丰富环境联合褪黑素对SAMP8小鼠学习记忆功能及脑神经细胞凋亡的影响

Effects of enriched environment combined with melatonin on learning and memory function and brain neuron apoptosis in SAMP8 mice

背景:脑老化后神经细胞凋亡增多,其机制不明确。近年研究表明,褪黑素可调节多种凋亡因子的表达,丰富环境作为一种治疗阿尔茨海默病的有效方案已投入临床使用,然而二者合用是否有更好的疗效及其各自的作用机制目前尚不清楚。目的:探讨丰富环境与褪黑素联合干预对SAMP8小鼠学习记忆功能及脑神经细胞凋亡的影响。方法:选用3月龄雄性SAMP8小鼠24只,利用随机数字表法分成4组:对照组、丰富环境组、褪黑素组和联合处理组,每组6只。对照组和褪黑素组小鼠饲养于标准环境,丰富环境组和联合组饲养于丰富环境,直至小鼠满4月龄;满4月龄次日,褪黑素组、联合处理组小鼠开始皮下注射褪黑素8 mg/(kg·d),对照组、丰富环境组皮下注射等量生理盐水,共30 d。给药结束后继续饲养于原环境至11月龄,采用筑巢实验和Morris水迷宫实验评估小鼠学习记忆能力;行为学实验结束后,采用NeuN免疫荧光/TUNEL双标染色及尼氏染色观察海马CA1区细胞凋亡情况,采用Western blot法检测海马中凋亡相关蛋白Caspase-3、Bax、Bcl-2、P53的表达。结果与结论:①与对照组相比,其他3组小鼠的筑巢实验得分、Morris水迷宫跨平台次数、海马CA1区NeuN免疫反应性、尼氏染色阳性细胞数量、海马中Bcl-2蛋白表达显著增加(P<0.05),联合处理组上述指标值显著多于丰富环境组、褪黑素组(P<0.05);②苏木精-伊红染色显示,与对照组相比,其他3组小鼠海马CA1区神经细胞形态和排列较佳;③与对照组相比,其他3组小鼠的Morris水迷宫逃避潜伏期、海马CA1区免疫荧光/TUNEL双阳性细胞数量及Caspase-3、Bax、P53蛋白表达水平显著降低(P<0.05),联合处理组上述指标值显著低于丰富环境组、褪黑素组(P<0.05);④结果表明,丰富环境和褪黑素对SAMP8小鼠的学习记忆功能、脑神经细胞凋亡具有改善作用,其机制可能与调控小鼠海马神经细胞P53/P21通路来减少细胞凋亡有关;且将丰富环境和褪黑素联合应用的疗效很可能高于单方案治疗。

BACKGROUND:Neuronal apoptosis increases after brain aging and the mechanism is not clear.Recent studies have shown that melatonin can regulate the expression of a variety of apoptosis factors and enriched environments have been used as an effective treatment for Alzheimer’s disease.However,whether the combination of the two has better efficacy and their respective mechanisms of action remain unclear.OBJECTIVE:To investigate the effects of enriched environment combined with melatonin intervention on learning and memory function and brain neuron apoptosis in SAMP8 mice.METHODS:Twenty-four 3-month-old male SAMP8 mice were randomly divided into control group,enriched environment(EE)group,melatonin(MT)group,and enriched environment combined with melatonin(EE+MT)group,with 6 mice in each group.Mice in the control and MT groups were fed in a standard environment and those in the EE and EE+MT groups were fed in an enriched environment until they were 4 months old.At the age of 4 months,mice in the MT and EE+MT groups were subcutaneously injected with melatonin(8 mg/kg/d)for 30 days and those in the control and EE groups were given the same amount of normal saline for 30 days.After treatments,all the animals were kept in the original environment until 11 months of age,and their learning and memory abilities were evaluated by nest building test and Morris water maze test.After the completion of behavioral experiments,the apoptosis of hippocampal CAI cells was observed by immunofluorescence/TUNEL double standard staining and Nissl staining.The expression levels of apoptosis-related proteins Caspase-3,Bax,Bcl-2,and P53 were detected by western blot assay.RESULTS AND CONCLUSION:Compared with the control group,there was a significant increase in the score of the nest building test,cross-platform frequency,NeuN immunoreactivity in the hippocampal CA1 region,the number of Nissl-positive cells and the expression level of Bcl-2 protein in the hippocampus in the EE,MT,and EE+MT groups(P<0.05).These indicators were significantly higher in the EE+MT group than the EE and MT groups(P<0.05).Hematoxylin-eosin staining results indicated that compared with the control group,the EE,MT,and EE+MT groups showed better morphology and arrangement of nerve cells in the hippocampal CA1 region.Compared with the control group,Morris water maze escape latency,the number of immunofluorescence/TUNEL double positive cells in the hippocampal CA1 region and the expression levels of Caspase-3,Bax and P53 proteins in the hippocampus were significantly decreased in the EE,MT,and EE+MT groups(P<0.05).These indicators in the EE+MT group were significantly lower than those in the EE and MT groups(P<0.05).Overall,both enriched environment and melatonin can improve learning and memory function and brain neuron apoptosis in SAMP8 mice.The mechanisms may be related to the regulation of P53/P21 pathway in mouse hippocampal neurons to reduce apoptosis.Moreover,the combined application of enriched environment and melatonin is likely to be more effective than a single regimen treatment.