BMSC-Exos Attenuate Inflammation And Pyroptosis Post Cerebral Ischemia Reperfusion Injury By Modulating Microglia Phenotype

Background:Stroke is characterized by high incidence,mortality,recurrence,and disability rates,which is also the most common cause of death and disability.Stroke can be classified into two pathological phenotypes:ischemic stroke and hemorrhagic stroke.Ischemic stroke accounts for approximately 80%stroke cases.However,the clinically treatment methods for ischemic stroke are limited,mainly mechanical thrombectomy or drug thrombus.Blood reperfusion may induce deteriorated tissue damage and dysfunction.This pathophysiological process is named as cerebral ischemia-reperfusion(CI/R)injury.Hence,how to alleviate CI/R injury and restore neurological deficit post CI/R has become a key issue of cerebrovascular disease rehabilitation.Recent studies have also demonstrated that BMSC-Exos exert applaudable neuroprotective effects by exerting anti-inflammatory effects in several neurological diseases,but the specific mechanism of BMSC-Exos in CI/R injury is not clear.NLRP3 inflammasome,as the initiating factor of aseptic inflammatory response in the central nervous system after cerebral ischemia,induces cell death and inflammatory response in the central nervous system,leading to nerve cell damage.We have previously reported that BMSC-Exos can alleviate oxygen-glucose deprivation/reoxygenation(OGD/R)-induced inflammation and pyroptosis in PC12 cells.It is reported that Exos can inhibit pro-inflammation M1 microglia.Based on the aforementioned evidence,we hypothesized that BMSC-Exos could alleviate CI/R injury-induced inflammation and pyroptosis by modulating microglia polarization.Object:Explore whether BMSC-Exos could alleviate CI/R injury-induced inflammation and pyroptosis by modulating microglia polarization in vitro and in vivo.Methods:Extract exosomes by density gradient centrifugation and determine the characteristics of exosome.TTC staining and Brain Water Content assay were used to detect brain injury.mNSS,CatWalk system and MWM test were used to evaluate neurobehavioral deficits.Western blot analysis and immunofluorescence staining detect the levels of NLRP3 inflammasome and pyroptosis associated proteins.Immunofluorescence staining,Flow Cytometry,and RT-qPCR analysis was utilized to detected microglia phenotype in vitro and in vivo.Flow Cytometry was used to evaluate pyroptotic cell death.Results:The results of Flow Cytometry,Western blot,TEM and NTA exhibits that BMSC was isolated successfully,and BMSC-Exos conforms to the characteristics of exosome;Intravenous administration of BMSC-Exos reduces the infarct area and alleviates short-term neurobehavioral deficits after MCAO(middle cerebral artery occlusion,MCAO);BMSC-Exos intervention improved short term neurobehavior,walking parameters,cognitive and memory function as evaluated by mNSS test,CatWalk system and MWM test;Western blot analysis and immunofluorescence staining suggested that BMSC-Exos suppress inflammation and pyroptosis of neuron;Immunofluorescence staining,Flow Cytometry and RT-qPCR demonstrate that BMSC-Exos could inhibit pro-inflammatory M1 phenotype and promote anti-inflammatory M2 phenotype in vivo and in vitro;BMSC-Exos restrain PC12 pyroptosis by BV2 skewed polarization toward M2 phenotype.Conclusion:BMSC-Exos alleviate neuron inflammation and pyroptosis by shifting microglia toward the anti-inflammatory M2 phenotype.