大鼠亚慢性铝暴露N6-甲基腺苷甲基化和N6-甲基腺苷RNA结合蛋白1分析

Analysis of N6-methyladenosine methylation and N6-methyladenosine RNA binding protein 1 in rats with subchronic aluminum exposure

目的观察亚慢性铝暴露对大鼠海马组织N6-甲基腺苷(m^(6)A)甲基化水平及N6-甲基腺苷RNA结合蛋白1(YTHDF1)表达的影响。方法24只SPF级雄性SD大鼠,随机分为对照组(生理盐水)和低(10μmoL/kg)、中(20μmoL/kg)、高(40μmoL/kg)剂量Al(mal)3组,每组6只,腹腔注射隔天染毒,持续90 d。染毒结束后,采用Morris水迷宫实验测试大鼠逃避潜伏期、目标象限停留时间和穿越平台次数,评估学习记忆能力;处死后,取脑组织称重计算脑体比,取海马组织采用酶联免疫吸附法测定m^(6)A甲基化水平,免疫印迹法测定YTHDF1蛋白相对表达量。结果染毒期间无大鼠死亡。染毒结束后,对照组和低、中、高剂量组大鼠脑体比分别为(0.46±0.06)%、(0.44±0.04)%、(0.49±0.06)%和(0.51±0.07)%,差异无统计学意义(P>0.05)。大鼠逃避潜伏期的时间与组间不存在交互作用(P>0.05),大鼠逃避潜伏期随训练时间增加而缩短(P<0.05);与对照组、低剂量组比较,高剂量组第3~5天大鼠逃避潜伏期延长(均P<0.05)。与对照组比较,低、中、高剂量组大鼠目标象限停留时间缩短,高剂量组大鼠穿越平台次数减少(均P<0.05)。与对照组比较,中、高剂量组大鼠海马组织m^(6)A甲基化水平升高,YTHDF1蛋白相对表达量降低(均P<0.05)。结论亚慢性铝暴露可能通过升高m^(6)A甲基化水平、抑制YTHDF1蛋白表达导致学习记忆能力损害。

Objective To explore the effects of subchronic aluminum exposure on the level of N6-methyladenosine(m^(6)A)methylation and the expression of N6-methyladenosine RNA binding protein 1(YTHDF1)in the hippocampus of rats.Methods Twenty-four healthy male SD rats were randomly divided into the control group(normal saline),the low dose group[10μmol/kg Al(mal)_(3)],the medium dose group[20μmol/kg Al(mal)_(3)]and the high dose group[40μmol/kg Al(mal)_(3)],with 6 rats in each group.The Al(mal)3 solution was administered via intraperitoneal injection on alternate days for 90 days.Escape latency,target quadrant dwell time and platform crossing times were tested to evaluate the learning and memory ability of the rats by the Morris water maze test after exposure.The brain tissue was weighted and the brain-to-body weight ratio was calculated after euthanasia.The level of m^(6)A methylation and the expression of YTHDF1 were determined by enzyme-linked immunosorbent assay and western blot assay,respectively.Results All rats survived during aluminum exposure period.The brain-to-body weight ratios of the control group and the low,medium and high dose groups were(0.46±0.06)%,(0.44±0.04)%,(0.49±0.06)%and(0.51±0.07)%,respectively,with no statistically significant differences(P>0.05).The escape latency of rats in the high dose group was longer than that in control and low group during the third to fifth day(both P>0.05).The escape latency of rats in all groups was shortened with the increase of training days(P<0.05).The target quadrant dwell time of rats in low,medium and high dose groups were lower than that in control group,and the platform crossing times of rats in high dose group were lower than that in control group(all P<0.05).The methylation level of m^(6)A and expression level of YTHDF1 in hippocampus of rats in medium and high dose groups was higher than that in control group(both P<0.05).Conclusion The learning and memory impairment caused by subchronic aluminum exposure may be related to the increase of m^(6)A methylation level and the decrease of YTHDF1 expression.