Construction Of Curcumin Loaded Exosome And Its Mechanism In Cerebral Ischemia Reperfusion Rats

ObjectiveCerebral ischemia reperfusion injury usually leads to cell apoptosis,oxidative stressand and other phenomena.Mitochondria play a key role in various cellular processes.In the case of cerebral ischemia,mitochondria produce excessive ROS,and excess ROS accumulation will cause further ischemic damage by activating the mitochondrial-mediated apoptosis pathway,so it is important to improve cerebral recovery from ischemic stroke by reducing the accumulation of ROS.Curcumin is a natural antioxidant isolated from plant turmeric.It has antioxidant,scavenging free radical,anti-apoptotic,and neuroprotective effects,but due to its chemical instability and difficulty crossing the blood-brain barrier(BBB)greatly limits its application.As a natural carrier system,exosomes have high biocompatibility and more importantly,they can effectively pass BBB.Therefore,in this study,macrophage-derived exosomes(Ex)were selected as a carrier,and cur was loaded with macrophages to improve the stability and targeting of cur.At the same time,the effects and mechanisms of Ex-cur on reducing cerebral ischemia-reperfusion in rats were explored by TTC staining,neural function score,immunofluorescence and western blot experiments.MethodsThe linear,precision,repeatability,and recovery of the UV quantitativeanalysis method for cur established in this study meet the measurement requirements.And the optimal formula for curcumin exosomes loading was screened by orthogonal experiment.Curcumin loaded exosomes(Ex-cur)were prepared by gradient supercentrifugation,and the morphology,particle size,Zeta potential,marker protein,stability,blood-brain barrier penetration and encapsulation rate of Ex-cur were investigated.Immunofluorescence and in vivo imaging were used to evaluate the targeting of Ex-cur.The neuroprotective effect of Ex-cur on ischemia-reperfusion rats was investigated by TTC staining,neurological function score and immunofluorescence test.Then,the effects of Ex-cur on intracellular ROS accumulation,mitochondrial membrane potential and cytochrome c release in ischemic regions of rats were investigated by immunofluorescence and protein western blot experiments.Finally,the expression of Ex-cur on mitochondrial induced apoptosis related proteins(Bax,cleaved caspase-9,and cleaved caspase-3)was investigated by western blot.ResultsThe linearity,precision,repeatability,and recovery rate of the UV quantitative analysis method for cur established in this study all met the requirements of the measurement.The optimal preparation method of Ex-cur was selected by orthogonal experiment,The cell and drug were incubated for 24 h,the concentration of drug action was 40 μg /m L,and the cell density was 85%.The prepared Ex-cur has complete structure,uniform morphology,uniform particle size,good stability,and strong ability to cross the blood-brain barrier.The results of immunofluorescence and in vivo imaging experiments show that Ex-cur has good targeting ability,can reach the ischemic area,and colocalizes well with neuronal cells.The results of TTC staining and neural function scores showed that Ex-cur can reduce the infarct volume and improve neural function in rats with ischemia-reperfusion,indicating that Ex-cur can protect the brain injury of ischemia-reperfusion rats.The results of immunofluorescence andWestern blot experiments showed that Ex-cur can effectively reduce the accumulation of intracellular ROS,increase the membrane potential of mitochondria,and inhibit the release of cytochrome c in mitochondria,thereby reducing the damage caused by cerebral ischemia-reperfusion Damage caused by mitochondria.Western blot experiments showed that Ex-cur can down-regulate Bax,cleaved caspase-9,cleaved caspase-3 the expression of apoptosis-related proteins,indicating that Ex-cur can reverse mitochondrial dysfunction and inhibit I / R ischemia Apoptosis induced by reperfusion injury.ConclusionsThe macrophage derived exosomes prepared in this paper are loaded with cur.In rats with cerebral ischemia and reperfusion,Ex-cur can target the infarcted area of the ischemic brain and reduce the accumulation of intracellular ROS in the focal area,it can increase the membrane potential of mitochondria and inhibit the release of cytochrome c in mitochondria.These findings provide useful experimental evidence for the potential clinical treatment of Ex-cur to restore brain I / R injury.