探讨改良开心散（MKXS）改善条件早老素1/2条件双敲除（PScDKO）阿尔茨海默病（AD）小鼠模型记忆和突触损伤的作用机制。具体来说,将60只PScDKO小鼠（3-3.5月龄）及其年龄匹配的野生型（WT）同窝动物随机分为三组：WT组（n=20），PScDKO组(n=20)，和PScDKO+MKXS组(n=20)。WT和PScDKO组的小鼠用标准食物喂养，PScDKOMKXS组的小鼠用含有MKXS（2.55g·kg〜（-1））的食物喂养60天。采用新的物体识别任务来检测小鼠的识别记忆，和Westernblot检测小鼠海马突触相关蛋白(HPC)的蛋白水平,例如NR1，NR2A，NR2B,p-αCaMKⅡ,tau,还有p-tau.基于免疫组织化学观察小鼠HPCCA1中的小胶质细胞形态。采用实时定量PCR（qRT-PCR）检测小鼠HPC中促炎因子和突触相关蛋白的mRNA水平，包括COX-2，iNOS，IL-1β,IL-6,TNF-α,PSD95,NR1,NR2A,NR2B,MAP2IL-1β的蛋白水平,TNF-α,采用酶联免疫吸附试验(ELISA)检测IL-6水平。采用免疫共沉淀法(Co-IP)检测PSD95与αCaMKⅡ、PSD95与p-αCaMKⅡ的相互作用。结果表明，PScDKO+MKXS对新对象表现出显著较高的偏好指数和识别指数，较低的p-tau蛋白水平（ser396/404）和COX-2，iNOS，TNF-α,IL-1β,和HPC中的IL-6，NR1，NR2A的蛋白质水平更高，NR2B,和p-αCaMKⅡ和NR1，NR2A的mRNA水平，NR2B,PSD95和MAP2，αCaMKⅡ与PSD95的相互作用和p-αCaMKⅡ与PSD95的相互作用强于PScDKO组。免疫组化染色显示MKXS抑制小胶质细胞的活化。总之,MKXS通过抑制神经炎症调节αCaMKⅡ-PSD95蛋白结合改善AD小鼠记忆和突触损伤。
To investigated the mechanisms underlying the effects of modified Kaixin San(MKXS) on improving memory and synaptic damage of Alzheimer\'s disease(AD) mouse model with conditional presenilin 1/2 conditional double knockout(PS cDKO). Specifically, 60 PS cDKO mice(3-3.5 months old) and their age-matched wild-type(WT) littermates were randomized into three groups: WT group(n=20), PS cDKO group(n=20), and PS cDKO+MKXS group(n=20). Mice in WT and PS cDKO groups were fed with standard chow and those in PS cDKO+MKXS group were given chow containing MKXS(at 2.55 g·kg~(-1)) for 60 days. Novel object reco-gnition task was employed to detect the recognition memory of mice, and Western blot to detect the protein levels of synapse-associated proteins in the hippocampus(HPC) of mice, such as NR1, NR2 A, NR2 B, p-αCaMKⅡ, tau, and p-tau. Microglial morphology in the HPC CA1 of mice was observed based on immunohistochemistry. Quantitative real time-PCR(qRT-PCR) was employed to detect the mRNA levels of the pro-inflammatory factors and synapse-associated proteins in the HPC of mice, including COX-2, iNOS, IL-1β, IL-6, TNF-α, PSD95, NR1, NR2 A, NR2 B, and MAP2. The protein levels of IL-1β, TNF-α, and IL-6 were tested by enzyme-linked immunosorbent assay(ELISA). The interaction between PSD95 and αCaMKⅡ and between PSD95 and p-αCaMKⅡ was tested by co-immunoprecipitation(Co-IP). The results showed that PS cDKO+MKXS demonstrated significantly higher preference index and recognition index of the new objects, lower protein level of p-tau(ser 396/404) and mRNA levels of COX-2, iNOS, TNF-α, IL-1β, and IL-6 in HPC, higher protein levels of NR1, NR2 A, NR2 B, and p-αCaMKⅡ and mRNA levels of NR1, NR2 A, NR2 B, PSD95, and MAP2, and stronger interaction of αCaMKⅡ with PSD95 and interaction of p-αCaMKⅡ with PSD95 than the PS cDKO group. Immunohistoche-mical staining showed that MKXS inhibited the activation of microglia. In conclusion, MKXS improves memory and synaptic damage in mice with AD by modulating αCaMKⅡ-PSD95 protein binding through inhibition of neuroinflammation.
