Study On Mechanism Of Cistanches Herba Against Alzheimer’s Disease Based On Network Pharmacology

Objective:In this study,APP/PS1 mice were used as Alzheimer’s disease(AD)animal models to evaluate the effects of cistanche phenylethanoid glycosides on learning and memory disorders in dementia mice and the related neuroprotective mechanisms,and to compare the anti-AD effects of cistanche phenylethanoid glycosides and its two main components.The main research results:(1)Using network pharmacology method,we explored the active components and possible pathways and related targets with the treatment potential of AD in cistanche deserticola,and predicted and screened the possible pathways and related targets of cistanche deserticola to improve Alzheimer’s disease.(2)Effects of cistanche phenylethanoid glycoside(Ph Gs),echinacoside(ECH)and verbascoside(ACT)on the learning and memory ability of AD mice and the regulation of brain histopathological changes.(3)The effects of Ph Gs,ECH and ACT on the activation of glial cells,neuroinflammatory response and related transduction pathways in APP/PS1 double transgenic mice.(4)Effects of Ph Gs,ECH and ACT on p-CAMKII/CAMKII protein and related synaptic proteins in APP/PS1 transgenic mice.Methods:(1)The TCMIP V 2.0(http: //www.tcmip.cn/TCMIP/index.Php/Home/)database and literature review were used to predict the main active components and targets of Cistanche deserticola,and the AD-related targets were predicted based on Gene Cards(https: //www.genecards.org/)database.Constructing a common target screening and interaction network of drugs and diseases.The enrichment results were analyzed and screened using gene ontology(GO)functional enrichment and genome encyclopedia(KEGG)pathway enrichment analysis to unearth the active components,possible pathways and related targets with AD treatment potential in Cistanches Herba,and to predict and screen the possible pathways and related targets for Cistanches Herba to improve Alzheimer’s disease.(2)Sixty six-month-old APP/PS1 Transgenic mice(Tg)and 13 fellow-month-old Wide Type mice(WT)were randomly divided into five groups(n = 12): Tg group,Tg+Ph Gs group(60 mg/kg),Tg+ ECH group(60mg/kg),Tg+ACT group(60 mg/kg)and Tg+memantine hydrochloride group(3 mg/kg).The mice in the WT and Tg groups were orally administered with equal volumes of normal saline every day for three consecutive months.The improvement effects of Ph Gs,ECH and ACT on the nesting ability and learning-memory ability disorders of AD mice were observed through the nesting test,Morris water maze test,dark avoidance test and platform test.(3)The effects of Ph Gs,ECH and ACT on neurons in the brain tissue of transgenic mice were evaluated by HE staining.The effects of Ph Gs,ECH and ACT on Aβ deposition in the brain tissue of mice were observed by Th-T staining and immunohistochemistry.The expression levels of astrocyte and microglia activation marker proteins(GFAP,Iba-1)and M1 microglia activation marker(CD11b)in the mouse brain were detected by immunohistochemistry with Ph Gs,ECH and ACT.(4)The contents of IL-1β,TNF-α,TGF-β1 and IL-4 in the cortical tissue of mice were detected by ELISA kit.The expression levels of marker proteins(i NOS and Arginase-1)and TLR4,NFκB pathway-related proteins(NFκB p65,p-NFκB p65)of the M1/M2 phenotype in hippocampal and cortical microglia were detected by Western blot.(5)The content of glutamic acid in the mouse cortical tissue is determined by an ELISA kit;The expression levels of p-CAMKII/CAMKII proteins and synaptic related proteins(Homer-1,SAP102,SYP)in the cortex and hippocampus of mice in each group were detected by Western blot.Results:(1)237 targets of Cistanche deserticola were collected,and 32 targets shared with AD were obtained.,and a PPI network was constructed using a STRING database to select APP,PTGS2,CASP3,TLR4,NFκB1 and other central targets,which were predicted to be possible targets of Cistanches Herba for improving Alzheimer’s disease.To further explore the correlation of common targets,a PPI network was constructed.Common targets involving multiple biological processes,cellular components and molecular functions were imported into the DAVID database for GO and KEGG enrichment analyses.According to the enrichment results of KEGG pathways,the significantly enriched pathways included Alzheimer’s disease,glutamatergic synapses,dopaminergic synapses,NF-κB signaling pathway,calcium signaling pathway,and Toll-like receptor signaling pathway.This study focused on the Toll-like receptor signaling pathway and NF-κB signaling pathway,and explored the mechanism of phenylethanoid glycosides(Ph Gs,ECH and ACT)of cistanche in the prevention and treatment of AD.(2)When the nesting experiment was carried out for 12 h and 24 h;Compared with wild-type mice,the nesting ability of mice in the transgenic group was significantly reduced.After treatment with Ph Gs,ECH and ACT,the nesting ability of AD mice was significantly increased.Morris water maze test showed that compared with the wild-type mice,the escape latency of the transgenic mice was significantly prolonged and the number of platform crossing times was significantly reduced.After treatment with Ph Gs,ECH and ACT,the escape latency of AD mice was significantly shortened,and the number of platform crossing times was significantly increased.These results indicated that Ph Gs,ECH and ACT could improve the long-term learning and memory disorders in APP/PS1 transgenic AD mice.The results of the dark avoidance test showed that compared with the wild-type mice,the escape latency of the transgenic mice was significantly shortened,and the number of errors was significantly increased.After treatment with Ph Gs,ECH and ACT,the escape latency of AD mice tended to be prolonged and the error times tended to be decreased,with the error times of the ECH and ACT groups significantly reduced.Platform test results showed that compared with wild-type mice,the latency of mice in the model group was shorter,and the number of errors was increased.After treatment with Ph Gs,ECH and ACT,the latency of AD mice tended to increase,and the error times were significantly reduced.(3)HE staining results showed that after treatment with Ph Gs,ECH,and ACT,the nuclei of AD mice in the cortex and hippocampus tended to be normal in color,and the cells showed complete morphology and were full in volume.The Th-T staining results showed that Ph Gs,ECH and ACT could reduce Aβ deposition in the brain tissue of mice in the transgenic group.Immunohistochemical experiments showed that compared with the transgenic mice,Ph Gs,ECH and ACT could reduce the aggregation of Aβ,the expression of GFAP,Iba-1 and CD11 b in the brain of AD mice.(4)Western Blot results showed that Ph Gs,ECH and ACT could increase the level of Arginase-1 in the brain of APP/PS1 mice to a certain extent,and reduce the level of i NOS in the brain of APP/PS1 mice to a certain extent.Ph Gs,ECH and ACT could significantly reduce the level of TLR4 in the hippocampus of APP/PS1 mice and reduce the expression level of p-NF-κB p65/NF-κB p65 in the brain of APP/PS1 mice.ELISA results showed that Ph Gs,ECH and ACT could significantly reduce the content of IL-1β in mouse cortex,and ACT could significantly increase the content of TGF-β1 in mouse cortex.(5)The results of ELISA showed that Ph Gs,ECH and ACT could significantly reduce the glutamic acid content in the cortical tissue of AD mice.Western blot results showed that Ph Gs,ECH and ACT could upregulate the expression of p-CAMKII/CAMKII in the cortex and hippocampus of APP/PS1 mice to a certain extent.Ph Gs,ECH and ACT can up-regulate the expressions of Homer-1 and SAP102 in the cortex and hippocampus of AD transgenic mice.Conclusion:(1)Based on network pharmacology results and existing literature research basis,cistanche phenylethanoid glycosides(Ph Gs,ECH and ACT),as the main active components of cistanche,may be the main material basis of cistanche in the treatment of AD,and it is predicted that the key pathways for the treatment of AD by cistanche may be the Toll-like receptor signaling pathway and NF-κB signaling pathway.(2)Ph Gs,ECH and ACT can improve the spatial cognition and memory dysfunction as well as the brain histopathological changes of APP/PS1 double transgenic mice.At the same therapeutic dose,the therapeutic effects of ACT were better than those of Ph Gs and ECH in the Morris water maze test and the dark avoidance test,and the therapeutic effect of ACT was slightly worse than that of Ph Gs and ECH in the platform test results,but there was no significant difference.In addition,the ACT treatment group had a better effect on the improvement of brain histopathology in AD mice.(3)Ph Gs,ECH and ACT can inhibit Aβ deposition and reduce glial cell activation,regulate microglia polarization type,reduce the production of inflammatory factors,and play a neuroprotective role.The phenotypic effects of the three drugs on microglia M1/M2 showed that among the treatment groups with the same doses of Ph Gs,ECH and ACT,although the promotion effects of Ph Gs and ECH on M2-type microglia of APP/PS1 mice in this study were not as good as that of ACT,but they also had certain treatment effects.In addition,Ph Gs,ECH and ACT inhibit the TLR4/NF-κB inflammatory pathway by regulating glial cell activation,and regulate the transcription events of inflammatory factors by the downstream transcription factor NF-κB p65 to reduce the neuroinflammatory response.Among them,among the Ph Gs,ECH and ACT treatment groups at the same dose,ACT could more significantly inhibit the TLR4/NF-κB signaling pathway and thus protect neurons from glial cell-mediated neuroinflammation.(4)Ph Gs,ECH and ACT reduced the level of glutamic acid in the cortical tissue of AD mice and increased the expression level of synapse-related proteins,thus playing a neuroprotective role.Among them,among the Ph Gs,ECH and ACT treatment groups at the same dose,ACT could more significantly increase the expression of synapse-related proteins in the brain of APP/PS1 mice.