Based On Network Pharmacology Strategy And UHPLC-Q-Exactive Orbitrap-MS To Investigate The Active Components And Mechanism Of SPLAnti-CIRI

Objective:To investigate the main active component of total lignans of Syringa pinnatifolia Hemsley Lignans（SPL）against cerebral ischemia and reperfusion injury（CIRI）,and further investigate the effect of this active ingredient on CIRI and its underlying mechanism.Methods:1.Studied the effect of the SPL medicated plasma on oxygen glucose deprivation/reoxygenation（OGD/R）hippocampal neuron cell（HT22）cells by plasma pharmacological method.40 adult Wistar rats were randomly divided into 4 groups（n=10 per group）:control group（pretreated with CMC-Na,intragastrically）,SPL groups pretreated with SPL at three dosages（6.25 mg/kg,13.50 mg/kg and 27.00 mg/kg,intragastrically,for 7 d）.The plasma containing SPL was prepared.HT22 cells were divided into 6 groups:Control group（10%blank plasma+HT22 cells）,OGD/R model group（10%blank plasma+HT22 cells+OGD/R）,Edaravone group（200μM Edaravone+HT22 cells+OGD/R）,SPL low dose group（10%plasma with low dose SPL+HT22 cells+OGD/R）,SPL medium dose group（10%plasma with medium dose SPL+HT22 cells+OGD/R）,SPL high dose group（10%plasma with high dose SPL+HT22 cells+OGD/R）.HT22 cells were incubated with above medicated medium respectively for 12h and treated with OGD/R conditions（deprived oxygen and glucose for 6 hour,then quickly reoxygenated for 12 hours）.Observed the effect of SPL medicated plasma on the survival rate and related oxidative stress indexes of OGD/R HT22 cells,reflecting the protective effect of SPL on CIRI in vitro.2.Network pharmacology-based strategy to investigate the active components and mechanism of SPL Anti-CIRINCBI Pubmed and CNKI databases were used to explore the active ingredients of SPL,Swiss target prediction was used to predict the target of SPL.Gene Cards,Drugbank,OMIM databases were applied to retrieve the target of the CIRI.Obtain key targets of SPL anti-CIRI:mapped the component-targets interaction networks and CIRI-targets interaction networks through STRING 11.5.The two interaction networks were integrated by“Merge”in Cytoscape 3.9.1,calculated the“Degree”value via“Network Analyzer”,screened for the targets with degree 2 times larger than average and the targets which BC,DC greater than the median value.Subsequently,Cytoscape 3.9.1 was used to construct the networks of“component-key target”to find the main active ingredients and key targets of SPL anti-CIRI.Done the Gene Ontology（GO）analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis for the key targets by DAVID database,draw histogram and bubble charts on the analysis results by the bioinformatics website.Cytoscape 3.9.1 was used to construct network of"component-key target-pathway".Combining relevant literatures,searched for the related pathways of SPL against CIRI from “component-key target-pathway”.3 The study of effect and mechanism of SPL active ingredient,（-）Secoisolariciresinol,（SECO）,on OGD/R HT22 cellsBased on the results of the experiments"2",investigated the anti-CIRI effects and underlying mechanism of SECO,one of the main active ingredients of SPL.3.1 High Performance Liquid Chromatography（HPLC）determined the contents of SECO in SPL;3.2 UHPLC-Q-Exactive Orbitrap-MS was used to determine whether SECO was the main blood component of SPL;3.3 The pharmacological effects of SECO were studied on OGD/R HT22HT22 cells were cultured in vitro and randomly divided into 6 groups:Control group（DMEM）,Model group（OGD/R+DMEM）,Edaravone group（OGD/R+DMEM+200μM Edaravone）,SECO low,medium,and high dose group（OGD/R+DMEM+40μM,80μM,160μM SECO）.The HT22 cells in above groups（except Control group）were quickly reoxygenated for 12 hours after oxygen and glucose deprivation for 6 hours.All the groups were given corresponding medium above respectively,continued incubating for 12h.The levels of Lactate dehydrogenase（LDH）,malondialdehyde（MDA）,Superoxide dismutase（SOD）and glutathione peroxidase（GSH-Px）in the HT22 cells were detected by test kits.The level of reactive oxygen species（ROS）in the HT22 cells were determined by DCFH-DA fluorescent probe technique;detected the production of the inflammatory factors Interleukin-1β（IL-1β）,Interleukin-6（IL-6）,and tumor necrosis factor-α（TNF-α）by ELISA;The apoptosis of HT22 cells was detected by flow cytometry,the activity of Caspase-3 was detected by test kits,the relative protein expression levels of the apoptotic protein B cell leukemia/lymphoma-2（Bcl-2）/Bcl-2-associated X protein（BAX）was detected by Western Blot.The relative protein expression levels of p-PI3K/PI3K,p-AKT/AKT,and p-FOXO-3α/FOXO-3a were detected by Western Blot.3.4 The study of the mechanism of SECO in protecting OGD/R HT22 cells:HT22 cells were cultured in vitro and randomly divided into 4 groups:Control group,OGD/R model group,SECO group（OGD/R+160μM SECO）,SECO+PI3K/Akt pathway inhibitor group（OGD/R+160μM SECO+20μM LY 294002）.The HT22 cells in each group was operated according to the experimental method in 3.3.RESULT:1 The effect of the SPL medicated plasma on OGD/R HT22 cellsCompared with the OGD/R model group,the cell survival rates of the SPL containing plasma groups was significantly increased（P<0.05 or P<0.01）,the morphology of HT22 cells was significantly improved,LDH and MDA of HT22 cells were significantly reduced（P<0.05 or P<0.01）,SOD and GSH-Px activities were significantly increased（P<0.05 or P<0.01）.2 47 lignans were collected by"CNKI"and"NCBI Pubmed"databases.479 targets of47 lignans and 1507 CIRI-related targets were predicted,51 key targets were screened out by network analysis finally.The lignans with high degree in"components-key targets"network are:（-）-secoisolariciresinol（Degree=26）,balanophonin（Degree=25）,（-）-cubebin（Degree=21）,predicting these three components maybe the main active ingredients of SPL against CIRI.The research shows,key targets with higher degree are:CDK2（Degree=35）,CDK1（Degree=26）,predicting the both maybe the key targets of SPL against CIRI.Through analyzing the"components-targets-pathway"network,bubble chart,and combining with relevant literature,we found the underlying pathways of SPL anti-CIRI are:PI3K-Akt signaling pathway（Degree=24）and Fox O signaling pathway（Degree=15）.3 The effect and mechanism of SECO anti-CIRI3.1 In the result of HPLC,SECO showed a good linear relationship with the peak area in the range of 0.028～0.280 mg·m L^-1,and the linear equation is Y=2 838.6X+59.354（r²=0.999 7）.The average recovery rate was 99.27%,RSD was 1.55%（n=9）.The content of SECO in SPL was measured:30.36%.3.2 The result of UHPLC-Q-Exactive Orbitrap-MS showed SECO is the main blood component of SPL.3.3 The study of the pharmacological effects of SECO in OGD/R HT22Compared with the control group,the cells viability in OGD/R model group was significantly reduced（P<0.01）;LDH,MDA and ROS in OGD/R HT22 cells were significantly increased（P<0.01）,while the activities of SOD and GSH-Px were significantly decreased（P<0.01）;IL-1β,IL-6 and TNF-αwere significantly increased（P<0.01）.The apoptosis rates of HT22 cells significantly increased（P<0.01）,the ratio of Blc-2/BAX in the OGD/R HT22 cells was significantly decreased,and the activity of Caspase-3 was significantly increased（P<0.01）.The relative protein expression levels of p-PI3K/PI3K,p-Akt/AKT,and p-Fox O-3a/Fox O-3a were significantly decreased（P<0.01）.Compared with the OGD/R model group,the cell survival rates of the SECO groups were significantly increased（P<0.05 or P<0.01）.LDH,MDA and ROS of HT22cells were significantly decreased（P<0.05 or P<0.01）,the activities of SOD and GSH-Px were significantly increased（P<0.05 or P<0.01）;IL-1β,IL-6 and TNF-αwere significantly decreased（P<0.05 or P<0.01）;the apoptosis rates of HT22 cells was significantly decreased（P<0.05 or P<0.01）,the relative expression level of Blc-2/BAX in the HT22 cells was significantly increased,and the activity of Caspase-3 was significantly decreased（P<0.05 or P<0.01）.The relative expression levels of p PI3K/PI3K,p Akt/Akt,and p Fox O-3a/Fox O-3a were significantly increased（P<0.05 or P<0.01）.3.4 The mechanism of SECO protecting OGD/R HT22 cellsCompared with the SECO group,after added the inhibitor LY294002,the pharmacological effects（protective effects）of SECO on the OGD/R HT22 cells in"3.3"were reversed.CONCLUSION:SECO maybe the main active ingredient of SPL against CIRI,which could alleviate oxidative stress,inflammatory response,apoptosis of OGD/R HT22,and its underlying mechanism is related to the activation of PI3K/AKT/Fox O signaling pathway.