A Network-based Method For Mechanistic Investigation And Neuroprotective Effect On Post-treatment Of Senkyunolid-H Against Cerebral Ischemic Stroke In Mouse

Background and objective:Cerebral ischemic stroke(CIS)belongs to stroke in traditional Chinese Medicine,which has high incidence rate,high mortality rate,high disability rate and high recurrence rate.It is one of the most serious diseases threatening human life and health.Published studies showed that the lactones of Ligusticum chuanxiong are the main active and metabolic components in traditional Chinese medicine Ligusticum chuanxiong They have the pharmacological effects of anti-oxidative damage,anti-inflammatory,anti-coagulant,anti-platelet aggregation,vasodilation,cytotoxin and so on Senkyunolide-H(SEH)belongs to senkyunolide lactones,which has good liposoluble and water-soluble properties and can enter into the blood and cerebrospinal fluid rapidly.Therefore,this study aims to employ network pharmacology to reveal potential mechanism of SEH against CIS on a system level,and to confirm the therapeutic effects and mechanism of SEH on CIS by models of cerebral ischemia-reperfusion(I/R)in vivo and in vitro.And this study provides innovative ideas for the development of traditional Chinese medicine in the treatment of CIS.Methods:1.We predicted the mechanism of SEH in the treatment of CIS via network pharmacology.The aims to predict the targets of SEH with pharmapper server and to obtain CIS related targets with OMIM database and disgenet database.The key targets of the treatment of SEH on CIS were selected by network merging and topological parameter analysis with Cytoscape 3.5.1.The key targets were analyzed by the database of DAVID and KEGG for the function enrichment and pathway analysis.2.The aim to verify the effect and mechanism of SEH intervention on I/R injury via in vivo experiments.140 male ICR mice(8 weeks old)were randomly divided into 5 groups(n=28 in each group),namely,sham operation group,SEH+sham operation group(40mg/kg),model group,low-dose SEH group(20mg/kg),high-dose SEH group(40mg/kg).The model of middle cerebral artery occlusion and reperfusion(MCAO/R)in mice was made.After 0?6 and 24 hours of ischemia-reperfusion(n=14 in each group;7 animals for histological analysis and 7 animals for biochemical analysis),the mice were killed by cervical dislocation method and their brains were taken for reserve;the differences of nerve function and infarct volume in each group were compared by neurobehavioral score and TTC staining;the NeuN in CA1 area of hippocampus in ischemic side of mice was detected by immunohistochemistry;the protein expressions of PI3K,p-PI3K p85(Tyr 458),Akt,p-Akt(Ser 473),NF-?B,Bcl-XL and Cleaved-Caspase3 in the ischemic penumbra of mice were determined by western blotting.3.The aim to verify the effect and mechanism of SEH intervention on I/R injury via in vitro experiments.The model of oxygen glucose deprivation and reperfusion(OGD/R)in PC 12 cells was established.The normal control group,the model group without drug administration,groups of different concentrations of SEH(25,50,100?M)and the inhibitor groups(LY294002 10?M)were set up.The cell activity was detected by CCK-8 method,and the expressions of the target proteins in each group were observed by western blotting.Results:1.Through network pharmacology analysis,62 key targets of the SEH treatment on CIS were obtained by screening topological indices and analyzed for Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment.GO analysis indicated that SEH might have a role in treating CIS via regulating some biological processes including regulation of transcription from RNA polymerase ? promoter,epidermal growth factor receptor signaling pathway,phosphatidylinositol-mediated signaling,and some molecular function,such as transcription factor and protein phosphatase binding,nitric oxide synthase regulator activity.Meanwhile,KEGG analysis indicated the mechanisms might be concerned with PI3K/Akt,ErbB,neurotrophin,FOXO and estrogen signaling pathways.Among them,the regulation of PI3K-Akt signaling pathway might be one of the most crucial mechanisms.2.We found that compared with MCAO model group,the nerve function score and infarct volume of SEH groups were significantly reduced in vivo experiments;with the prolongation of ischemia-reperfusion time,the number of NeuN-positive neurons in CA1 area of hippocampus in mouse ischemic side was gradually reduced,but the number of NeuN-positive neurons in SEH groups were significantly higher than that of MCAO model groups;with the prolongation of ischemia-reperfusion time,the level of Bcl-XL protein and the ratio of p-PI3K p85/PI3K and p-Akt(Ser 473)/Akt protein levels in ischemic penumbra area were significantly higher than those in the model group;in addition,the expression of Cleaved-Caspase3 and NF-?B protein in the model group increased with the prolongation of reperfusion time,but those of the SEH treatment groups were decreased than the model group(p<0.05).There was no significant difference between the sham operation group and SEH treatment sham operation group(p>0.05).3.CCK-8 results showed that the cell viability of OGD group was significantly lower than that of normal control group,but in OGD/R group treated with 50?M and 100?M SEH,the cell viability gradually increased with the increase of SEH concentration,and especially in the group of 100?M SEH.The results of western blotting showed that after OGD/R,the expression of Bcl-XL protein and the ratio of p-PI3K p85/PI3K and p-Akt(Ser 473)/Akt protein level were significantly decreased,the level of Cleaved-Caspase3 and NF-?B protein were increased,but after 100?M SEH treatment,the expression of Bcl-XL protein and the ratio of p-PI3K p85/PI3K and p-Akt(Ser 473)/Akt protein level were significantly increased,and the level of Cleaved-Caspase3 and NF-?B protein were decreased,but the effect of 100?M SEH was inhibited by 10?M LY294002(p<0.05).Conclusion:1.SEH carries a therapeutic potential in CIS involved multiple targets and pathways,and the regulation of PI3K-Akt signaling pathway might be one of the most crucial mechanisms.2.SEH can improve the neurological function,reduce the volume of cerebral infarction and cell apoptosis induced by MCAO/R in mice.3.SEH can improve cell viability and reduce the apoptosis of PC 12 cells induced by OGD/R.4.SEH treatment on CIS may be through the regulation of PI3K/Akt and NF-?B signaling pathway to increase the anti-apoptosis capacity.