| Objective: A network pharmacology and molecular docking approach was used to investigate the targets and related signaling pathways of Sulforaphane(SFN)in the treatment of Acute Carbon monoxide poisoning(ACOP)brain injury and to clarify its potential mechanism of action.Based on the results of the network pharmacology study,the AMPK signaling pathway was selected and a rat model was established to elucidate the molecular mechanism of SFN in the treatment of ACOPinduced brain injury.Methods: The 2D structure of lycopene was obtained by searching Pubchem,the predicted targets of compounds were obtained through Pharmmapper website,the genes related to ACOP brain injury were obtained through Gene Cards and OMIM databases,after that,the common targets of SFN and diseases were obtained by using Draw Venny Diagram online program,the STRING database was used to construct The protein interaction(PPI)network was constructed using the STRING database,the drug-target network map was constructed using Cytoscape 3.7.1software,and GO enrichment analysis and KEGG pathway analysis were performed using the Metascape database.Molecular docking of the simulated compound SFN with the core target AMPK was performed using Auto Dock software and finally visualized and analyzed by Pymol software.One hundred and thirty-five SPF-grade male SD rats were divided into normal control group(NC group),acute carbon monoxide poisoning group(ACOP group)and lycopodium sulfide(SFN)group according to random number table method,with 45 rats in each group.The SFN group was given intraperitoneal injection of sulforaphane aqueous solution [20mg/(kg.d),1 time/d],and the ACOP and normal control rats were given the same volume of saline.After 7d of the intervention,each group of rats was scored neurobehaviorally using the Morris water maze test.The histomorphological changes of cerebral cortex in each group were observed by hematoxylin-eosin(HE)staining,ultrastructural changes of cerebral cortex in each group were observed by transmission electron microscopy,neuronal damage in cerebral cortex and morphological and quantitative changes of Nisin vesicles were observed by Nisin staining,the expression levels of AMPK,DRP1,MFN2 and Caspase9 were detected by q-PCR,and the expression levels of P-AMPK,MFN2,DRP1,Caspase3 and Caspase9 were detected by immunohistochemistry.The expression levels of PAMPK,MFN2,DRP1,Caspase3 and Caspase9 in the brain tissues of rats were measured by q-PCR and immunohistochemistry and Western blotting.Results: Through screening,a total of 81 effective target genes of SFN and 36 common targets of SFN and ACOP brain injury were obtained,and relevant signaling pathways were obtained by GO function and KEGG pathway analysis,involving cancer pathway,hepatitis B pathway,prolactin signaling pathway,autophagy-animal signaling pathway,interleukin-17 signaling pathway,estrogen signaling pathway,drug metabolism,lifespan regulation signaling pathway,the serotonergic synapses,and transcriptional dysregulation in cancer.The molecular docking between AMPKα and SFN was selected,and the results showed that lycopene and AMPKα could bind to each other through hydrogen bond,hydrocarbon bond,electrostatic interaction force and van der Waals force,among which the hydrogen bond with two amino acid residues of GLY-99 and VAL-96 was the tightest.The results of animal experiments showed that the escape latency and swimming distance of the ACOP group were significantly longer than those of the NC group,and the platform dwell time was significantly shorter.Compared with the ACOP group,the escape latency and swimming distance were significantly shortened and the platform dwell time was prolonged in the SFN intervention treatment rats,which was statistically significant(P<0.05).HE staining of neuronal cells in the NC group showed intact cell structure,clear outline,blue nuclei and varying degrees of purplish red cytoplasm,neatly arranged and uniformly colored.In the ACOP group,the neuronal cells were disorganized,with nuclei fixed and coloration deepened,while in the SFN group,normal and abnormal cells were intermingled.SFN treatment effectively protected the ultrastructural and functional integrity of cortical tissue and reduced brain edema.Nissl staining under light microscopy showed that the number of Nissl vesicles in the ACOP group was significantly lower than that in the NC group,and the number of Nissl vesicles increased after SFN treatment(P<0.05).The q PCR results showed that the m RNA levels of all target genes in the NC group were stably maintained at baseline levels,with no significant differences between time intervals.Compared with the NC group,DRP1 and Caspase9 m RNA levels were increased and AMPK and MFN2 m RNA levels were decreased in the ACOP group(P<0.05).In addition,DRP1 and Caspase9 m RNA levels were significantly decreased and AMPK and MFN2 m RNA levels were significantly increased in the SFN group compared with the ACOP group(P<0.05).the mean optical density values of P-AMPK and MFN2-positive cells and their protein expression levels in the ACOP group tended to decrease at the early stage of intoxication(<7 d),and DRP1,The mean optical density values of Caspase3 and Caspase9 positive cells and their protein expression levels tended to increase at the early stage of intoxication(<7 d),which were significantly higher than those of the normal control group at the same time point(P<0.05).Although the protein expression trends of P-AMPK,MFN2,DRP1,Caspase3 and Caspase9 in the SFNtreated group were consistent with those in the ACOP group,the expression levels of two proteins,P-AMPK and MFN2,were significantly higher in the treated group than in the ACOP group at the same time point(P<0.05),and the expression levels of three proteins,DRP1,Caspase3 and Caspase9 The expression levels of DRP1,Caspase3 and Caspase9 were significantly lower than those of the ACOP group at the same time point(P<0.05).Conclusion: Sulforaphane exerts therapeutic effects on ACOP-induced acute brain injury through multiple targets and pathways,of which AMPK signaling pathway is one of the most important ones.Early application of SFN can effectively inhibit apoptosis,improve mitochondrial ultrastructure damage,and regulate the expression of mitochondrial fusion splitting protein through activation of AMPK signaling pathway to inhibit apoptosis,which in turn exerts neuroprotective effects in acute brain injury of ACOP. |
