Engineered Exosomal Mir-193b-3p Delivery Attenuates Nenroinflammation In Early Brain Injury After Subarachnoid Hemorrhage And Its Mechanism

Part Ⅰ Analysis of miRNAs expression profile of plasma exosomes in patients with subarachnoid hemorrhageObjectiveAneurismal subarachnoid hemorrhage(SAH)is a serious type of stroke with high mortality and disability.Identifying circulating pathophysiologic changes are helpful to improve theranostics of SAH.In this study,we reported and verified the differences of circulating exosomal miRNAs expression profile in SAH patients.Methods1.Next generation sequencing(NGS):We extracted exosomes from plasma of three aSAH patients and three healthy controls using the extraction kit.The morphology of exosomes was evaluated using transmission electron microscopy(TEM).The identity of exosomes was further validated by quantitating the exosome membrane-associated markers CD63 and Alix using Western blot analysis.Total exosomal RNA was used to prepare small RNA library.And then we performed the single-end sequencing(36bp or 50bp)on an Illumina Hiseq 2500.The raw reads were subjected by ACGT101-miR.miRNA differential expression was analyzed using the medical statistical analysis.2.Validation of NGS data by quantitative real-time polymerase chain reaction(qRT-PCR):Expression of exosomal miRNAs from a total of 20 serum samples was analyzed,including samples from patients with SAH(n=10)and samples from healthy control subjects(n=10).Total RNA was extracted from exosomes using QIAzol Lysis Reagent(Qiagen,Germany)according to the manufacturer’s instructions.Total RNA was reverse transcribed to cDNA,and qRT-PCR was carried out.The results of PCR reactions of miRNAs expression in plasma of SAH patients was analyzed by the statistical analysis.3.Animal model preparation:A SAH mice model was established by injection of blood into the suprachiasmatic cistern.To examine the conservation of miRNA expression,it is necessary to observe whether the miRNA exhibited significantly altered expression levels in mice after SAH when compared with human.Results1.Exosome had a spherical shape with a size of 30-100 nm surrounded by a membrane using TEM.The expression of exosome characteristic proteins CD63 and Alix were both positive.2.NGS of these samples yielded 9 to 15 million reads aligned to the reference human genome sequence.These reads corresponded to>50,000 different RNA sequences.From a list of 2139 known miRNAs,only 746 were considered as being expressed,with raw read counts≥1 in at least one sample.To further assess the differences between the groups,we performed a global statistical analysis to detect true differentially expressed miRNA sequences(adjusted p-value<0.05,|log2(fold change|>1 and expression level is not low).This approach yielded 6 miRNAs that were significantly differentially expressed in aSAH patients and control plasma exosome samples.These were:hsa-miR-369-3p,hsa-miR-136b-3p,hsa-miR-410-3p,hsa-miR-195-5p,hsa-miR-486-3p,and hsa-miR-193b-3p.3.To evaluate the miRNA NGS results,expression of exosomal miRNAs from a total of 20 serum samples were analysed,including samples from patients with SAH(n=10)and samples from health control subjects(n=10).Technical confirmation of the significance of the observed differences in miRNA expression with additional qRT-PCR assays with a cohort(10 samples post-SAH and 10 samples of normal controls)confirmed that hsa-miR-369-3p and hsa-miR-410-3p exhibited significantly down-expression,whereas hsa-miR-193b-3p and hsa-miR-486-3p exhibited significantly up-expression after SAH when compared with controls.4.Through SAH mice model validation,we found that the miRNA expression trend in plasma exosomes was the same as that in SAH patients.Increased expression of plasma exosomal miR-193b-3p and miR-486-3p remained statistically significant relative to controls in this analysis,whereas the expression levels of miR-369-3p and miR-410-3p were decreased with controls.However,they were both decreased with controls in brain tissues of the mice.Conclusion1.The circulating exosomal miRNA expression profiles showed a distinct pattern between the aSAH patients and healthy controls.2.We found the consistency of plasma exosomal miRNAs between SAH patients and SAH m models.3.The differential miRNAs may be involved in the pathophysiological progression of SAH,and may serve as potential targets for the treatment of SAH.Part Ⅱ Surface functionalized exosomes as targeted drug delivery vehicles for subarachnoid hemorrhage therapyObjectiveThe blockage function of blood-brain barrier(BBB)prevents the entrance of drugs to CNS.In this study,Rabies virus glycoprotein(RVG)has been engineered for the exosomal surface as targeted delivery of miRNAs drugs into the CNS.Methods1.The extraction and culture of mouse bone marrow mesenchymal stem cells(BMSCs):Bone marrow from 6-week-old adult male mice was mechanically harvested from femurs and tibia,BMSCs were cultured in cells petri dishes.2.RVG-Lamp2b overexpression lentivirus production:RVG-lamp2b-mus gene fragment was synthesized by PCR method,and then RVG-Lamp2b-mus gene sequence was added into the plasmid.After bacterial culture,a large number of plasmids were extracted,and detected by qRT-PCR to determine whether the plasmid vector was constructed successfully.293T cells were transfected with RVG-lamp2b overexpression plasmid to harvest lentivirus.RVG-Lamp2b overexpression lentivirus was then ready for further analysis.3.RVG-Lamp2b overexpression lentivirus was used to infect BMSCs.Exosomes were purified from cell culture supernatants in serum-free medium of BMSCs.Western blot was used to detect Iamp2b to determine whether RVG-lamp2b fusion protein was successfully expressed in exosomes.The morphology of exosomes was evaluated using TEM.The identity of exosomes was further validated by quantitating the exosome characteristic proteins to confirm the success of exosome extraction.4.Electroporation technology was used to transfect miR-193b-3p mimics into exosomes.The quantification of miR-193b-3p in exosomes was performed using qRT-PCR to confirm the success of electroporation.5.Unmodified/Exos and RVG/Exos were loaded with FAM-labeled miR-193b-3p The slides from brains in the SAH model were observed at 2 h after injection into the tail vein.The fluorescence of FAM in the slides of SAH model was observed.Results1.The result of DNA sequencing showed that RVG-Lamp2b overexpression plasmid vector was successfully constructed,and lentiviral vectors were successfully produced2.We used western blot analysis to measure protein levels of Lamp2b in these four groups to validate whether the lentiviral vectors were successfully infected into BMSCs Compared with the positive control,blank control and positive control,the expression of Lamp2b in exosomes secreted by BMSCs transfected with RVG-Lamp2b overexpression lentivirus was significantly increased.3.Exosome had a typical biconcave shape using TEM.Compared with BMSCs,the expression of exosome characteristic proteins CD63 and Alix were increased,while Golgiosome labeled protein GM-130 was not expressed4.MiR-193b-3p levels in isolated exosomes that were loaded with miR-193b-3p mimics or scrambled miRNAs via electroporation were measured using qRT-PCR analysis.We found that levels of miR-193b-3p in exosomes transfection with miR-193b-3p mimics were significantly higher than in those transfected with the negative control5.We found substantially more FAM-labeled Exos in the inferior basal temporal lobe of mice in the RVG/Exos group than that in the unmodified/Exos group,and almost no FAM could be observed in the inferior basal temporal lobe of the miR-193b-3p group.Conclusion1.The exosomes with RVG-Lamp2b fusion protein secreted by BMSCs through genetic engineering.2.RVG modified exosomes can cross BBB into the SAH cortex and transport miRNA drugs specifically into the CNS.Part Ⅲ:Brain-targeted exosomal exosomal miR-193b-3p delivery attenuates neuroinflammation in early brain injury after subarachnoid hemorrhage in miceObjectiveTo determine the role and mechanism of RVG/Exos/miR-193b-3p in early brain injury after SAH in mice.Methods1.Animal groups:Target validation groups:Sham group and 4 SAH groups arranged by time:12,24,48,and 72 hours after SAH.Controlled experiments groups:Sham group,SAH groups,RVG/Exos groups,RVG/Exos/Scr groups,RVG/Exos/miR groups.2.Animal model preparation and drug administration:A SAH mice model was established by injection of blood into the suprachiasmatic cistern.The drug was administered through the tail vein.3.Detection methods and indicators:The expression levels of downstream target genes Hdac3,Class I Hdac genes,apoptosis and inflammatory response genes were detected by qRT-PCR.The protein levels of HDAC3 and p65 was detected by Western blot.The concentrations of inflammatory factors in brain tissue were detected by ELISA.Dry-wet test was used to evaluate brain edema.The BBB permeability was assessed by Evan’s blue staining.Neurodegeneration was detected using Fluoro-Jade C(FJC)staining.Results1.We found that,in comparison with the Sham group,the mRNA levels of Hdac3 reached peak levels at 12-24 h after SAH,and protein levels of HDAC3 reached a peak at 24 h after SAH.We also found that,in comparison with the sham group,expression levels of class I HDACs(HDACs 1,2,8)mRNA did not reach peak levels before 24 h after SAH.2.Rela mRNA levels were not significantly different after the Exos/miR-193b-3p treatment.HDAC3 levels were lower after Exos/miR-193b-3p treatment,whereas acetylation status of p65(ac-p65)protein levels were higher in the nucleus.3.Caspase-3 gene expression was elevated after SAH and lower after Exos/miR-193b-3p treatment using qRT-PCR.Interestingly,we found that Bcl-2 expression was lower after SAH and was dramatically greater after Exos/miR-193b-3p treatment.The mRNA levels of IL-1β,IL-6,and TNF-α were higher after SAH,and Exos/miR-193b-3p significantly reduced the expression of proinflammatory cytokines by qRT-PCR.Levels of IL-1p,IL-6,and TNF-α in the cerebral hemisphere were measured using ELISA.We found that inflammatory cytokine levels were substantially higher with biphasic peaks after SAH.The data suggested that Exos/miR-193b-3p significantly lowered expression levels of IL-1β,IL-6,and TNF-α in the brain after SAH.4.The SAH group showed lower neurological scores than the sham group.Exos/miR-193b-3p markedly improved neurological scores compared with the SAH and Exos/Scramble miRNA groups at 24 h post-SAH.Similarly,the degree of brain edema was significantly less pronounced in the Exos/miR-193b-3p group than in the SAH and Exos/Scramble miRNA groups.The numbers of FJC-positive cells were lower in the Exos/miR-193b-3p group and were higher in the SAH and Exos/Scramble miRNA groups compared with the sham group.SAH produced the amount of Evan’s blue extravasation into the brain,which revealed the BBB injury.Also,enhanced Evan’s blue extravasation was found in the Exos group and Exos/Scramble miRNA group as compared with the sham group.However,the BBB permeability was significantly attenuated by the Exos/miR-193b-3p treatment.ConclusionOur data suggested that miR-193b-3p in a SAH mouse model alleviates inflammatory responses,inhibits neuronal apoptosis,maintains BBB integrity,reduces brain edema,reduces neuronal degeneration,and improves the prognosis.Inhibition of inflammation by miR-193b-3p affects the ac-p65 and reduces the expression of HD ACS.These findings suggest that RVG/Exos/miR-193b-3p may weaken neuroinflammation by suppressing the expression and the activity of HDAC3 and increasing NF-κB p65 acetylation after SAH.