Objectives: The liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily, and LXR-β is an important receptor for cholesterol content in brain cells. LXR-β/retinoic X receptor (RXR-α)/ATP binding cassette transporter A1 (ABCA1) cholesterol transmembrane transport system is closely related to the occurrence and development of Alzheimer's disease (AD). LXR agonist TO901317 can affect the accumulation of β- amyloid protein in the brain tissue of APP/PS1 double transgenic AD mice. However, the molecular mechanism is not clarified in detail. This study aims to evaluate the effects of LXR agonist TO901317 on the cognitive function of AD mice fed with high cholesterol diet, and to explore its possible mechanism from the perspective of cholesterol metabolism.
Methods: Twenty four male 6-month-old APP/PS1 double transgenic AD mice were randomly divided into 4 groups, 6 mice in each group: a control group (fed with normal diet), a cholesterol rich diet (CRD) group, a TO901317 group (fed with CRD combined with TO901317), and a GSK2033 group (fed with CRD combined with TO901317 and LXR antagonist GSK2033). The mice were fed with pellet feed made of high cholesterol feed, mixed with lard, egg yolk powder, and cod liver oil twice a day. TO901317 and GSK2033 were dissolved and diluted to a final concentration at 0.03%. The drugs were given to the mice daily through gastric tube according to their body weight. Meanwhile, the mice in the drug group were fed with high cholesterol diet . After feeding for 3 months, Morris water maze was used to observe the changes of spatial exploration and memory ability of AD mice in each group. The contents of TC, LDL, and HDL in serum of mice in each group were detected by cholesterol enzyme colorimetry, and the differences among the groups were compared. The expression of Aβ42 in the brain of AD mice was detected by ELISA. Western blotting was used to detect the protein levels of LXR-β, RXR-α, ABCA1, and Caveolin-1 in the brain of each group.
Results: Morris water maze results showed that the times, distance and the duration of mice crossing the platform in the CRD group were significantly decreased compared with the control group (all P<0.05), while these three figures in TO901317 group were significantly increased compared with the CRD group (all P<0.05). Compared with the TO901317 group, there was a decrease of these figures in the GSK2033 group (all P<0.05). The serum TC and LDL levels in the CRD group were significantly higher than those in the control group, while HDL levels were significantly lower (all P<0.001). The figures of the TC and LDL contents level in the TO901317 group were lower than those in the CRD group, while HDL levels were higher (all P<0.001). Compared with TO901317 group, the contents of the TC and LDL in GSK2033 group were significantly increased, while HDL content was significantly decreased (all P<0.001). ELISA results showed that the production of Aβ42 peptides in the brain of CRD group was the highest while the content in the TO901317 group was significantly decreased (P<0.001), which was the lowest among the groups. The figure in the control group was close to the GSK2033 group. Western blotting results showed that the protein levels of LXR-β, RXR-α, and ABCA1 in the CRD group were significantly decreased compared with the control group, but the protein level of Caveolin-1 was increased (all P<0.01). After TO901317 treatment, the protein levels of LXR-β, RXR-α and ABCA1 were significantly increased, while the protein level of Caveolin-1 was decreased partially (all P<0.001). In the GSK2033 group, the effect of TO901317 on AD mice was partially reversed by GSK2033. Compared to TO901317 group, the protein levels of LXR-β, RXR-α, and ABCA1 showed a decrease trend, while the protein level of Caveolin-1 showed an increase state (all P<0.05).
Conclusions: High cholesterol diet leads to severer spatial exploration, learning and memory impairment in transgenic AD mice, while the LXR agonist TO901317 attenuates this effect. The mechanism may be that TO901317 promotes cholesterol efflux by activating LXR-β/RXR-α/ABCA1 transmembrane transport system, reduces the expression of Caveolin-1, improves the composition of lipid raft, and ultimately reduces the production of Aβ42 in the brain.
目的: 肝脏X受体(liver X receptor，LXR)是核受体超家族中的一员，其中LXR-β是脑细胞内胆固醇含量的重要感受器，而LXR-β/类视黄醇X受体(retinoic X receptor，RXR)-α/ATP结合盒转运子A1(ATP binding cassette transporter A1，ABCA1)胆固醇跨膜转运体系与阿尔茨海默病(Alzheimer’s disease，AD)的发生、发展密切相关。LXR激动剂TO901317能影响APP/PS1双转基因小鼠脑组织中β淀粉样蛋白(β-amyloid protein，Aβ)的生成，但其具体机制尚未阐明。本研究旨在观察LXR激动剂TO901317对高胆固醇饲料饲喂的AD小鼠的认知功能的影响，并从胆固醇代谢的角度探讨其可能的机制。方法: 选取24只雄性6月龄APP/PS1双转基因AD小鼠，并随机分为4组，即普通饲料(control)组、高胆固醇饮食(cholesterol rich diet，CRD)组、CRD联合LXR激动剂TO901317饲喂组(TO901317组)以及CRD、TO901317联合LXR拮抗剂GSK2033饲喂组(GSK2033组)，每组各6只。以CRD为基础饲料混合猪油、蛋黄粉、鱼肝油等加工制成的颗粒饲料，按每天两次饲喂小鼠；药物组在CRD的同时给予药物，TO901317及GSK2033溶解后均稀释至最终浓度0.03%，并按照小鼠体重通过胃管每日灌胃给药，各组总治疗时间为3个月。饲喂3个月后，采用Morris水迷宫实验观察各组小鼠空间探索和记忆能力的变化；酶标仪比色法检测各组小鼠血清中TC、LDL和HDL的含量；ELISA法检测各组AD小鼠脑组织中Aβ42的表达情况；蛋白质印迹法检测各组小鼠脑组织LXR-β、RXR-α、ABCA1和Caveolin-1蛋白质水平的变化。结果: Morris水迷宫实验结果显示：与control组相比，CRD组小鼠穿越平台的次数、距离和时间均显著降低(均P<0.05)；相较于CRD组，TO901317组小鼠穿越平台的次数、距离和时间显著增加(均P<0.05)；而相较于TO901317组，GSK2033组上述指标均降低(均P<0.05)。酶标比色仪检测结果显示：CRD组小鼠血清中TC和LDL含量较control组显著增加，而HDL却显著减少(均P<0.001)；TO901317组中TC和LDL含量较CRD组减少，HDL含量增加(均P<0.001)；GSK2033组小鼠血清中TC和LDL含量较TO901317组却显著增加，HDL含量显著减少(均P<0.001)。ELISA结果显示：4组中CRD组小鼠脑内的Aβ42生成量最多；与CRD组比较，TO901317组小鼠脑内的Aβ42量显著减少(P<0.001)，4组中含量最低；control组与GSK2033组含量接近。蛋白质印迹法结果显示：CRD组中LXR-β、RXR-α、ABCA1蛋白质水平较control组显著降低，Caveolin-1蛋白质水平却明显升高(均P<0.01)。而经TO901317处理后，AD小鼠脑组织中LXR-β、RXR-α、ABCA1蛋白质水平明显升高，Caveolin-1蛋白质水平降低(均P<0.001)；在GSK2033组中，TO901317对AD小鼠的影响被GSK2033部分逆转，相较于TO901317组，小鼠脑内的LXR-β、RXR-α、ABCA1蛋白质水平降低，Caveolin-1蛋白质水平升高(均P<0.05)。结论: CRD导致转基因AD小鼠产生更加严重的空间探索障碍和学习记忆能力障碍，而LXR激动剂TO901317减轻了这一效应。其机制可能为TO901317通过激活LXR-β/RXR-α/ABCA1跨膜转运体系促进胆固醇外排，降低Caveolin-1的表达并改善脂筏的构成，最终降低脑内Aβ42的生成。.
Keywords: ABCA1 transmembrane transport system; Alzheimer’s disease; Aβ 42; TO901317; lipid rafts; liver X receptor.