淫羊藿苷芳香脂质体构建及其提高AD小鼠认知能力的研究

Construction of icariin aromatic liposomes and their improvement of cognitive ability in AD mice

关键词:  淫羊藿苷  芳香脂质体  阿尔茨海默病  认知功能  抗炎能力。

Key words:  Icariin  Aromatic liposomes  Alzheimer"s disease  Cognitive function  Anti-inflammatory capacity.

摘要:

目的 本研究旨在研究一种包含淫羊藿苷(ICA)的芳香脂质体(ICA-BO@Lips),并评估其在阿尔茨海默病模型小鼠中的认知改善和神经保护作用。方法 采用薄膜分散法制备ICA-BO@Lips,通过粒度分析仪和投射电镜检测芳香脂质体粒径、PDI、电位和外观；采用HPLC检测ICA的载药量和包封率。通过RT-PCR测定TNF-α、IL-1β、IL-12A、COX-2、INOS-2和IL-6的mRNA表达；选用APP/PS1 小鼠为 AD 动物模型,通过对尾静脉注射 ICA-BO@Lips,通过行为学和病理切片综合分析其对AD动物模型认知功能改善作用和相关机制；通过 Morris 水迷宫和筑巢实验对小鼠行为学进行考察；对小鼠脑部切片进行HE染色、尼氏染色和免疫组化分析。结果 测得ICA-BO@Lips粒径为(105.54±1.31) nm,PDI 为 0.22±0.028,电位为(-16.88±1.24)mV证明该脂质体分散均匀,脂质表面带负电荷体；透射电镜显示,该脂质体表现为光滑类球形且无粘连；HPLC测定ICA的载药量和包封率分别为(2.10±0.024)%和(77.88±0.32)%。RT-PCR测得在10 μmol/L的ICA-BO@Lips处理后,可以显著抑制小胶质细胞炎症因子的mRNA表达；Morris 水迷宫和筑巢实验结果显示ICA-BO@Lips显著改善了AD小鼠的认知功能；能够降低Aβ沉积,保护神经元形态,提升小鼠海马和皮层区 BDNF水平,调节促凋亡蛋白 Bax、Caspase-3 及抗凋亡蛋白 Bcl-2 等相关因子的表达；未对主要脏器产生明显的病理变化,显示出良好的安全性。结论 本研究开发的ICA-BO@Lips在AD动物模型中展示了显著的认知改善和神经保护作用,具有潜在的临床应用价值。

Abstract:

ABSTRACT: OBJECTIVE This study was conducted to investigate an aromatic liposome containing icariin (ICA) (ICA-BO@Lips) and to evaluate its cognitive improvement and neuroprotective effects in mice models of Alzheimer's disease. METHODS ICA-BO@Lips was prepared by thin film dispersion method. Particle size, PDI, potential and appearance of aromatic liposomes were detected by particle size analyzer and electron microscopy. The drug loading and encapsulation rate of ICA were determined by HPLC. The mRNA expression of TNF-α, IL-1β, IL-12A, COX-2, INOS 2 and IL-6 was determined by RT-PCR. APP/PS1 mice were selected as the AD animal model. The effect of APP/PS1 mice on the cognitive function of the AD animal model and the related mechanism were comprehensively analyzed by the behavioral and pathological sections after injection into the tail vein ICA-BO@Lips. The behavior of mice was investigated by Morris water maze and nesting experiment. HE staining, Nissl staining and immunohistochemical analysis were performed on mouse brain sections. Results The measured ICA-BO@Lips particle size was 105.54±1.31 nm, PDI was 0.22±0.028, and potential was -16.88±1.24 mV, indicating that the liposome was uniformly dispersed and negatively charged on its surface. Transmission electron microscopy showed that the liposome was smooth and spherical without adhesion. The drug loading capacity and encapsulation rate of ICA measured by HPLC were 2.10± 0.024% and 77.88± 0.32%, respectively. RT-PCR showed that treatment with 10 μmol/L ICA-BO@Lips could significantly inhibit the mRNA expression of inflammatory cytokines in microglia. The results of Morris water maze and nesting experiment showed that ICA-BO@Lips significantly improved the cognitive function of AD mice; It can reduce the deposition of Aβ, protect the morphology of neurons, increase the level of BDNF in hippocampus and cortex, regulate the expression of pro-apoptotic protein Bax, Caspase-3 and anti-apoptotic protein Bcl-2 and other related factors. There were no obvious pathological changes to major organs, showing a good safety. CONCLUSION ICA-BO@Lips developed in this study showed significant cognitive improvement and neuroprotective effects in AD animal models, which has potential clinical application value.