| BackgroundThe brain damage following subarachnoid hemorrhage（SAH）is composed of early brain injury（EBI）and cerebral vasospasm（CVS）.EBI is defined as the blood-brain barrier（BBB）disruption and neurocyte apoptosis after SAH.The inflammation and apoptosis process of endothelial cells in the microvessels within brain or the artery within subarachnoid space will lead to EBI and CVS,respectively.In several brain diseases,the activated high-mobility group box1 protein（HMGB1）/Toll like receptor 4（TLR4）signal pathway can initiate the inflammatory reaction and apoptotic cascades in the endothelial cells.Inhibition of the activity of HMGB-1/TLR4 pathway will lead to significant neuroprotective effects.However,the role of HMGB-1/TLR4 in the development of EBI and CVS following SAH is still to be clarified.Stem cell transplantation in the treatment of brain injury after stroke has become one of the hotspots in the neuroscience field,however,its mechanism remains to be further elucidated.Among them,the protective effects of stem cells by secreting exosomes may be one of the important mechanisms.The exosome（Exo）derived from bone marrow mesenchymal stem cells（BMSCs）is composed of some biomacromolecules,such as micro RNA,c DNA and peptide.It plays an obvious anti-inflammatory and anti-apoptotic protective role in the treatment of a variety of brain disease models.However,whether the exosomes of BMSCs have neuroprotective effects after SAH and its correlation to the HMGB-1/TLR4 inflammatory signaling pathway is still to be clarified.In this study,the proteomics,morphology and nuclear magnetic resonance techniques had been applied（in vivo or in vitro）to explore the mechanism of HMGB-1/TLR4 signal pathway and its target proteins in BBB disruption and delayed CVS following SAH.Additionally,whether the exosomes of BMSCs could effectively reduce EBI and CVS by suppressing the activity of HMGB-1/TLR4 inflammatory signaling pathway was also detected.Methods1.Collection of Exos from BMSCsAfter the identification,the 5th generation cultured BMSCs were used to collect the Exoes by using modified precipitation method.The transmission electron microscope,Western blot and Nanoparticles Tracking Analysis（NTA）techniques were applied to identify the collected BMSCs-Exo.2.The roles of BMSCs-Exo in BBB disruption and its mechanismThe male Sprague Dawley rats（300-350 g）were used to establish the SAH model using endovascular perforation method.The animals were divided into three groups:Sham,SAH+PBS and SAH+Exo groups.The 200μg BMSCs-Exo was immediately injected into cisterna magna after SAH model establishment.At 24 h after SAH,the mortality,neurological function,weight and SAH score each group were analyzed;the brain edema was evaluated using MRI,and morphological alteration of the tight junctions（TJs）between endothelial cells was observed using transmission electron microscope.To explore the potential protective mechanism,mi R129-5p inhibitor,mi R129-5p antagomir,was used to assess the brain water content,Evans blue content and the Ig G distribution in hippocampus.Moreover,q-PCR,immunofluorescence staining and Western blot were applied to detect the activities of HMGB-1/TLR4 pathway and its downstream target protein,such as TLR4,TNF-ɑ,p53 and NF-κB.The expression level and distribution of TJs each group,such as Occludin and ZO-1,were also evaluated using immunofluorescence staining and Western blot methods.3.The effects of BMSCs-Exo on alleviating CVS and its mechanismTo observe the anti-vasospasm role of BMSCs-Exo,histology dying and MRI methods were used to measure the diameters and wall thickness of basilar artery.In addition,the activities of HMGB-1/TLR4 pathway and its downstream target protein,pro-inflammatory and pro-apoptosis factors,such as NF-κB（p65）,p53 and Caspase-3,were also detected using immunohistochemistry staining and Western blot methods.Results1.The Exo from BMSCs were successfully collectedThe identification results indicated that the 5th BMSCs were positively stained with CD44,CD90 marker antibodies and negatively stained with CD45 and CD11,which demonstrated the authentic identification of BMSCs in this study.Additionally,using transmission electron microscope,the morphology of BMSCs-Exo showed the typical“plate”type.The levels of CD-9,CD-63 and TSG101 markers for the BMSCs-Exo were abundantly expressed,although GM-130 was rarely expressed.The NTA method indicated that the diameter of BMSCs-Exo was about 100 nm.2.BMSCs-Exo alleviated BBB disruption through suppressing HMGB-1/TLR4 pathway activityFollowing SAH,BMSCs-Exo treatment could significantly reduce the mortality,neurological deficits and brain edema,additionally,preserved the normal function and morphology of tight junction between endothelial cells.However,the application of mi R129-5p antagomir could reverse the protective role of BMSCs-Exo in alleviating brain edema.Moreover,after SAH,the HMGB-1/TLR4 pathway was activated,the expression level of HMGB-1,TLR4 and its downstream target protein,such as NF-κB（p65）,TNF-ɑ,p53 and Caspase-3 were markedly enhanced,which could be significantly quenched by using BMSCs-Exo.In addition,BMSCs-Exo could also maintain the BBB integrity through preserving the levels and location of tight junction protein（Occludin and ZO-1）between endothelial cells.3.BMSCs-Exo attenuated CVS severity by quenching HMGB-1/TLR4 pathway activityAt 72h after SAH,the results of histology dying and MRI methods indicated that the diameters of basilar arteries were significantly decreased,meanwhile,the wall thickness of basilar arteries were markedly increased,which demonstrated there was severe CVS in the arteries within subarachnoid space.The HMGB-1/TLR4 signal pathway in the endothelial cells of basilar arteries was activated,and the levels of pro-inflammatory and pro-apoptosis factors,such as NF-κB（p65）,p53,Caspase-3,were enhanced,and the number of apoptotic endothelial cells were also increased.The BMSCs-Exo treatment could significantly suppress the activity of HMGB-1/TLR4 signal pathway and attenuate the CVS severity of basilar arteries.Conclusion1.BMSCs-Exo was successfully enriched and labeled by fluorescence staining.It was further confirmed that BMSCs-Exo could enter into the endothelial cells of the microvessels to play the role of neurovascular protection.2.Following SAH,the activated HMGB-1/TLR4 signal pathway in endothelial cells of the microvessels and the basilar arteries led to the EBI and delayed CVS through increasing the expression levels of pro-inflammatory and pro-apoptosis factors,such as NF-κB（p65）,p53,Caspase-3.3.BMSCs-Exo attenuated the EBI and CVS severity through suppressing the activities of HMGB-1/TLR4 signal pathway and its downstream pro-inflammatory and pro-apoptosis factors by the mi R129-5p within BMSCs-Exo. |
