开心散加减方通过抑制神经炎症调控αCaMKⅡ-PSD95蛋白结合改善阿尔茨海默病模型小鼠记忆障碍的机制研究

Modified Kaixin San improves memory and synaptic damage of mice with Alzheimer′s disease by modulating αCaMKⅡ-PSD95 protein binding through inhibition of neuroinflammation:a study of mechanism

该文探究开心散加减方(MKXS)通过调节神经炎症反应改善早老素1/2条件性双基因敲除(PS cDKO)阿尔茨海默病(AD)模型小鼠记忆障碍和突触损伤的作用机制。将60只3~3.5月龄PS cDKO小鼠及同窝野生型(WT)对照小鼠,随机分为3组:野生型(WT)组、模型(PS cDKO)组、模型+开心散加减方(PS cDKO+MKXS)组,每组20只。野生型组和模型组小鼠给予普通饲料喂养,模型+开心散加减方组小鼠给予含MKXS(给药剂量为2.55 g·kg^(-1))的饲料喂养60 d。新异物体测试检测小鼠识别记忆;蛋白印迹法(Western blot)检测小鼠海马(HPC)中磷酸化Ca^(2+)/CaM依赖性蛋白激酶Ⅱα(p-αCaMKⅡ)、N-甲基-D-门冬氨酸受体(NMDAR)各亚单位(NR1、NR2A、NR2B)、tau蛋白(tau)的蛋白表达情况;免疫组织化学染色观察HPC CA1区小胶质细胞形态;实时荧光定量PCR(qRT-PCR)检测小鼠HPC中环氧合酶-2(COX-2)、诱导型一氧化氮合酶(iNOS)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子α(TNF-α)、NR1、NR2A、NR2B、突触后致密蛋白95(PSD95)和微管相关蛋白2(MAP2)的mRNA水平;ELISA检测小鼠HPC中IL-1β、IL-6和TNF-α的蛋白含量;免疫共沉淀(Co-IP)检测Ca^(2+)/CaM依赖性蛋白激酶Ⅱα(αCaMKⅡ)及p-αCaMKⅡ与PSD95蛋白相互作用。结果显示,与模型组小鼠相比,MKXS明显增加小鼠对新物体的偏好指数、识别指数,降低小鼠HPC中p-tau(ser 396/404)的蛋白表达水平和COX-2、iNOS、TNF-α、IL-1β、IL-6 mRNA的表达量,增加小鼠HPC中NR1、NR2A、NR2B、p-αCaMKⅡ蛋白和PSD95、NR1、NR2A、NR2B、MAP2 mRNA的表达量,并且MKXS增强了小鼠HPC中PSD95与αCaMKⅡ、PSD95与p-αCaMKⅡ蛋白相互作用。免疫组织化学染色结果显示,MKXS抑制小胶质细胞激活。综上所述,MKXS对阿尔茨海默病模型小鼠记忆障碍改善作用可能通过抑制神经炎症反应调节αCaMKⅡ与PSD95蛋白结合,进而恢复突触功能受损而实现。

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.