| Purpose:Our previous studies had found that the brain-targeted liposomes of Cyclovirobuxin D(CVBD)enhanced brain targeting by intranasal administration.Then,the pharmaceutical and pharmacokinetic evaluation,and brain-targeted ability in vitro had been well investigated.Here,middle cerebral artery occlusion(MCAO)induced cerebral ischemia-reperfusion injury(CIRI)rat model and oxygen and glucose deprivation/reoxygenation(OGD/R)induced cell model were established for:(1)the therapeutic effect of CVBD brain-targeted liposomes on MCAO rats;(2)the protective effect of CVBD brain-targeted liposomes in OGD/R-injured HT22 cell;(3)the mechanism of the anti-CIRI effect of CVBD braintargeted liposomes.Methods:1.Pharmacological evaluation of CVBD brain-targeted liposomes in the treatment of CIRIThe CIRI rat model was induced by MCAO.MCAO rats were then randomly divided into four groups:sham group,model group,nimodipine group and CVBD group.Pharmacological evaluation was assessed in vivo by general indexs,neurobehavioral scores,triphenyltetrazolium chloride(TTC)staining,histopathological staining(HE staining and Nissl staining),small animal magnetic resonance imaging,biochemical assay and Western blot.2.Protective effect of CVBD brain-targeted liposomes in OGD/R-injured HT22 cellThe safe dosage and administration dosage of CVBD liposomes in OGD/R-injured HT22 cell were screened by live cell real-time dynamic imager and CCK8 method.Then,the protective effect of CVBD liposomes on OGD/R-injured HT22 cell was investigated by cell fusion degree,cell proliferation curve and cell viability.The ROS level was also detected as the pharmacological index in vitro.3.The anti-CIRI mechanism of CVBD brain-targeted liposomes in OGD/R-injured HT22 cellOGD/R-injured HT22 cell was infected by mRFP-GFP-LC3 adenovirus.The autophagosome and autophagy flow were observed by laser confocal microscopy,and autophagy-related protein expressions(LC3,p62 and Beclin 1)were analyzed by Western blot.Meanwhile,the classic autophagy inhibitor,chloroquine,was used to explore the autophagy-regulated mechanism of CVBD brain-targeted liposomesin treating CIRI.Results:1.Pharmacological evaluation of CVBD brain-targeted liposomes in the treatment of CIRICIRI rat model was successfully established by MCAO.CVBD liposomes effectively alleviated the weight loss,neural function scores,size of cerebral ischemic necrosis and pathological damage of brain,promoted the number of normal nissl bodies and blood flow of injured vessel.In addition,CVBD liposomes significantly lowered serum LPO and MDA levels,enhanced CAT and SOD levels and increased the oxidative stress-related protein expressions of Nrf2,HO-1,GCLM and NQO1.Besides,CVBD liposomes remarkably promoted autophage-related protein expression of LC-3B.2.Protective effect of CVBD brain-targeted liposomes in OGD/R-injured HT22 cellOGD/R model of HT22 cell was successfully established.Based on the model,the administration dosages of CVBD were 37.5 μM(CVBD-H group),18.75 μM(CVBD-M group)and 9.38 μM(CVBD-L group).CVBD liposomes effectively protected OGD/Rinjured HT22 cells by reducing neuronal cell necrosis and lysis,increasing cell viability and cell fusion.Further,CVBD liposomes dose-dependently amellivated ROS levels in OGD/Rinjured HT-22 cells.3.The anti-CIRI mechanism of CVBD brain-targeted liposomes in OGD/R-injured HT22 cellOGD/R-injured HT22 cell was successfully infected by mRFP-GFP-LC3 adenovirus.CVBD liposomes increased the autophagy level in HT22 cells with OGD/R damage.CVBD liposomes improved the protein expressions of LC3 and Beclin 1,and decreased the protein expression of p62.Conclusion:CVBD brain-targeted liposomes inhibited CIRI in vivo and in vitro via reguating oxidative stress and enhanceing autophagy level. |
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