Study On Neuroprotective Effect Of Stem Cell Derived Exosome Treated With Muscone On Ischemic Stroke Rats

Objective:In this study,bone marrow mesenchymal stem cells（BMSCs）-derived exosome（Exo）（Mus treated-Exo）treated with muscone（Mus）were used to explore the possible mechanism of neuroprotective effect of Mus treated-Exo on ischemic stroke rats.Methods:（1）BMSCs were cultured by whole bone marrow adherent method,and the cell morphology was observed by inverted microscope.The phenotype CD29,CD90,CD34 and CD45 expression of P₃ BMSCs were identified by flow cytometry.（2）10μM muscone was co-cultured with BMSCs for 36 h,and BMSCs culture medium was collected.Mus treated-Exo was obtained from BMSCs culture medium by ultracentrifugation.The morphology of Mus treated-Exo was identified by transmission electron microscope,the particle size of exosome was identified by nano-flow meter,and the expression of exosome phenotypic membrane proteins CD9,CD63 and CD81 were identified by Western Blot.（3）The rat model of middle cerebral artery occlusion（MCAO）was established,and the rats were randomly divided into MCAO model group,control exosome group（Ctrl-Exo）,muscone treated exosome group（Mus treated-Exo）and muscone group（Mus）.2 hours after cerebral ischemia,the rats were reperfused and injected into the femoral vein,and the MCAO model group was injected with the same volume of PBS.（4）After 24 hours of cerebral ischemia,the neuroprotective effect of Mus treated-Exo on ischemia-reperfusion rats was explored by neurofunction score,TTC staining,TUNEL staining,immunofluorescence staining and Western Blot.Results:（1）BMSCs showed typical fibroblast-like or spindle-shaped morphology under microscope.The results of flow cytometry showed that the expression rate of CD29,CD90,a positive marker protein on the surface of P₃generation BMSCs was 98.9%and 99.0%,respectively.The expression rate of negative marker protein CD34,CD45 on the surface of BMSCs 0.47%and 0.38%,respectively.（2）The results of transmission electron microscopy showed that Mus treated-Exo was doubly concave disk-shaped and the membrane structure was intact.Nano-flow analysis showed that the particle of Mus treated-Exo was mainly distributed between 50-150 nm,with an average size of 76.11±17.60 nm.The results of Western Blot identification showed that Mus treated-Exo highly expressed phenotypic markers CD9,CD63 and CD81.（3）Neurological deficit:（1）The results of Bederson score showed that compared with MCAO model group,Ctrl-Exo group,Mus treated-Exo group and Mus group all improved the neurological function of rats in different degrees.Among them,the improvement of neurological function in Mus treated-Exo treatment group was the most obvious（P<0.001）,and the effect was significantly better than that in Ctrl-Exo group and Mus group（P<0.05,P<0.01）.（2）The results of Grip test score showed that compared with MCAO model group,the motor function of rats in each treatment group was improved in varying degrees,and the motor function in Mus treated-Exo treatment group was the most obvious（P<0.001）,which was significantly higher than that in Ctrl-Exo treatment group and Mus treatment group（P<0.05,P<0.01）.（4）TTC staining:（1）Compared with MCAO model group,the cerebral infarction volume of rats in each treatment group decreased in different degrees,and the decrease in cerebral infarction volume in Mus treated-Exo treatment group was the most significant（P<0.001）,and was significantly lower than that in Ctrl-Exo,Mus treatment group（both P<0.05）.（2）Compared with the MCAO model group,the cerebral hemispheric swelling in each treatment group was improved in varying degrees,and the therapeutic effect of Mus treated-Exo was the most significant（P<0.001）.（5）TUNEL staining:compared with the MCAO model group,the number of neuronal apoptosis in each group decreased in different degrees after Ctrl-Exo,Mus treated-Exo,Mus treatment,especially in the Mus treated-Exo treatment group（P<0.001）,and there was significant difference compared with the Ctrl-Exo,Mus treatment group（P<0.05,P<0.01）.（6）Immunofluorescence staining:compared with the MCAO model group,the number of microglia activation markers CD68 and Iba-1 in each treatment group decreased in different degrees,especially in the Mus treated-Exo treatment group（both P<0.001）,and there was significant difference compared with the Ctrl-Exo,Mus treatment group（P<0.05,P<0.01）.（7）Western Blot:Compared with the MCAO model group,the expression of pro-apoptotic protein Bax,Cleaved-caspase 3,pro-inflammatory factor IL-6,COX-2 in the brain tissue of each treatment group decreased in varying degrees,of which the Mus treated-Exo treatment group decreased the most significantly（P<0.01,P<0.001）,and the therapeutic effect was more significant than that of the Ctrl-Exo,Mus treatment group（P<0.05,P<0.01）.The expression of anti-apoptotic protein Bcl-2 in brain tissue of rats in each group increased in varying degrees,and the expression of anti-apoptotic protein Mus treated-Exo in the treatment group was the most significant（P<0.001）.And there was statistical significance compared with Ctrl-Exo,Mus treatment group（P<0.05,P<0.01）.Conclusion:（1）Mus treated-Exo can effectively improve the neurological function and reduce the volume of cerebral infarction in MCAO rats,and the therapeutic effect is better than that of Ctrl-Exo and Mus.（2）Mus treated-Exo can effectively inhibit neuronal apoptosis and microglial activation in MCAO rats,and reduce the expression of pro-apoptotic protein Bax,Cleaved-caspase 3 and pro-inflammatory factor IL-6,COX-2,promote the expression of anti-apoptosis protein Bcl-2 in MCAO rats,and its therapeutic effect is better than that of Ctrl-Exo and Mus.The therapeutic effect of Mus treated-Exo on ischemic stroke rats may be achieved by inhibiting apoptosis and inflammation.