine and SYP,which may improve thelearning and memory impairment in offspring mice induced by arsenic exposure.Key words arsenic;D-serine;D-amino acid oxidase;learning and memory长期饮水砷暴露引起的神经毒性备受国内外学者关注，流行病学调查结果显示，饮水砷暴露可使儿童智力水平降低，影响学习能力1-2 ，动物【基金项目】国家自然科学基金项目（No.81872570)；辽宁省自然科学基金项目（No.2021-MS-347)；沈阳医学院大学生创新创业项目(No.20229023)*牛泽华和李金蕊为本文共同第一作者通信作者王艳，E-mail：z i j i n g 15 t o m.c o m6文献标识码】A【文章编号】10 0 8-2 344（2 0 2 3)0 5-0 454-0 4实验也证明，长期砷暴露会对仔鼠学习记忆产生影响3-4，然而其具体机制目前尚未阐明。研究表明，D-丝氨酸是学习记忆关键受体N-甲基-D-天冬氨酸受体（N-methyl-D-aspartate receptor，NM-DAR）的内源性激动剂，是维持突触可塑性所必需的5。D-氨基酸氧化酶（D-amino acid oxi-dase，D A A O）为D-丝氨酸的降解酶6 ，我们前期实验数据结果显示，砷暴露后可以引起仔鼠学习记忆下降以及仔鼠海马中DAAO水平升高7 。沈阳医学院学报Journal of ShenyangMedicalCollege第2 5卷第5期2 0 2 3年9月研究表明，抑制DAAO可增加大鼠海马NMDAR诱发的放电活动，提高认知功能【8 。本研究通过对砷暴露仔鼠给予DAAO抑制剂6-氯苯并d】异恶唑-3-醇（6-chlorobenzod i s o x a z o l-3-o l,CBIO）进行干预，探讨DAAO在砷所致仔鼠学习记忆损伤中的作用，以期为地方性砷中毒的防治工作提供理论依据。1材料与方法1.1主要试剂亚砷酸钠（NaAsO,）、C BI O、D-丝氨酸和L-丝氨酸标准品（美国Sigma公司），Trizol 试剂盒和 SYBR Premix Ex Taq II Real-time RT-PCR试剂盒（日本TaKaRa公司）。1.2实验动物分组及染毒SPF级昆明种小鼠由辽宁长生生物技术有限公司提供，动物生产许可证号SCXK（辽）2 0 2 0-0 0 0 1，动物使用许可证号SYXK（辽）2 0 19-0 0 0 5。雌鼠2 4只，雄鼠12 只，体重（2 52）g，适应性饲养1周，雌雄2：1合笼，查出阴栓时定为受孕日。妊娠第1天，将孕鼠随机分为饮蒸馏水组和染砷组（含60mg/LNaAsO,水），自由饮水，每组各12 只。于仔鼠出生后6 周按如下分组给予CBIO进行腹腔注射干预，将饮蒸馏水组窝别的仔鼠随机分为对照组和30 mg/kgCBIO组，将染砷组窝别的仔鼠随机分为6 0 mg/LNaAsO,组和6 0 mg/LNaAsO,+30mg/kgCBIO组，对照组和6 0 mg/LNaAsO,组仔鼠腹腔注射0.5%羧甲基纤维素钠，干预组仔鼠腹腔注射30 mg/kgCBIO，连续干预2 周后取脑组织后分离海马，每个实验项目选取每组6 只仔鼠（来自不同窝别）。本研究经沈阳医学院伦理委员会批准，符合动物伦理学要求（批号：SYYXY2020092301）。1.3UHPLC-MS/MS法测定D-丝氨酸和L-丝氨酸水平采用乙腈沉淀法制备组织样品，色谱条件：色谱柱Agilent Poroshell EC-C18 column（10 0mm2.1mm，1.9m）；进样量10 l；柱温35；流速0.3ml/min；梯度洗脱：流动相A含0.1%甲酸的甲醇溶液，流动相B含13mmol/L的醋酸铵水溶液，0 5min流动相A2%2.5%；56min流动相A2.5%；6 6.5m in 流动相A2.5%60%；6.5 12 m in 流动相A60%2%；停止时间12min。质谱条件：采用多反应监测（MRM），正离子扫描模式GasTemp325；G a s Flo w 6L/min;Sheath Gas Temp 350;Sh e a th G a sFlow 12 L/min。455.1.4Real-time RT-PCR 法检测DAAO、突触素（SYP）和突触后致密物95（PSD95）mRNA 的表达水平将海马组织根据Trizol试剂盒操作要求，提取总RNA后将其逆转录为cDNA。配制Real-timePCR反应体系，应用ABI7500实时定量PCR仪（美国ABI公司），测定各样本等质量总RNA的mRNA的Ct值，以GAPDH基因Ct值为内参，采用2-AACt方法计算目的基因的相对表达量。各基因片段扩增引物序列均由上海生工生物工程股份有限公司提供，见表1。表1各基因片段扩增引物序列基因引物序列DAAO5-TTTTCTCCCGACACCTGGC-35-TGAACGGGGTGAATCGATCT-3PSD955-ATCCTGTCGGTCAATGGTGTT-35-AGTCCTTGGTCTTGTCGTAGTC-3SYP5-TGCCAACAAGACGGAGAGT-35-CGAGGAGGAGTAGTCACCAA-3GAPDH5-CAATGTGTCCGTCGTGGATCT-35-TCCTCAGTGTAGCCCAAGATG-31.5统计学方法采用SPSS22.0软件进行统计学分析，符合正态分布的计量资料采用均数土标准差表示，多组间比较采用单因素方差分析（ANO-VA），组间两两比较采用SNK-q检验，P0.05），Na A s O,组仔鼠海马D-丝氨酸和L-丝氨酸水平均明显下降（P0.05)；与NaAsO,组比较，NaAsO,+CBIO组仔鼠海马D-丝氨酸水平明显升高（P0.05)。见图1。A400-300200-100-片段长度(bp)159259142124*#ControlCBIONaAsO2NaAsO2+CBIO3讨论而给予D-丝氨酸能够明显改善LTP的诱导与维456B250-200150-100-50-0与对照组比较，*P0.05；与NaAsO,组比较，P0.05），NaAsO,组仔鼠海马DAAOmRNA水平升高而SYP和PSD95mRNA表达水平下降（P0.05）；与NaAsO,组比较，NaAsO,+CBIO组仔鼠海马DAAOmRNA表达水平下降而SYPmRNA表达水平升高（P0.05）。见图2。A2.01.5-1.00.5-0.0-B1.5-1.00.50.0C1.5-1.0-0.5-0.0与对照组比较，*P0.05；与NaAsO,组比较，P0.05图2 砷暴露及CBIO干预对仔鼠海马DAAOmRNA（A）、SYPmRNA（B）、PSD 95m RNA（C）表达水平的影响（n=6）沈阳医学院学报Journal of ShenyangMedical College?第2 5卷第5期2 0 2 3年9月*在中枢神经系统发育过程中，离子型谷氨酸受体NMDAR在学习记忆中起着关键性作用，而D-丝氨酸则是其活化必不可少的内源性共激动剂，调节突触可塑性9，L-丝氨酸是D-丝氨酸的前体物质【10 。研究显示，通过降解D-丝氨酸可阻断长时程增强（long-term potentiation，LT P）的产生，ControlCBIOControlCBIOControlCBIOControlCBIONaAsO2NaAsO2+CBIO#NaAsO2NaAsO2+CBIO#NaAsO2NaAsO2+CBIONaAsO2NaAsO2+CBIO持。本课题组前期研究结果提示，砷暴露会使仔鼠海马中D-丝氨酸和NMDAR表达水平明显降低7 。本研究结果显示，砷暴露后仔鼠海马D-丝氨酸和L-丝氨酸水平均明显降低，CBIO干预后D-丝氨酸水平明显升高，表明砷暴露影响了D-丝氨酸和L-丝氨酸水平，而给予干预剂后可以改善砷所致的D-丝氨酸水平改变。DAAO是催化D-丝氨酸降解的主要酶12 。有研究表明，抑制DAAO水平可改善学习记忆损伤大鼠的认知功能8 。研究显示，CBIO是一种DAAO的高效抑制剂13。本研究结果显示，砷暴露仔鼠海马DAAOmRNA表达水平明显升高，表明砷暴露会影响DAAO基因水平的表达，给予DAAO抑制剂CBIO干预后DAAOmRNA水平明显降低，由此可进一步影响D-丝氨酸水平，进而可能改善砷引起的学习记忆损伤。突触是神经元间信息传递的关键结构，SYP和PSD95为突触的标志性蛋白。Chen等【14 研究显示，对小鼠的学习和记忆行为障碍改善后，其海马SYP和PSD95表达水平明显增加。研究显示，在培养的谷氨酸能突触中，内源性D-丝氨酸与PSD95共定位，参与谷氨酸能突触发育15。本研究结果显示，砷暴露后仔鼠海马SYP和PSD95mRNA表达水平均降低，CBIO干预后SYP mRNA表达水平升高，PSD95mRNA表达水平具有升高趋势，提示砷暴露可影响突触相关基因的表达水平，而CBIO干预后由于抑制了DAAO的水平，可使D-丝氨酸水平升高，从而可能引起突触相关蛋白的改变。综上所述，砷暴露可使仔鼠海马D-丝氨酸水平降低和DAAO转录水平升高，抑制DAAO水平可使D-丝氨酸和突触相关蛋白SYP水平升高，从而可能改善砷暴露所致的仔鼠学习记忆损害，但其中具体机制有待于进一步探讨。参考文献：1 Vahter M,Skroder H,Rahman SM,et al.Prenatal and childhoodarsenic exposure through drinking water and food and cognitive沈阳医学院学报Journal of Shenyang Medical College第2 5卷abilities at 10 years of age:A prospective cohort study J.EnvironInt,2020,139:105723.2 Wasserman GA,Liu X,Parvez F,et al.A cross-sectionalstudy of water arsenic exposure and intellectual function inadolescence in Araihazar,Bangladesh J.Environ Int,2018,118:304-313.3 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