Effects Of Olfactory Stimulation With Food And Drug Homology On Learning And Memory In AD Model Rats

ObjectiveAt present,China is accelerating into an aging society,and the incidence and mortality of Alzheimer’s disease(AD)in middle-aged and elderly people in China are increasing,which brings great economic and care pressure to families,and also poses new challenges to the society on how to care for these patients.It is essential to seek new and effective prevention and treatment methods.However,the treatments approved by the US Food and Drug Administration(FDA)are not only expensive,having many side effects and only relieving symptoms.Olfactory dysfunction is confirmed to occur in the early stages of AD,and olfactory stimulation has been widely used in the prevention of cognitive impairment.Therefore,the interaction network between essential oil components used for olfactory stimulation and ADrelated targets was constructed to predict potential targets and mechanisms of action for treatment,and to provide theoretical support for later experimental verification.This study aims to investigate the effect of olfactory stimulation on the learning and memory ability of rats with Aβ1-42-induced AD,and to explore the possible mechanism,so as to provide new ideas and theoretical support for the treatments of AD.Methods1.The corresponding targets of essential oil components were obtained through TCMSP,Swiss Target Prediction and Pharm mapper databases.The AD-related targets were obtained through Gene Cards,OMIM,Pharm GKB,TTD and Drug Bank databases.The two targets were intersected by R software to screen the anti-AD targets of olfactory stimulation.2.STRING database and Cytoscape 3.8.0 software were used to construct the related protein interaction network,and R software was used to enrich the selected key targets,and then the results were visualized.3.Molecular docking: The target proteins were searched in Uni Prot database,and the corresponding files were downloaded from PDB database and Pub Chem website.The molecular docking simulation was carried out by Py MOL2.4,Chem Draw,Auto Dock Tools1.5.6 and Auto Dock Vina1.1.2 software.4.Neonatal SD rats were randomly divided into sham group,AD model group,olfactory stimulation group,olfactory stimulation combined with enriched environment group and memantine treatment group.The rats in the two stimulation groups received corresponding stimuli from the third day after birth until the end of the behavioral test.The AD model was established by intracerebroventricular injection in neonatal SD rats at the age of five weeks.Memantine was administrated by oral gavage(10mg/kg/day)from the second day after operation until the rats were killed at the end of the behavioral test.5.The spontaneous activity test,Y maze,Morris water maze and multifunctional closed maze were used to detect the learning and memory ability of rats.6.Immunofluorescence was used to detect the expression of DHE and Nrf2 in the hippocampus and cortex.7.Western Blot was used to detect the expression of oxidative stress marker proteins Nrf2,Keap1,HO-1,NQO1,HIF1 A and synapse-associated proteins NMDAR,PSD95,n NOS,SYP and GAP-43.8.Biochemical kits were used to detect the expression of oxidative stress markers GSH and GSSG.The expressions of cholinergic system markers Ach and ACh E were detected.9.Transmission electron microscopy was used to observe the synaptic ultrastructure in the CA1 region of hippocampus.Results1.A total of 381 targets corresponding to essential oil components and 9128 AD-related targets were screened out through relevant databases,and 311 intersection targets were obtained.The PPI network contained 310 nodes and 3824 edges,and the average degree value of nodes was 24.7,and 40 key targets were screened.The key targets were mainly enriched in the biological process of cellular oxidative stress response.Sixty-eight signaling pathways were identified by KEGG enrichment analysis.2.The binding energy scores of molecular docking showed that all the five compounds had good binding effects with the target proteins Nrf2,HO-1 and HIF1 A.3.Results of the spontaneous activity test: compared with the sham group,the AD model group showed no significant differences in the total time and distance of activity and the time and distance of activity in the central region.Compared with the AD model group,the olfactory stimulation group showed no significant difference in the above experimental results.4.Y-maze results: There was no significant difference in the number of arm entries among the five groups of rats.Compared with the sham group,the spontaneous alternation response rate was significantly decreased in the AD model group(P < 0.01).However,the rate of spontaneous alternate-response in the olfactory stimulation group and memantine treatment group was significantly higher than that in the AD model group(P < 0.01).5.Results of the multifunctional closed maze: Compared with the sham group,the AD model group had a significantly increased water-seeking latency(P < 0.01),while the olfactory stimulation group had a reduced waterseeking latency(P < 0.05).6.Morris water maze results: Compared with the sham group,the AD model group had a significantly increased escape latency(P < 0.01),a significantly reduced number of platform crossings(P < 0.05),and significantly reduced the time spent in the target quadrant and the proportion of moving distance in the target quadrant(P < 0.05).Compared with the AD model group,olfactory stimulation reduced the escape latency(P < 0.01)and increased the number of crossing the platform(P < 0.01)and the time spent in the target quadrant(P < 0.01).7.Effect of olfactory stimulation on oxidative stress: Compared with the sham group,the expression of DHE fluorescent positive substances was stronger in the hippocampus and cortex of the AD model group rats(P < 0.01).Compared with the AD model group,the expression of DHE fluorescence positive substances in the hippocampus and cortex of the olfactory stimulation group was weaker(P < 0.01).Compared with the sham group,the expression of Nrf2(P < 0.05),HO-1(P < 0.01)and NQO1(P < 0.05)protein in the hippocampus of the AD model group was decreased,while the expression of Keap1(P < 0.01)was increased.Olfactory stimulation up-regulated the protein expression of Nrf2(P < 0.05),HO-1(P < 0.01)and NQO1(P < 0.05),and decreased the protein expression of Keap1(P < 0.01).Compared with the sham group,the expression level of GSH in the AD model group decreased(P < 0.01),and the expression level of GSSG increased(P < 0.01).Olfactory stimulation increased GSH expression(P < 0.01)and decreased GSSG expression(P < 0.01)compared with the AD model group.8.The effect of olfactory stimulation on synaptic function and neurotransmitters: compared with the sham group,the number of synapses in the AD model group was less,while the number of synapses in the olfactory stimulation group was more than that in the AD model group.In terms of morphological structure,the postsynaptic density increased and the synaptic cleft was blurred in the AD model group.Olfactory stimulation could reverse these morphological changes to a certain extent.Compared with the sham group,the expression of NMDAR2a(P < 0.05),NMDAR2b(P < 0.01),PSD95(P < 0.05),SYP(P < 0.01)and GAP-43(P < 0.05)in the hippocampus of the AD model group was decreased,and the expression of n NOS(P < 0.05)was promoted.Olfactory stimulation reversed this pathological change.Compared with the sham group,the expression of Ach in the AD model group was lower but not statistically significant,and the expression of ACh E was increased in the AD model group(P < 0.01).Olfactory stimulation reduced ACh E expression compared with the AD model group(P < 0.01).Conclusions1.Forty core targets of olfactory stimulation against AD were screened by network pharmacology.2.Olfactory stimulation reduces oxidative stress by activating Nrf2 signaling pathway to improve cognitive impairment in AD animal models.3.Olfactory stimulation improves cognitive impairment in AD animal models by regulating the expression of synaptic function-related proteins and neurotransmitters.