| Background: Ischemic stroke accounts for more than 80% of all strokes and is a common disease with high mortality and disability rates worldwide.Currently,thrombolysis for revascularization is the mainstay of stroke treatment,but it has limitations such as short treatment time window and reperfusion injury.Effective poststroke drugs are essential,while the presence of the central nervous system blood-brain barrier prevents most drug molecules from entering the brain,making it a challenge for stroke treatment.Exosomes(Exo)are extracellular vesicles secreted by cells which can act as drug delivery vehicles to deliver substance molecules across the blood-brain barrier for the treatment of central nervous system diseases.Baicalein(Bai),a Chinese herbal extract,has multiple neuroprotective effects against ischemic stroke,but suffers from poor water solubility and low bioavailability.Objective: To prepare engineered baicalein-loaded exosomes;to investigate the effectiveness of drug-loaded exosomes for drug delivery across the blood-brain barrier;and explore the neuroprotective mechanism of drug-loaded exosomes against ischemic stroke.Methods: The kit extracted SD rat serum exosomes,loaded baicalein into exosomes using ultrasound,and characterized the morphology,particle size and surface markers of exosomes or drug-loaded exosomes by transmission electron microscopy,nanoparticle tracking analysis technique,and western blot.Healthy KM mice were selected to construct a middle cerebral artery occlusion(MCAO)model,and baicalein and baicalein-loaded exosomes(Exo+Bai)were intraperitoneal injected into the mice for subsequent studies,respectively.The experiments were divided into 6 groups: shamoperated group(Sham group),middle cerebral artery occlusion group(MCAO group),PBS solvent group(PBS group),exosome group(Exo group),baicalein group(Bai group),and baicalein-loaded exosomes group(Exo+Bai group);cerebral infarct volume was determined by TTC staining and neurological deficits were assessed by Zea-Longa neurological function score;fluorescently labeled exosomes were studied for intracerebral uptake;western blot was used to detect the expression levels of M1/M2 phenotypic markers in microglia of each group of mice and to determine the expression levels of apoptosis-related proteins in ischemic brain tissue.Finally,a network pharmacology method was used to search baicalein and ischemic stroke related targets from relevant databases,and the intersection of the two was used to produce a Venn diagram to obtain the potential targets of baicalein on ischemic stroke;protein interaction network construction and core gene screening,GO enrichment analysis and KEGG pathway enrichment analysis were performed on the intersection targets to predict the possible targets and related pathways of baicalein on neuroprotective effects in ischemic stroke.Results: First,according to the results of the previous experiments of our group and the western blot results,we successfully extracted and purified the serum exosomes and identified them in this study.The maximum absorption peak of baicalein was measured at 272 nm using an ultra-micro spectrophotometer,and the standard curve of baicalein concentration was successfully prepared and fitted;the precision test results showed that the assay method has good precision and can be used for the determination of baicalein content.The encapsulation rate of baicalein-loaded exosomes was25.64%±0.78%,which was calculated from the standard curve.Intracerebral uptake experiments showed that red fluorescently labeled exosomes were observed in the brain tissue sections of MCAO mice at 6h,18 h and 24 h of reperfusion,indicating that the exosomes were able to cross the blood-brain barrier and distribute in the brain tissue.Further intraperitoneal injection of Exo+Bai into MCAO mice revealed that the Exo+Bai group was more effective in improving brain infarct volume(P<0.01)and reducing neurological scores(P<0.001)in MCAO mice compared with the Bai group by TTC staining and Zea-Longa neurological scores.Western blot experiments showed that compared with the Bai group,the Exo+Bai group more significantly increased the expression of microglia M2 phenotype marker Arg-1,CD206 in ischemic brain tissue(P<0.05)and decreased the expression of M1 phenotype marker CD86,i NOS(P<0.05).Western blot experiments also showed that compared with Bai group,the Exo+Bai group effectively decreased the expression of apoptosis-related proteins Bax and Cyt C(P<0.05)and elevated the expression of Bcl2(P<0.05)in ischemic brain tissue of MCAO mice.Finally,a total of 82 potential target genes of baicalein for ischemic stroke were retrieved by database search using network pharmacology;a protein interaction network was constructed for these intersecting genes,and the top 10 core gene targets were screened as AKT1,VEGFA,TP53,SRC,CASP3,HIF1 A,EGFR,MAPK3,PTGS2 and ESR1;GO enrichment analysis and KEGG pathway enrichment analysis showed that these targets are mainly involved in regulating biological processes and molecular functions such as hypoxia,redox,apoptosis and PI3 K signaling,and may be enriched in PI3K-AKT signaling pathway.Conclusion: Baicalein-loaded serum exosomes can deliver baicalein to the brain across the blood-brain barrier and improve the effective bioavailability of baicalein;compared with baicalein treatment alone,baicalein-loaded exosomes administration was more effective in reducing brain infarct volume and improving neurological function scores in MCAO mice,and may exert anti-inflammatory effects by modulating the change of microglia polarization phenotype from M1 to M2,and further improve brain ischemia-reperfusion injury by inhibiting apoptosis;network pharmacological analysis showed that baicalein has multi-targeted effects on ischemic stroke by regulating hypoxia,redox,apoptosis and angiogenesis,and the mechanism may be mediated by PI3K-AKT signaling pathway. |
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