Mechanism Of Floralozone Improving Cognitive Dysfunction In Vascular Dementia Rats By Reducing TRPM2 Expression And Activating NMDAR Signaling Pathway

BackgroundVascular dementia（VD）refers to the severe cognitive dysfunction syndrome caused by ischemic stroke,hemorrhagic stroke and cerebrovascular diseases resulting in hypoperfusion of brain areas such as memory,cognition and behavior,and is the second largest type of dementia after Alzheimer’s disease.Studies have shown that chronic cerebral ischemia can increase the risk of VD and aggravate the disease course of VD.Therefore,it has become a hot topic to explore the pathogenesis of VD through chronic cerebral ischemia model.Floralozone,an essence derived from HERBA LAGOTIS,has been shown to improve atherosclerosis in rats caused by high-fat diet.However,whether it will play a beneficial role in VD remains unclear.ObjectiveThe purpose of this study was to investigate the effect of Floralozone on cognitive function in VD rats induced by chronic cerebral ischemia and its underlying mechanism.Methods（1）The drug target of Floralozone was retrieved through BATMAN-TCM and Swiss Target Prediction databases;Gene Cards,OMIM,and DRUGBANK were used to identify vascular dementia disease targets;Protein-Protein Interaction networks（PPI）was constructed using STRING11.5 database and Cytoscape3.9.0 software,and the potential functional modules and core networks of proteins in PPI were explored;Gene ontology（GO）enrichment analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway enrichment analysis were performed by Metascape database;the“drug-target-pathway”network diagram was constructed with Cytoscape3.9.0 software;PPI and drug-target-pathway network were verified by molecular docking technology.（2）Cell experiment:The brains of suckle rats born 0-24 h were taken out in a sterile environment and hippocampal tissue was rapidly stripped.The hippocampal tissue was cut into fragments and digested with 0.25%trypsin（without EDTA）for 10 min.Then,DMEM/F12 medium was used for culture.The primary nerve cells cultured for 7 days were used in the experiment.The experiment was divided into 8 groups:（1）Control group;（2）VD model group;（3）Floralozone group;（4）VD+Floralozone（25μmol/L）group;（5）VD+Floralozone（50μmol/L）;（6）VD+Floralozone（100μmol/L）group;（7）VD+m NGF（0.1μmol/m L）group;（8）VD+Floralozone（100μmol/L）+m NGF（0.1μmol/m L）group.The protein expression of TRPM2 and NR2B in primary nerve cells was detected by immunofluorescence and western blot.（3）Animal experiment:The vascular dementia model was established by ligation of bilateral common carotid arteries.The neurologic and learning functions of the rats were evaluated by muscle strength test,Bederson score and Morris Water maze test.Hematoxylin-eosin staining and Neu N staining were used to observe the pathological changes of hippocampal neurons;TUNEL staining kit was used to detect the apoptosis of hippocampal neurons in rats;the number and length of dendritic spines of rat hippocampal neurons were observed by Golgi staining;the contents of SOD,MDA and GSH in serum of rats were detected by Elisa kit;the protein expression level of TRPM2 and NR2B in rat hippocampus was detected by immunofluorescence and Western Blot.（4）TRPM2^-/-mice experiment:the experiment was divided into four groups:（1）C57control group;（2）TRPM2^-/-mice group;（3）TRPM2^-/-mice+Floralozone（100 mg/kg/day）group;（4）TRPM2^-/-mice+m NGF（9μg/day）group.The protein expression level of NR2B was detected by immunohistochemistry.Results（1）Network pharmacology results showed that:AKT1,CASP3,NMDAR2B,TRPM2,TRPV1,GRIN1,AR,MAPK14,PSEN1,GRIN2,CGRM1,HTR3A,PTGS2 and other target proteins may be the core targets of Floralozone in the treatment of VD.GO enrichment analysis showed that Floralozone may through regulating nerve receptor activity and postsynaptic neurotransmitter activity,behavior,and translation initiation and other biological processes play to the role of the treatment of VD,KEGG pathway analysis showed that Floralozone may be affected by neural activity ligand receptor interaction,dopaminergic synapses,apoptosis play a role of resistance to VD,etc.（2）In vitro experiment results showed that:（1）H₂O₂can induce the decrease of NR2B protein expression level and the increase of TRPM2 protein expression level in primary nerve cells;（2）Floralozone significantly decreased the protein expression of TRPM2 and increased the protein expression of NR2B in primary nerve cells.（3）In vivo experimental results showed that:（1）Floralozone could significantly improve the neurological function of VD rats;（2）The Morris water maze experiment showed that Floralozone significantly improved spatial learning and memory in VD rats;（3）Floralozone can improve endothelial dysfunction in rats by reducing oxidative stress level;（4）Floralozone has protective effect on hippocampal neurons of VD rats;（5）The decrease of dendritic spines of hippocampal neurons in VD rats was significantly reversed by Floralozone;（6）Floralozone significantly up-regulated NR2B protein expression and down-regulated TRPM2 protein expression in hippocampus.（4）The results of TRPM2^-/-mouse experiment showed:the effect of Floralozone on VD was closely related to the decrease of TRPM2 protein expression.ConclusionFloralozone can improve cognitive dysfunction in VD rats induced by chronic cerebral ischemia by reducing TRPM2 expression and activating NMDAR signaling pathway.