Studies On The Protective Effect And Mechanism Of Nuciferine On Ischemic Brain Injury In Rats

Objective:The middle cerebral artery occlusion（MCAO）was used to establish the model of cerebral ischemia in rats.The effect of nuciferine on anti-ischemic stroke was evaluated by behavioral score,pathology and molecular detection.Metabolomics technology was used to analyze the changes of endogenous small molecules in serum samples and the differential metabolites related to ischemic stroke was found.Then,we explored the anti-ischemic stroke mechanism of nuciferine（Nuci）.Methods:1.SD rats were divided into 6 groups randomly including the sham group,the MCAO model group,the EGb 761 group(100 mg·kg^-1),and the Nuci group(low,medium,and high doses were 10,20,and 40 mg·kg^-1 respectively).MCAO ischemic stroke model rats were administered with Nuci and positive drug（EGb 761）as intervention drugs,neurological deficit scores,infarct volume and cerebral water content were detected to evaluate the pharmacodynamics.2.UPLC-Q-TOF/MS metabolomics was used to analyze the effect of Nuci on the endogenous substances in the serum of MCAO model rat,and multivariate statistical analysis was used to confirm the differential metabolites and related genes and enzymes,then the metabolic pathways the metabolites involving in were predicted.3.The expression of NOD like receptor 3（NLRP3）protein in the ischemic brain was detected by Western blot.The levels of tumor necrosis factor-α（TNF-α）,interleukin-18（IL-18）and interleukin-1β（IL-1β）were detected by enzyme-linked immunosorbent assay（ELISA）.Results:1.In the experiment of evaluating the effect of nuciferine,after the MCAO model was established,the neurological deficit scores,infarct volume and cerebral water content in the model group were significantly higher than those in the sham group（P<0.001）,indicating that the MCAO modeling was successful.After administration of Nuci and EGb 761,the scores of rats were decreased.Compared with the MCAO group,Nuci-H、Nuci-M and EGb761 groups were able to significantly reduce the scores（P<0.01 or P<0.05）,but there was no significant difference in the Nuci-L group（P>0.05）.Compared with MCAO group,infarct volume of rat in the Nuci-H、Nuci-M and EGb 761 groups were significantly reduced（P<0.05,P<0.01 or P<0.001）.After administration of Nuci-H and EGb 761,cerebral water content were significantly reduced（P<0.05）.2.The metabolites in the serum samples unmodeled,after modeling and after administration of Nuci were detected respectively by the UPLC-Q-TOF/MS technology.QI software,HMDB and literature were used to screen the differential metabolites between different groups.Based on the identified differential metabolites,the metabolic pathways and related enzymes and genes were identified by Cytoscape and Gen CLi P software.The content of L-Lactic acid,Isobutyrylglycine,Sebacic acid,Linoleic acid,Glucosylgalactosyl hydroxylysine,Docosahexaenoic acid and Oxoglutaric acid in the serum samples of MCAO model rat were up-regulated compared to the sham group.While the content of Hydroxyphenylacetylglycine,N-Methylnicotinium,N2-Succinyl-L-ornithine,Tauroursodeoxycholic acid,Sphingosine-1-phosphate,Sphinganine-1-phosphate,2-Hydroxyestradiol-3-methyl ether,Glutamylphenylalanine,Vitamin D2-3-glucuronide,Neoxanthin,Stearoylcarnitine and Petroselinic acid were down-regulated in the serum samples of MCAO model rat compared to the sham group.Linoleic acid,Tauroursodeoxycholic acid,Sphingosine-1-phosphate,Sphinganine 1-phosphate,2-Hydroxyestradiol-3-methyl ether,Vitamin D2-3-glucuronide,Neoxanthin,Stearoylcarnitine,Glucosylgalactosyl hydroxylysine,Docosahexaenoic acid and Petroselinic acid were up-regulated in serum samples after Nuci-H was administrated.While L-Lactic acid,Hydroxyphenylacetylglycine,Isobutyrylglycine,N-Methylnicotinium,N2-Succinyl-L-ornithine,Sebacic acid,Glutamylphenylalanine and Oxoglutaric acid in serum samples were down-regulated after Nuci-H was administrated.Further analysis revealed that phospholipase A2（PLA2）,12/15-lipoxygenase（12/15-LOX）,sphingosine kinase 1/2（SPHK1/2）,isocitrate dehydrogenase enzymes（IDH）,glutamate oxaloacetate transaminase 1（GOT1）,alanine glyoxylate transaminase 2（AGXT2）,cytochrome 2B（CYP2B）,fatty acid desaturase（FADS）,dihydrolipoamide dehydrogenase（DLDH）and glutamate-pyruvate transaminase（GPT）may be the main participants involving in the anti-ischemic stroke process of Nuci.3.The results of ELISA showed that the levels of TNF-α,IL-18 and IL-1βin serum significantly increased after MCAO（P<0.01）.Compared with the MCAO group,Nuci-H,Nuci-M and EGb 761 were abled to significantly reduce the content of TNF-α、IL-18（P<0.01or P<0.05）.Nuci and EGb 761 were abled to significantly reduce the level of IL-1β（P<0.01）.It suggested that the inflammatory state was relieved after administration.Western Blot results showed that,the expression of NLRP3 in the MCAO group was significantly higher than those in the sham group（P<0.01）;compared with the MCAO model group,Nuci can significantly reduce the expression of NLRP3 in ischemic brain tissue（P<0.01 or P<0.05）.Conclusion:Metabolomics technology and pharmacodynamics analysis were used to clarify that Nuci may exert anti-ischemic stroke effect and protect rats from ischemic stroke mainly by affecting the Sphingolipid metabolism,energy metabolism（glycolysis,gluconeogenesis and tricarboxylic acid cycle）and metabolic stress.The results of molecular pharmacology showed that the anti-stroke mechanism of Nuci may be related to the inhibition of inflammation and the NLRP3 signaling pathway,down-regulation of NLRP3 expression,and decrease the contents of TNF-α,IL-18 and IL-1β.These findings provide an new insight and reference of the anti-ischemic stroke mechanism of Nuci.