| Background:Alzheimer disease(AD)is one of the major causes of dementia,incidence increased year by year,resulting in heavier burden on society.Its main pathological features include amyloid protein(A?),a large number of senile plaques deposited outside the cells,and the loss of neurons.Bu Yang Huan Wu Tang(BYHWT)is a classic and famous prescription in TCM which has the function of replenishing the blood and removing the blood stasis.It has a definite effect on the treatment of the cerebrovascular accident in clinical practice.Our previous research showed that BYHWT could improve the ability of learning and memory in mice AD,which maybe related with the RAGE/LRP1 cell signaling pathway.This study will continue to do some research for the function mechanism of BYHWT,in order to certain whether it can affect the capillaries endothelial cells effect BMEC LRPland RAGE to adjust A? central metabolism,thereby preventing and treating AD.Methods:BYHWT extract was prepared according to the clinical administration of decoction.In vivo APP/PS1 transgenic mice which were 7 months old were selected as AD model,using PCR to validate APP and PS1 gene expression.APP/PS1 mice were randomly divided into 5 groups(n=20)which were the model group,the positive drug group,the BYHWT high,medium and low dose groups,Another 20 C57BL/6 mice were selected as the blank control group.The blank control group and model group were given the same volume of saline,the positive drug group was given naipaiqi0.001g·kg-1,high,medium and low dose groups were given 37.06,18.53,9.26g·kg-1 BYHWT(raw drug)separately.The mice were fed for 35 days and then done the Morris water maze experiment to test the ability of learning and memory of the mice.In this study,hematoxylin-eosin(HE)staining,transmission electron microscopy and Tunel staining were used to observe the morphology and apoptosis of hippocampus of each group of mice,the morphological changes of microvessels in hippocampus of mice were observed by transmission electron microscope,the expression of apolipoprotein LRP1,RAGE,ApoJ and VCAM-1 was detected by immunohistochemistry[IHC).In vitro Mice brain microvascular endothelial cells were cultured by the cell nutrient fluid of 24?mol/L A?25?35 in vitro to simulate the AD cell mode,intervened by the 150?mol/L BYHWT.The general morphology of cells was observed by light microscope,the ultrastructure of the cells was observed by electron microscope,the method of ELISA was used to detect the inflammatory factors of IL-1??IL-6?TNF-? and the secretion of A?25-35,flow cytometry to test apoptosis rate,method of WB to test the protein expression of RAGE,LRP1,ICAM,VCAM-1,ApoJ,ApoE and NF-?B of the cells.Results:In vivo APP/PS1 expression was positive in RNA of the double transgenic mice.Morris water maze experiment showed that,in the experiment of the space exploration,compared with the mice of the model group,the number of mice crossing the platform was significantly lower,and the residence time of target quadrant was significantly prolonged,which were from the BYHWT high,medium dose groups,and the positive drug group.In the experiment of the positioning navigation,the escape latent period of mice was significantly shortened which were from the BYHWT high,medium dose groups,and the positive drug group.HE staining showed that,the arrangement of cells in the hippocampal CA1 region of the mice from the blank group was regular,while the arrangement of cells from the model group was disordered,with fewer cell layers,and uncleared nucleolus.The arrangement of cells in the hippocampal CA1 region of the mice from the BYHWT group was regular,with round or oval nucleolus and more cell layers.TEM observed that,hippocampal capillary deformed and narrowed of the model group,with incomplete endothelial cells,large vacuoles outside the cavity,and synaptic fusion.After treatment of BYHWT,the morphology of hippocampal neurons was gradually restored,the capillary structure was clear,and the synaptic morphology was good.The Tunel method which was used to detect apoptosis showed that,the ratios of cells in hippocampus of high and middle dose groups increased significantly,compared with the model group.IHC displayed that,the expression of LRP1 and ApoJ in hippocampus of high and middle dose groups increased significantly and the expression of RAGE and VCAM-1 decreased obviously,compared with the model group.In vitro The experiment in vitro showed that,abnormal changes of cell morphology appeared in model group,compared with normal cells,for example,cell rounding,slower proliferation,karyopyknosis,perinuclear space broadening,abnormal nuclear cytoplasmic ratio,rough endoplasmic reticulum expansion,mitochondrial vacuolar degeneration,microvilli decreased or abscised,and etc.Compared with the model group,the cells in the BYHWT low,medium,high dose groups and positive drug group had a certain degree of morphological recovery,among them,the middle and high dose group recovered significantly and high dose group and positive drug group were the closest to the normal group.High and middle dose groups could cut down the content of IL-1?,IL-6,TNF-?,A?25-35 in cells of the rat induced by A?,so as to alleviate the inflammatory reaction induced by A? in rat BMEC cells,and at same time,high and middle dose groups could reduce the apoptosis of BMEC cells induced by A?.Compared with the model group,the expressions of RAGE protein from the three concentrations were significantly down regulated,with dose dependent relationship,high concentration group had the most obvious effect(P<0.01).The three concentration groups had significant effect on the expression of LRP1 protein,with dose dependent relationship,low concentration group had obvious effect(P<0.01).The expressions of NF-?BP65 protein from the three concentrations were significantly down regulated(P<0.05).In the three concentration group,the expression of APOE protein was up-regulated only in the high concentration group(P<0.05),low and medium concentration group had no effect.Compared with the model group,the expressions of ICAM-1 and VCAM-1 protein from the three concentrations were significantly down regulated,with dose dependent relationship,the middle concentration group(P<0.05)and high concentration group(P<0.01)had the most obvious effect,which differences were statistically significant.The expressions of NF-?BP65 protein from the three concentrations were significantly down regulated,with dose dependent relationship,the middle and high concentration group(P<0.01)had the most obvious effect.Conclusions:The BYHWT high and middle dose can improve the learning and memory ability of AD mice,recover the morphology of CA1 area in hippocampus of mice,improve the morphology of endothelial cells in hippocampus,and the mechanism maybe related with which BYHWT can enhance the expression of RAGE and ApoJ protein in hippocampus of AD mice and weaken the expression of RAGE and NF-KBp65 protein in hippocampus of AD mice.In vitro experiments showed that BYHWT could regulate the metabolism of A? by regulating the RAGE/LRP1 transporter,effect the ICAM-1 and VCAM-1 protein mediated vascular endothelial inflammation by inhibiting NF-?BP65 signaling pathway,and improve the morphological changes of mice brain microvascular endothelial cells induced by A?,thereby protecting the brain microvascular endothelial cells injury. |
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