内质网应激在甲氨蝶呤相关神经毒性和认知障碍发生中的作用

Effect of endoplasmic reticulum stress in the occurrence of Methotrexate - related neurotoxicity and cognitive impairment

目的研究甲氨蝶呤（MTX）与认知障碍的关系，并探讨其神经毒性发生的可能机制。方法健康雄性SD大鼠30只，体质量180～220g。采用随机数字表法将实验动物分成3组：对照组、60mg／kgMTX（MTX60）组和100mg／kgMTX（MTX100）组，每组10只。MTX60与MTX100组大鼠分别予60mg／kg、100mg／kgMTX；对照组大鼠予等体积的9g／L盐水处理。MTX预处理后用Morris水迷宫实验评价不同时间点3组大鼠的学习记忆能力；3组实验大鼠分别于完成Morris水迷宫实验后取海马组织，应用Westernblot检测CCAAT/增强子结合蛋白同源蛋白（CHOP）、半胱氨酸天冬氨酸蛋白酶（caspase）12和活化caspase-3（cleavedcaspase-3）的表达水平，分别用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法（TUNEL）和HE染色检测海马神经细胞凋亡水平和病理改变。结果MTX处理后，大鼠在Morris水迷宫probe空间探索试验阶段，MTX60组和MTX100组大鼠平台象限时间较对照组明显减少，差异有统计学意义[（27．30±3．98）S和（21．63±4．22）s比（33。30±6．31）s，F=13．94，P〈0．05]；且MTX100组平台象限时间短于MTX60组，差异有统计学意义（P〈0．05）。与对照组比较，MTX6。组和MTX100组大鼠海马CA1区域的神经细胞出现变性；MTX60和MTX100组大鼠海马CAl区域TUNEL阳性细胞数较对照组明显增加，差异有统计学意义[（4．72±0．12）％和（9．124±0．12）％比（1．11±0．49）％，F=95．272，P〈0．05]；且MTX，00组阳性细胞数多于MTX60组，差异有统计学意义（P〈0．05）。MTX。和MTX100组海马组织CHOP、caspase-12和cleavedcaspase-3蛋白表达水平较对照组明显增加，差异均有统计学意义（CHOP：2．98±0．31和4．15±0．61比0．38±0．12，F=232．74，P〈0．05；caspase-12：0．33±0．04和0．45±0．06比0．14±0．02，F=120．70，P〈0．05；cleavedcaspase-5：0．354-0．04和0．44±0．06比0．05±0．03，F=198．64，P〈0．05）；且MTX100组蛋白表达水平高于MTX60组，差异均有统计学意义（P均〈0．05）。结论MTX可以诱导大鼠出现认知障碍，并且内质网应激介导的海马神经细胞凋亡可能在MTX相关认知障碍发生机制中发挥重要作用。

Objective To investigate the relationship between Methotrexate （MTX） and its cognitive dysfunc- tion, and to explore the possible mechanism of neurotoxicity induced by MTX. Methods Thirty healthy male SD rats weighting 180 -220 g were divided into 3 groups using random number table：control group ,60 mg/kg MTX （MTXro） group and 100 mg/kg MTX （MTX100 ） group, with 10 rats in each group. The rats in MTX60 group, MTX100 group re- ceived 60 mg/kg MTX and 100 mg/kg MTX,respeetively. The rats in control group accepted the same volume of 9 g/L saline injection as MTX group. Spatial memory of rats was evaluated by using Morris water maze test at different time points after pretreatment with MTX. After the Morris water maze test, the hippocampus were harvested and the expres- sions of C/EBP homologous protein （CHOP）, cysteinyl aspartate specific proteinase 12 （caspase- 12） and cleave cys- teinyl aspartate specific proteinase 3 （cleaved caspase -3 ） were detected by using Western blot. Meanwhile, cell apop- tosis and pathological change of hippocampal neurons were detected by terminal dexynucleotidyl transferase - mediated dUTP nick end labeling（TUNEL） assay and HE staining respectively. Results In the Morris water maze test,the time in platform quadrant of rats in MTXro group and MTXI0o group was shorter than that of rats in control group during probe training [ （27.30 ± 3.98 ） s and （21.63 ± 4.22） s vs （33.30 ± 6.31 ） s, F = 13.94, P 〈 0.05 ], and the time in target quadrant of MTX600 group was shorter than that of MTXr0 group （ P 〈 0.05 ）. Compared with the control group, there were degenerated neurons in hippocampus cornu ammorlis 1 （ CA1 ） area in MTX60 group and MTXi00 group. The number of TUNEL- positive cells of the hippocampus CA1 area increased significantly in MTXro group and MTX600 group rats [ （4.72 ±0.12）% and （9.12 ±0. 12）% vs （1.11 ±0.49）% ,F =95. 272,P 〈0.05] ,and the TUNEL - positive cells of rats in MTXI0o group were more than those of MTX6o group（ P 〈0.05 ）. The expressions of CHOP, caspase -12 and cleaved caspase - 3 also increased compared with the control group （ CHOP：2.98 ± 0.31 and 4. 15 ± 0.61 vs 0.38 ±0.12,F =232.74,P 〈0.05;caspase - 12：0.33 ±0.04 and 0.43 ±0.06 vs 0. 14 ±0.02,F = 120.70,P 〈 0.05 ; cleaved caspase - 3：0.35 ± 0.04 and 0.44 ± 0.06 vs 0. 05 --- 0.03, F = 198.64, P 〈 0.05）, and the protein ex-pression levels of rats in MTX10o group were higher than MTX60 group （ all P 〈 0.05 ）. Conclusions MTX can induce cognitive impairment in rats, and endoplasmic reticulum stress mediated hippocampal neurons apoptosis may play an important role in the mechanism of MTX - induced cognitive impairment in rats.