Bushen Yizhi Anti-Aging Prescription Improve Cognitive Function In APP/PS1 Mice Via The Inhibition Of TLR4/MyD88/NF-κB And The Polarization Of M2

Objective:APP/PS1 double transgenic mice are considered as one of the commonly used animal models for the study of Alzheimer’s disease(AD).This study was designed to adopt the APP/PS1 double transgenic mice(AD mice),to which Bushen Yizhi Anti-Aging Prescription(BSP)was given.The therapeutic effect of BSP on cognitive function in AD mice was observed and the immunomodulatory effect of BSP on mononuclear macrophages and microglia of AD mice were further explored.Methods:In this study,totally 20 male 8 months AD mice were randomly assigned to AD model group(AD group)and BSP intervention group(BSP group),and sex-matched8 months mice were used as wild-type group(WT group),with 10 animals in each group.Mice in each group was given oral administration for 8 weeks after 7 days of adaptive feeding in the clean grade animal room.AD mice were given the diet containing hawthorn and Chinese yam daily,BSP mice was given the equivalent amount of diet containing BSP daily,while WT group was given the same amount of normal diet daily.The Water maze test and Y maze test were performed at the end of administration.After the behavioral test,the mice were sacrificed,and their spleens were taken for the preparation of mononuclear macrophage suspension.Then,the brain tissue was isolated for extracting proteins and for making frozen sections.The activatingmarkerIba-1ofmicrogliaandsecreted pro-inflammatory/anti-inflammatory cytokines were detected by Western blot and immunofluorescence chemistry.Splenic M1/M2 macrophages were determined by flow cytometry.All experimental data were statistically analyzed by Graph Pad Prism5.0 statistical software.Results:1.During the water maze training period,the results showed that,compared with AD model group,the time and total distance from water entry point to platform of mice in BSP treatment group were significantly decreased with the increase of training days,(P<0.01,P<0.05,respectively).During the water maze test period,the results showed that the total time from water entry point to the platform(P<0.05),the total distance(P<0.05),and the time to enter the platform area for the first time(P<0.05)in the BSP treatment group were reduced compared with the AD model group.Y maze results showed that the number of spontaneous entries into the new arm of mice in BSP treatment group was significantly more than that in AD model group(P<0.05).2.The expression of Iba-1 in each group was detected by immunofluorescence double staining and Western blot.The results showed that the expression of Iba-1~+in the brain of mice in AD group was significantly increased(all P<0.01).The expression of Iba-1~+was significantly decreased after BSP intervention(all P<0.05),suggesting that the activation of microglia in the brain was inhibited.3.The results of immunofluorescence double staining showed that the co-expression of Iba-1~+i NOS~+and Iba-1~+IL-6~+in DG region of mice in AD model group was increased(P<0.05,P<0.01,respectively),while it was decreased after the treatment of BSP(P<0.001,P<0.05,respectively).The results of Western blot showed that the expression of i NOS and IL-6 in AD model group were significantly increased(P<0.001,P<0.05,respectively),and the expression of i NOS and IL-6 were decreased after the treatment of BSP(P<0.001,P<0.01,respectively).4.The results of immunofluorescence double staining showed that the co-expressions of Iba-1~+Arg-1~+and Iba-1~+IL-10~+in DG region of mice in AD model group were significantly decreased(P<0.01,P<0.001,respectively),while the co-expressions of Iba-1~+Arg-1~+and Iba-1~+IL-10~+were increased in BSP treatment group compared with AD model group(P<0.05,P<0.01,respectively).The results of Western blot showed that the expression of Arginase-1 and IL-10 in the BSP treatment group was increased(P<0.01,P<0.05,respectively).5.The expression of different phenotypes in splenic macrophages was tested by flow cytometry.The results showed that compared with the wild group,the expression of CD16/32 and IL-12 was significantly increased(both P<0.01),while the expression of CD206 and IL-10 was significantly decreased in AD model group(P<0.01,P<0.05,respectively).After the BSP treatment,the expression of CD16/32 and IL-12 were decreased(P<0.001 for both),and the expression of CD206 and IL-10 were increased(P<0.01,P<0.05,respectively).6.The protein expressions of TLR4,My D88 and NF-κB in BSP group were significantly decreased(P<0.01,P<0.01,P<0.001,respectively).BSP inhibited the expression of TLR4/My D88/NF-κB inflammatory signaling pathway compared with AD model group(P<0.01,P<0.01,P<0.001,respectively).Conclusion:This study showed that BSP improved the cognitive function of APP/PS1 double transgenic mice.In addition,BSP can inhibit the activation of central microglia in APP/PS1 double transgenic mice,reduce the secretion of pro-inflammatory cytokines and increase the secretion of anti-inflammatory cytokines,promote the transformation of M1 microglia cells/macrophages to M2,therefore,improving the inflammatory microenvironment,and alleviating brain injury.