The Role And Mechanism Of Butylphthalide In Improving Emotional And Cognitive Dysfunction After Ischemic Stroke

Objective:(1)Clarifying the effect of butylphthalide on emotional and cognitive dysfunction in ischemic stroke model rats.(2)Predicting the targets of butylphthalide in improving emotional and cognitive dysfunction after ischemic stroke based on network pharmacology.(3)Elucidating the underlying molecular mechanisms of butylphthalide in improving emotional and cognitive dysfunction after ischemic stroke.Chapter 1: introduction.Chapter 2: butylphthalide improves emotional and cognitive dysfunction in ischemic stroke model rats.Establishment of a rat middle cerebral artery occlusion(Middle cerebral artery occlusion,MCAO)model,intraperitoneal injection of butylphthalide(10 mg/kg once daily for 14 days)or vehicle.Laser speckle contrast imaging,neurological functional assessment,and 2,3,5-triphenyltetrazolium chloride staining were used to evaluate whether the model was successful.The results showed that the cerebral blood flow decreased,the percentage of cerebral infarction area and the neurological function score increased in the MCAO model rats.Anxiety-like behavior in rats was detected by open field test,and it was found that butylphthalide increased the percentage of time spent in the central area of MCAO model rats.Depression-like behavior in rats was detected by sugar preference test,and it was found that butylphthalide increased the percentage of sugar preference of MCAO model rats.The spatial memory ability of rats was detected by Morris water maze and Y maze,and it was found that butylphthalide reduced the latency to the platform,increased the percentage of residence time in the target quadrant and the spontaneous alternation rate in MCAO model rats.The novel object recognition test was used to detect the non-spatial recognition and memory ability of rats,and it was found that butylphthalide increased the recognition index of MCAO model rats.The three-chamber social interaction test was used to detect the social memory ability of the rats and it was found that butylphthalide increased the social recognition index of the MCAO model rats.Chapter 3: predicting the target of butylphthalide in improving emotional and cognitive impairment after ischemic stroke based on network pharmacology.The structure and SMILES of butylphthalide were obtained from the Pub Chem database.The drug targets of butylphthalide are predicted by Swiss Target Prediction,Pharm Mapper,Ch EMBL,Super Pred,and Target Net databases,and a total of 437 target genes were obtained.Dis Ge NET,Therapeutic Target Database,Mala Cards,and Comparative Toxicogenomics Database were used to obtain disease targets for post-ischemic stroke anxiety(PSA),post-ischemic stroke depression(PSD),and post-ischemic stroke cognitive impairment(PSCI);a total of355 PSA targets,385 PSD targets,and 248 PSCI targets were obtained.Then take the union to get the common target of drug-disease,a obtain65 common targets of butylphthalide-PSA,71 common targets of butylphthalide-PSD,and 53 common targets of butylphthalide-PSCI.The protein-protein interaction(PPI)analysis was performed through the STRING database,and the network was analyzed and visualized using Cytoscape software.The PPI network of the butylphthalide-PSA common targets has 65 nodes and 598 edges,and the node Degree value is 0-52.The PPI network of the butylphthalide-PSD common targets has 71 nodes and 659 edges,and the node Degree value is 0-54.The PPI network of the butylphthalide-PSCI common targets has 53 nodes and460 edges,and the node Degree value is 1-43.And in the PPI analysis of butylphthalide-PSA/PSD/PSCI,the top 15 nodes of Degree value are highly overlapping.Gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis was performed using the DAVID database.238,256,211 GO＿BP entries and 79,77,79 KEGG pathways were obtained in the GO and KEGG enrichment analysis of the common targets of butylphthalide-PSA/PSD/PSCI,respectively.The biological processes and pathways obtained by the enrichment analysis are mainly associate with the regulation of apoptosis,inflammation and oxidative stress.Chapter 4: molecular mechanism of butylphthalide in improving emotional and cognitive impairment after ischemic stroke.The levels of inflammatory factors;the expression of TLR4/NF-?B signaling pathway molecules;the expression of microglia markers;the expression of PANoptosis molecules;the expression of synapse-related proteins;the levels of oxidative stress-related indicators were detected by enzyme-linked immunosorbent assay,real-time fluorescence quantitative PCR,western blot etc.The results show that:(1)Butylphthalide down-regulated the levels of inflammatory factors TNF-α,IL-1β and IL-6 in plasma and brain tissue of MCAO model rats.(2)Butylphthalide down-regulated the levels of TLR4 and p-NF-κB p65 in the hippocampus of MCAO model rats.(3)Butylphthalide inhibited the expressions of Iba1,i NOS,CD68 and increased the expressions of CD206 and Arg-1 in the hippocampus.(4)Butylphthalide down-regulated the levels of PANoptosis molecules caspase-1,caspase-3,caspase-8,GSDMD and MLKL in the hippocampus of MCAO model rats.(5)Butylphthalide increased the level of PSD95 in the hippocampus of MCAO model rats.(6)Butylphthalide down-regulated the levels of ROS and MDA in the cortex and hippocampus of MCAO model rats.Conclusion: Butylphthalide effectively ameliorate the emotional and cognitive dysfunction in MCAO model rats.The mechanism could be that butylphthalide inhibits the activation of TLR4/NF-κB pathway,inhibits the inflammatory response,reduces the PANoptosis level of the hippocampus,and at the same time inhibits oxidative stress,enhances synaptic plasticity,and exerts neuroprotective effects.