Study On Mechanism Of Jingfang Granules In Treating Intracerebral Hemorrhage

Objective: 1.Explore the therapeutic effect of Jingfang Granules(JFG)on intracerebral hemorrhage(ICH)in rats 2.Exploring the mechanism of action of JFG in the treatment of ICH in rats through network pharmacology and metabolomics.Methods: 1.Collagenase Ⅳ was used to construct the ICH model in rats,and the protective effect of JFG on ICH in rats was evaluated by elevated body swing,beam balance,brain water content,MRI and Evans blue permeability.2.The active ingredients and targets of 11 medicinal materials in JFG were analyzed by the Traditional Chinese Medicine Systems Pharmacology(TCMSP)database,and the targets related to ICH were searched in OMIM,TTD and Genecard disease databases.The intersection of JFG and ICH targets were analyzed by PPI analysis to get the core targets,then the core targets were analyzed by GO functional annotation and KEGG pathway enrichment analysis,to reveal the targets and pathways of JFG in the treatment of ICH.3.Western Blot test was used to verify the core targets and pathways screened in network pharmacology.4.Metabonomics was used to analyze the hemorrhagic side brain tissue of rats treated with JFG,to study the changes of metabolites in rats after JFG intervention,and to explore the metabolic pathways involved by KEGG enrichment analysis.Results: 1.Collagenase Ⅳ successfully constructed the ICH model in rats.On days 3,7 and 14 after ICH,the beam balance test scores(P < 0.01,P < 0.001,P < 0.05)and the elevated body swing test scores(P < 0.05,P < 0.001,P < 0.001)of the model group were significantly increased compared with the sham group.At the same time,the beam balance test score on day 3 and day 7 after ICH was significantly decreased in highdose JFG group(P < 0.001,P < 0.01),and the elevated body swing test score on day 7and day 14 after ICH was significantly decreased in high-dose JFG group(P < 0.05,P< 0.05).The results of brain water content showed that on day 3 after ICH,compared with the sham group,the brain water content of the model group increased significantly(P < 0.01),and low-dose JFG administration significantly reversed this trend(P < 0.05).The Evans blue permeability test showed that on day 3 after ICH,compared with sham group,the Evans blue permeability of rats in model group was significantly increased(P < 0.01),and Evans blue permeability was significantly decreased after high dose of JFG(P < 0.05).2.The results of network pharmacological experiments indicate that JFG might through active components such as luteolin,(+)-Anomalin,Phaseol,quercetin as well as kaempferol,acted on pathways in cancer,lipid and atherosclerosis,fluid sheer stress and atherosclerosis,and so on,regulated 39 proteins such as AKT1,TNF,TP53,IL6,VEGFA,CASP3,IL1β,JUN,ESR1,MAPK3,played a protective role in cerebral hemorrhage.3.Western Blot assay results showed that compared with sham group,the expressions of TNF-α and IL-1β in model group were significantly increased(P < 0.05,P < 0.05),and the expressions of TNF-α and IL-1β were significantly decreased after low-dose JFG administration(P < 0.05,P < 0.01).Compared with the sham group,the Occludin and Claudin-5 expression of model group was significantly decreased(P < 0.01,P < 0.001),and the Occludin and Claudin-5expression of model group was significantly increased after high-dose JFG administration(P < 0.05,P < 0.05).The Occludin expression of JFG was significantly increased after low dose administration(P < 0.01).Compared with the sham group,the expression of MMP9 in the model group was significantly increased(P < 0.0001),while the expression of MMP9 protein in the high-dose JFG group was significantly decreased(P < 0.0001).4.The metabolomics of bleeding side brain tissue showed 222 different metabolites between the model group and the sham group,and 62 different metabolites between the JFG administration group and the model group.After the comparison of differential metabolites between the two groups,it was found that the contents of 44 differential metabolites were reversed after JFG administration,such as taurine,L-valine,trigonelline,and so on.Subsequently,the differential metabolic pathways of each group were analyzed,and it was found that taurine and taurine metabolism,valine,leucine and isoleucine biosynthesis,primary bile acid biosynthesis,purine metabolism and other metabolic pathways were involved in the changes of the above metabolites.Conclusion: JFG can play a protective role in intracerebral hemorrhage,relieve neurological injury,reduce the degree of cerebral edema,and protect the integrity of the blood-brain barrier after intracerebral hemorrhage.The mechanism may be related to JFG act on PI3K/AKT play anti-inflammatory and anti-blood-brain barrier damage,and regulation of metabolic pathways.