Study On Effect And Mechanism Of PF11 Pills Against Cerebral Ischemia-reperfusion Injury Based On Multi-omics

Ischemic stroke,caused by an insufficient blood flow of brain cerebral,has the characteristics of high rates of incidence,disability and mortality.The restoration of blood flow to ischemic brain tissue can salvage ischemic penumbra that remain neurological function and also cause the cerebral ischemia-reperfusion injury,such as cerebral edema,neuronal necrosis,free radical damage,and neuroinflammation.The important strategy in the treatment of ischemic stroke is to reduce cerebral ischemiareperfusion injury that promote the recovery of neurological function of patients,improve their prognosis and make a better life in the future.Pseudoginsenoside-F11,a characteristic ocotillol-type sapnion isolated from American ginseng,exhibits beneficial effects such as anti-inflammation,antioxidant effect and neuroprotective effect,et al.Studies have reported that PF11 treatment intravenously can reduce the cerebral infarction and neuronal damage in rats with cerebral ischemia-reperfusion by inhibiting intracellular calcium overload.Oral administration of PF11 can affect the process of microglia/macrophage polarization in brain tissue of rats with cerebral ischemia-reperfusion and reduce neuroinflammation.Oral administration of PF11 can improve neurological dysfunction in mice with cerebral ischemia-reperfusion by activating the BDNF/Trk B pathway.Our research found that PF11 pills have a good effect against myocardial ischemic.And the pharmacokinetic study showed that there was a high distribution of PF11 in brain tissue of rats after 3 hours of oral administration of PF11 pills which improved its dissolution rate and bioavailability in vivo.The role of PF11 pills against cerebral ischemiareperfusion injury has not been reported yet.PF11 possesses various pharmacological activities,and it can interfere diseases via multiple targets and multiple metabolic pathways.The investigation on the protective effects of PF11 against cerebral ischemiareperfusion injury from the joint analysis of protein level and metabolite level could contribute to explore more potential therapeutic targets and biomarkers that promote the diagnosis,treatment and prognosis of ischemic stroke.Therefore,on the basis of previous studies,the protective effects of PF11 pills against cerebral ischemia-reperfusion injury in rats was conducted and it was the first time that label free quantification proteomics and untargeted metabolomics were used to further investigate its underlying mechanism,analyze the changes of proteins and metabolites in rats after the onset of ischemic stroke and explore potential targets and biomarkers.The following studies were conducted:1.Study on the effect of PF11 pills against CI/RISD rats were pretreated with PF11 pills via intragastric(12.5,25,50 mg/kg)once a day for 14 days and the transient middle cerebral artery occlusion(t MCAO)was used in this experiment to establish classical cerebral ischemia-reperfusion model.Samples were collected after 3 days of administration of PF11 pills to analyze its therapeutic effects.The results showed that PF11 dose-dependently reduced the m NSS score of rats,decreased the infarct area of brain and alleviated brain edema,lowered the leakage of Evans blue and NSE,down-regulated the levels of TNF-α,IL-6 and IL-1β,increased the activity of SOD and decreased the contents of MDA.The above results indicated that PF11 pills protects cerebral ischemia-reperfusion injury by improving neurological dysfunction,reducing secondary damage,protecting the damaged blood-brain barrier,and reducing inflammation and oxidative stress.2.Study on the effect of PF11 pills against CI/RI based on label free quantification proteomicsThe composition and expression levels of proteins in the ischemic cerebral hemispheres of t MCAO rats were analyzed by Nano Elute UHPLC-tims TOF Pro platform based on LFQ proteomics and identified by Max Quant software and Uniprot database.A total of 319 differentially expressed proteins were identified in Sham,Model and PF11 pills groups,and the results of GO biofunction and KEGG signaling pathway enrichment analysis showed that differentially expressed proteins regulated axonal sheathing,neuronal cell myelination,brain development and other biological processes,affected the formation of neural spines,dendritic spines,myelin and neuronal synapses,and the generation of structural components of myelin,and were involved in c AMP signaling pathway,MAPK signaling pathway,Ras signaling pathway,Tight junction and other signaling pathways.Topological analysis of PPI network revealed that 10 targets such as RHOA,CAMK2 A,GRIN2B,SYN1 and MBP play important roles in the network.Molecular docking results showed that PF11 can form a stable binding conformation among RHOA,CAMK2 A,GRIN2B,SYN1 and MBP with the binding energies less than-7 kcal/mol.The above study showed that PF11 pills can regulate the differentially expressed proteins in t MCAO rats to maintain the stability of blood-brain barrier,promote the neurotransmission,and ameliorate the neurological impairment caused by ischemia-reperfusion.In addition,RHOA,CAMK2 A,GRIN2B,SYN1 and MBP may be the potential targets of PF11 pills against ischemic stroke.3.Study on the effect of PF11 pills against CI/RI based on untargeted metabolomicsThe metabolites in brain and serum of t MCAO rats were determined by untargeted metabolomics based on UPLC-Q/TOF-MS technology.The raw data were preprocessed by Mass Lynx V4.1 software and the differential metabolites were identified with the help of HMDB database and standards.A total of 18 potential biomarkers were identified,including L-phenylalanine,Ltyrosine,sphingosine-1-phosphate and Lyso PE(16:0/0:0),etc.Metabo Analyst 5.0online platform was applied in the analysis for metabolic pathway of potential biomarkers,and a total of 14 metabolic pathways were obtained.Among which phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,sphingolipid metabolism and tryptophan metabolism were the main metabolic pathways.The above study showed that PF11 pills can attenuate cerebral ischemiareperfusion injury mainly by regulating amino acid metabolism and lipid metabolism disorders in t MCAO rats.4.Integrated proteomics-metabolomics analysis of PF11 pills against CI/RICanonical correlation analysis(CCA)and Pearson correlation coefficient were used to conduct integrated proteomics-metabolomics analysis,and differential proteins and differential metabolites with Pearson correlation coefficient>0.6 and p<0.05 were marked as strongly correlated.The results showed that 11 differential metabolites(including phenylpyruvic acid,L-phenylalanine,L-tyrosine and L-tryptophan)were highly correlated to 25 differential proteins(including ICAM5,ACTN1,RHOA,CAMK2 A,GRIN2B and MBP,et al).In the proteomics study,MBP,RHOA,CAMK2 A and GRIN2 B were identified as potential targets of PF11 pills against ischemic stroke,and the integrated analysis showed that these four targets were strongly correlated to differential metabolites,which further provided evidence for the importance of these four targets.The above results suggested that PF11 pills can exert the protective effect against cerebral ischemia-reperfusion injury,regulate the level of metabolites in rats with ischemic stroke and improve metabolic disorders by affecting the expression of related differential proteins.In summary,the protective effects of PF11 pills against cerebral ischemiareperfusion injury in rats was conducted and it was the first time that label free quantification proteomics and untargeted metabolomics were used to further investigate its underlying mechanism.The proteomics study predicted 5 potential targets and 4potential signaling pathways.The metabolomics study predicted 18 potential biomarkers and 4 key metabolic pathways.The integrated analysis declared the correlations between differentially expressed proteins and differential metabolites,and provided some evidence for the importance of MBP,RHOA,CAMK2 A and GRIN2 B which were identified as potential targets of PF11 pills.This study provided a theoretical support for the further development and study of PF11 pills as class 1 new drug.