DAAO在砷暴露致仔鼠学习记忆损害中的作用

The role of DAAO in arsenic exposure induced learning and memory impairment in offspring mice

目的探究D-氨基酸氧化酶(D-amino acid oxidase,DAAO)在砷暴露致仔鼠学习记忆损害中的作用。方法将18只昆明种妊娠小鼠采用随机数字表法分为未处理组(蒸馏水,n=6)和染砷组[饮用水中含60 mg/L亚砷酸钠(NaAsO_(2)),n=12],自由饮水;自妊娠第1天至仔鼠断乳前经母鼠染砷,仔鼠断乳后至实验结束以自由饮水方式染砷。仔鼠出生后6周,根据母鼠的组别,将染砷组仔鼠分为NaAsO_(2)组和NaAsO_(2)+DAAO抑制剂6-氯苯并[d]异恶唑-3-醇(6-chlorobenzo[d]isoxazol-3-ol,CBIO)组,未处理组仔鼠为对照组;对照组和NaAsO_(2)组仔鼠腹腔注射0.5%羧甲基纤维素钠,NaAsO_(2)+CBIO组仔鼠腹腔注射60 mg/kg CBIO,连续干预2周。干预结束后,采用Y迷宫实验测定仔鼠空间学习记忆能力;仔鼠经心脏灌流后取脑组织,并分离海马,经苏木素-伊红(hematoxylin-eosin,HE)染色观察仔鼠海马组织神经细胞形态;超高效液相串联三重四级杆质谱法测定D-丝氨酸含量;实时荧光定量PCR测定突触素(synaptophysin,SYP),突触小体相关蛋白25(synaptosomal-associated protein 25,SNAP25),突触后致密物95(postsynaptic density 95,PSD95),DAAO和N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor,NMDAR)亚基NR1、NR2A、NR2B mRNA水平。结果对照组、NaAsO_(2)组、NaAsO_(2)+CBIO组仔鼠Y迷宫实验交替反应率[(58.06±3.78)%、(48.61±5.75)%、(56.25±6.76)%]比较,差异有统计学意义(F=4.87,P=0.023),且NaAsO_(2)组明显低于对照组和NaAsO_(2)+CBIO组(均P<0.05)。HE染色结果显示,对照组仔鼠海马组织CA1、CA3区神经细胞数量较多,状态良好;NaAsO_(2)组神经细胞数量较少,形态不规则,细胞形态轮廓不清晰;NaAsO_(2)+CBIO组神经细胞数量较多,细胞轮廓有所改善,组织损伤减轻。对照组、NaAsO_(2)组、NaAsO_(2)+CBIO组仔鼠海马D-丝氨酸含量及SYP、SNAP25、PSD95、DAAO、NR1、NR2A、NR2B mRNA水平比较,差异均有统计学意义(F=5.41、4.41、10.16、7.60、6.98、5.63、6.53、4.33,P=0.017、0.031、0.002、0.005、0.007、0.015、0.009、0.033);其中,NaAsO_(2)组仔鼠海马D-丝氨酸含量和SYP、SNAP25、PSD95、NR1、NR2B mRNA水平均明显低于对照组和NaAsO_(2)+CBIO组,NR2A mRNA水平明显低于对照组,DAAO mRNA水平明显高于对照组和NaAsO_(2)+CBIO组(均P<0.05)。结论砷暴露能够损害仔鼠学习记忆能力,降低海马D-丝氨酸含量以及NMDAR亚基和突触相关基因转录水平,上调DAAO转录水平;而抑制DAAO可使D-丝氨酸含量以及NMDAR亚基和突触相关基因转录水平升高,改善砷暴露所致的仔鼠学习记忆能力损害。

Objective To investigate the role of D-amino acid oxidase(DAAO)in arsenic exposure induced learning memory impairment in offspring mice.Methods Eighteen Kunming pregnant mice were randomly divided into a non-treatment group(distilled water,n=6)and an arsenic exposed group[60 mg/L sodium arsenite(NaAsO_(2))in drinking water,n=12]by randomized numerical table method.From the first day of pregnancy until weaning,the mother mice were exposed to arsenic.After weaning,the offspring mice were exposed to arsenic through free drinking water until the end of the experiment.Six weeks after birth,the offspring mice of the arsenic exposed group were divided into a NaAsO_(2)group and a NaAsO_(2)+DAAO inhibitor 6-chlorobenzo[d]isoxazol-3-ol(CBIO)group according to the group of mother mice.The offspring mice of non-treatment group as the control group.The offspring mice of the control group and NaAsO_(2)group were given intraperitoneal injection of 0.5%sodium carboxymethylcellulose,while the NaAsO_(2)+CBIO group was given intraperitoneal injection of 60 mg/kg CBIO for two consecutive weeks.At the end of the intervention,the spatial learning and memory abilities of the offspring mice were measured using a Y-maze experiment.After cardiac perfusion,brain tissue was taken from the offspring mice and the hippocampus was isolated.Hematoxylin-eosin(HE)staining was used to observe the morphology of the hippocampal neurons in the offspring mice.D-serine content was determined by ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry(UHPLC-MS/MS).The mRNA levels of synaptophysin(SYP),synaptosomal-associated protein 25(SNAP25),postsynaptic density 95(PSD95),DAAO,and N-methyl-D-aspartate receptor(NMDAR)subunits NR1,NR2A,NR2B were measured by real-time fluorescence quantitative PCR.Results There was a statistically significant difference in the offspring mice alternating response rate of the Y-maze experiment among the control,NaAsO_(2),and NaAsO_(2)+CBIO groups[(58.06±3.78)%,(48.61±5.75)%,(56.25±6.76)%,F=4.87,P=0.023],and the NaAsO_(2)group was significantly lower than the control and NaAsO_(2)+CBIO groups(P<0.05).The HE staining results showed that the control group had a higher number of neuronal cells in the CA1 and CA3 regions of the hippocampal tissue,and they were in good condition.The number of neuronal cells in the NaAsO_(2)group decreased,with irregular morphology and unclear cell contours.The NaAsO_(2)+CBIO group had a higher number of neuronal cells,improved cell morphology and contour,and reduced tissue damage.There were statistically significant differences in D-serine content and mRNA levels of SYP,SNAP25,PSD95,DAAO,NR1,NR2A,and NR2B in the hippocampus of the offspring mice from the control,NaAsO_(2),and NaAsO_(2)+CBIO groups(F=5.41,4.41,10.16,7.60,6.98,5.63,6.53,4.33,P=0.017,0.031,0.002,0.005,0.007,0.015,0.009,0.033).Among them,the D-serine content and mRNA levels of SYP,SNAP25,PSD95,NR1,and NR2B in the hippocampus of the NaAsO_(2)group were significantly lower than those in the control group and NaAsO_(2)+CBIO group,the mRNA level of NR2A was significantly lower than that in the control group,and the mRNA level of DAAO was significantly higher than that in the control and NaAsO_(2)+CBIO groups(P<0.05).Conclusions Arsenic exposure can induce the learning and memory impairment in offspring mice,reduce D-serine content in the hippocampus,as well as the transcription level of NMDAR subunits and synapse-associated proteins,up-regulate the transcription levels of DAAO;and inhibit DAAO,leading to increased D-serine content,the transcription levels of NMDAR subunits and synapse-associated proteins,improving arsenic exposure induced learning and memory impairment in the offspring mice.