丰富环境联合褪黑素对SAMP8小鼠学习记忆功能及DNA氧化损伤的影响

Effects of enriched environment combined with melatonin on learning and memory function and DNA oxidative damage in SAMP8 mice

目的探究丰富环境联合褪黑素对快速老化(SAMP8)小鼠学习记忆功能及DNA氧化损伤的影响。方法 6月龄SPF级健康雄性SAMP8小鼠24只,采用随机数字表法分为模型组、丰富环境组、褪黑素组和丰富环境+褪黑素组,每组6只;6只同源同月龄SAMR1小鼠为对照组。丰富环境组和丰富环境+褪黑素组小鼠饲养于丰富环境,同时褪黑素组、丰富环境+褪黑素组小鼠皮下注射褪黑素8 mg/(kg·d),模型组、对照组、丰富环境组小鼠皮下注射等体积0.9%氯化钠溶液,1次/d,共28 d。采用老化度评分评估小鼠老化情况;Morris水迷宫与Y迷宫实验评估小鼠学习记忆能力;苏木精-伊红染色法观察小鼠海马区细胞形态,免疫组织化学染色法检测小鼠海马区Aβ1-42蛋白水平;Western blot法、酶联免疫吸附法检测小鼠海马区DNA损伤标志蛋白磷酸化组蛋白H2A家庭成员X(γ-H2AX)、8-羟基脱氧鸟苷(8-OHdG)的水平。采用SPSS 25.0统计软件处理数据,多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果 (1)5组小鼠干预后老化度评分差异有统计学意义(F=126.4,P<0.01)。干预后,丰富环境组、褪黑素组、丰富环境+褪黑素组小鼠老化度评分均低于模型组(均P<0.05),且丰富环境+褪黑素组小鼠显著低于丰富环境组(P<0.05)。(2)5组小鼠逃避潜伏期的时间-组别交互作用、组别主效应、时间主效应均显著(F=11.2,799.9,121.8,均P<0.01)。第2~4天,丰富环境组、褪黑素组、丰富环境+褪黑素组小鼠逃避潜伏期均显著低于模型组(P<0.05)。5组小鼠目标象限停留时间与跨平台次数均差异具有统计学意义(F=70.38,48.83,均P<0.01)。丰富环境组、褪黑素组、丰富环境+褪黑素组小鼠目标象限停留时间与跨平台次数显著高于模型组(均P<0.05)。(3)5组小鼠总交替次数及正确交替率均差异具有统计学意义(F=291.328,113.482,均P<0.01)。褪黑素组和丰富环境+褪黑素组小鼠的总交替次数[(29.46±3.75)次,(32.57±3.52)次]和正确交替率[(53.16±3.47)%,(58.60±4.13)%]均明显高于模型组[(18.62±3.96)次,(43.61±3.92)%](均P<0.05)。(4)苏木精-伊红染色与免疫组织化学染色结果显示,与模型组比较,丰富环境组、褪黑素组、丰富环境+褪黑素组小鼠海马区细胞结构、形态有明显改善,Aβ1-42表达明显下调(均P<0.05)。(5)5组小鼠海马组织中γ-H2AX、8-OHdG蛋白表达水平差异均具有统计学意义(F=78.09,117.20,均P<0.01)。丰富环境+褪黑素组小鼠γ-H2AX、8-OHdG蛋白表达水平[(1.37±0.26),(4.79±0.35)pg/μg]显著低于丰富环境组[(2.83±0.25),(7.23±0.41)pg/μg]和褪黑素组[(2.43±0.22),(6.69±0.28)pg/μg](均P<0.05)。结论丰富环境与褪黑素均可改善SAMP8小鼠的学习记忆功能,两者联合效果更为显著,其机制可能与减少海马区DNA氧化损伤有关。

Objective To explore the effects of enriched environment combined with melatonin on learning and memory function and DNA oxidative damage in senescence accelerated mouse prone 8(SAMP8)mice.Methods Twenty-four 6-month-old SPF healthy male SAMP8 mice were randomly divided into model group,enriched environment group,melatonin group and enriched environment+melatonin group,with 6 mice in each group.Six homologous SAMR1 mice of the same age were used as the control group.The mice in the enriched environment group and the enriched environment+melatonin group were fed in the enriched environment.At the same time,the mice in the melatonin group and the enriched environment+melatonin group were subcutaneously injected with melatonin(8 mg/(kg·d))once a day for 28 d.The mice in the model group,the control group and the enriched environment group were subcutaneously injected with an equal volume of 0.9%sodium chloride solution once a day for 28 days.Aging score was used to evaluate the aging of mice.Morris water maze and Y maze tests were used to evaluate the learning and memory ability of mice.The cell morphology of hippocampus in mice was observed by hematoxylin-eosin staining,and the level of Aβ1-42 protein in hippocampus of mice was detected by immunohistochemical staining.The levels ofγ-H2A histone family member X(γ-H2AX)and 8-hydroxy-2 deoxyguanosine(8-OHdG)proteins in hippocampus of mice were detected by Western blot and Enzyme-linked immunosorbent assay.SPSS 25.0 statistical software was used to process the data.One-way analysis of variance was used for comparison among multiple groups,and LSD-t test was used for further pairwise comparison.Results(1)There was a statistical difference in aging scores among the 5 groups of mice after intervention(F=126.4,P<0.01).After intervention,the aging scores of mice in the enriched environment group,melatonin group,and enriched environment+melatonin group were lower than that in the model group(all P<0.05),and the score of the enriched environment+melatonin group was significantly lower than that in the enriched environment group(P<0.05).(2)The time and group interaction,group main effect and time main effect of the escape latency among the 5 groups of mice were statistically significant(F=11.2,799.9,121.8,all P<0.01).From day 2 to day 4,the escape latencies of mice in the enriched environment group,melatonin group and enriched environment+melatonin group were significantly lower than that in the model group(all P<0.05).There was a statistically significant difference in the target quadrant residence time and cross-platform times among the 5 groups(F=70.38,48.83,both P<0.01).The target quadrant residence time and cross-platform times of mice in the enriched environment group,melatonin group,and enriched environment+melatonin group were significantly higher than that in the model group(all P<0.05).(3)There were significant differences in the total number of alternations and correct rates among the 5 groups(F=291.328,113.482,both P<0.01).The total numbers of alternations and correct rates in melatonin group((29.46±3.75)times,(53.16±3.47)%)and the enriched environment+melatonin group((32.57±3.52)times,(58.60±4.13)%)were significantly higher than those in the model group((18.62±3.96)times,(43.61±3.92)%)(all P<0.05).(4)The results of hematoxylin-eosin staining and immunohistochemistry staining showed that compared with the model group,the cell structure and morphology of the hippocampus of mice in enriched environment group,melatonin group,and enriched environment+melatonin group were significantly improved,and the expression of Aβ1-42 was significantly reduced(all P<0.05).(5)There were statistically significant differences in the levels ofγ-H2AX and 8-OHdG proteins in the hippocampus of the 5 groups of mice(F=78.09,117.20,both P<0.01).The levels ofγ-H2AX and 8-OHdG of mice in the enriched environment+melatonin group((1.37±0.26),(4.79±0.35)pg/μg)were significantly lower than those in the enriched environment group((2.83±0.25),(7.23±0.41)pg/μg)and the melatonin group((2.43±0.22),(6.69±0.28)pg/μg)(all P<0.05).Conclusion Both enriched environment and melatonin can significantly improve the learning and memory function of SAMP8 mice,and the combined treatment effect is more significant.The mechanism may be related to the reduction of DNA oxidative damage in hippocampus.