Experimental Research Of The Neuroprotection Of Exosomes Drived From MiR-133b Modified MSCs And The Relevant Neuroprotective Mechanism On Intracranial Hemorrhage In Rats

Objective:In experiment 1,to investigate thetime-course of miR-133 b expressionin the perihematoma brain after ICH.In experiment 2,to collect exosomes drived from mi R-133 b modified MSCs.In experiment 3,to investigate if exosomes could transfer the mi R-133 b to braintissuesandtherelevant neuroprotective mechanism.Methods: In experiment 1,We extract the exosomes and identified them via Westen blot,and the expression of miR-133 b was tested by Real-time PCR.48 rats(50 rats were used,48 rats were survived after the surgery)were randomly assigned to 8 groups of 6 rats each,a sham group and 7 experimental groups arranged by time: 12 h,24 h,48 h,72 h,96 h,120 h and 168 h after ICH.All the rats in the experiment were killed at the indicated time point after ICH.The brain cortex of 6 rats in each group was extracted and used for Real-time PCR analysis.In experiment 2,according to the different adherent characteristic of cells,isolate andculture MSCs from the tibia and femur of aged 6 weeks SD rats.And the passage 6th cellswere used in the experiment.We used Western blot to identify the exosome protein markers,such as CD9,CD63 and CD81.We transfected the MSCs with mi R-133 b mimcs or miR-133 b negative control for 72 h.In experiment 3,72 rats(75 rats were used,72 rats were survived)were randomly divided into 4 groups(n = 18 per group): sham group,ICH + PBS group,ICH +miR-con group(exosomes +miR-con 100 ?g mixed in 0.5 ml PBS,72 h after ICH via t.v.),ICH + miR-133 b group(exosomes +miR-133 b 100 ?g mixed in 0.5 ml PBS,72 h after ICH via t.v.).At 96 h after ICH,the brain cortex of 6 rats per group were extracted for evaluating the level of mi R-133 b via Real-time PCR analysis,and 6 rats per group were used to for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)staining,fluoro-jade B(FJB)staining;Another 6 rats were used for western blot analysis.Results: In experiment 1,miR-133 b was both expressed in normal and perihematoma brain.Compared with the sham group,the level of miR-133 b expression in perihematoma brain were reduced significantly from 24 h after ICH,peaked at 72 h and then rebounded gradually.In experiment 2,the expression of miR-133 b was significantly increased compared with mi R-133 b transfected group and miR-con transfected group.(p<0.001)Wsetern Blot indicated that exosomes existed because CD9,CD63 and CD81 were expressed.In experiment 3,The Real-timePCR analysis indicated that the expression of miR-133 b in ICH+miR-133 b group was higher than that in ICH+mi R-con group(p<0.001).Compared with ICH+miR-con group,RhoA level was higher in ICH+mi R-133 b group both via Westen blot analysis(p<0.01).TUNEL staining showed that only a few TUNEL-positive apoptotic neurons were observed in the perihematoma brain in sham group,while the apoptotic index was found to be significantly higher in ICH+PBS group.As compared with ICH+miR-con group,ICH+miR-133 b group exerted significant rescue effects on neuronal apoptosis(P<0.01).FJB-positive cells were obviously increased both in cortex and perihematoma brain in ICH+PBS group,which was significantly attenuated by miR-133 b treatment(P<0.05)Finally,we detected that administration of miR-133 b obviously enhanced the phosphorylation activation of both ERK1/2 and CREB(p<0.01;p<0.05).Conclusions:Our findings suggested that exosomes drived from miR-133 b modified MSCs could highly expressed miR-133 b.As a vector,exosomes could transfer miR-133 b to brain tissuesafterICH.MiR-133 b then played a potential role of neuroprotection through targeting RhoA,which possiblyactivating the ERK1/2-CREB anti-apoptotic pathway.