Effect Of Exosomes Secreted From Bone Marrow Mesenchymal Stem Cells On Oxygen-Glucose Deprivation/Reoxygenation Injury In PC12 Cells And Underlying Mechanism

Background and aims:Cerebral ischemia reperfusion(I/R)injury can lead to severe dysfunction,and its treatment is difficult.NLRP3 inflammasome-mediated cell pyroptosis,which occurs with inflammation,is an important part of cerebral I/R injury.It has been reported in the literature that activating autophagy can inhibit inflammatory responses and thus protect tissues from I/R damage.Our previous study found that the protective effects of bone marrow mesenchymal stem cells(BMSCs)in cerebral I/R injury may be associated with the regulation of autophagy.Recent studies have demonstrated that exosomes secreted from BMSCs(BMSC-Exos)may play an essentlial role in the effective biological performance of BMSCs.BMSC-Exos not only play a role in tissue repair of BMSCs,but also avoid the risk of cell transplantation.It has been found that the protective mechanism of BMSC-Exos in myocardial I/R injury models is related to the alleviation of inflammation through activation of autophagy.However,the role of BMSC-Exos in cerebral I/R injury remains unclear.The purpose of this study was to determine the effect of BMSC-Exos on crebral I/R injury by in vitro test and to investigate whether the mechanism is related to pyroptosis and AMP activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)-dependent autophagic flux.Methods:1.Extraction and identification of BMSC-Exos:BMSCs were extracted by direct adherent method,supernatant was collected after culture,and BMSC-Exos were extracted by ultra-high speed centrifugation method.Western blot,transmission electron microscope(TEM)and nanoparticle tracking analysis(NTA)were used for identification.2.Building mould and grouping:PC 12 cells were subjected to oxygen-glucose deprivation/reoxygenation(OGD/R),divided into the control group,OGD/R group,OGD/R+BMSC-Exos group,OGD/R+MCC950 group,OGD/R+BMSC-Exos+pNLRP3 group,OGD/R+BMSC Exos+Cq group,OGD/R+BMSC Exos+compound C group.3.PC12 cells viability:Cell viability was determined with CCK-8,lactate dehydrogenase release(LDH)was determined with LDH assay and reactive oxygen species(ROS)was determined with DCFH-D A assay.4.PC 12 cells pyroptosis:After addition of NLRP3 inhibitor MCC950 and transfection with pNLRP3 overexpression plasmid,scanning electron microscopy,Hoechst 33342/propidium iodide(PI)double staining,immunofluorescence,western blot and enzyme-linked immunosorbent assay were used to detect cell pyroptosis.5.PC 12 cells autophagic flux:After addition of autophagy inhibitor Cq and AMPK inhibitor compound C,TEM,mRFP-GFP-LC3 adenovirus transfection,and western blott were used to detect autophagic flux and its influence on pyroptosis.Finally,co-immunoprecipitation(Co-IP)was used to detect the binding interaction between NLRP3 and LC3.Results:1.Identification of BMSC-Exos:the identification results of Western blot,TEM and NTA all suggested that the extracted BMSC-Exos conforms to the characteristics of exosomes.2.PC12 cells viability:BMSC-Exos could significantly improve the viability(p<0.05)and decrease LDH release(p<0.05)of PC12 cells after OGD/R.3.PC12 cells pyroptosis:In the BMSC-Exos treatment group,the cell contour became clearer and the surface villi increased and became denser;the PI staining proportion was decreased(p<0.05);ROS levels were decreased(p<0.05);the expression of NLRP3,cleaved caspase-1 and GSDMD-N and Interleukin 1?(IL-1?)was decreased(p<0.05).The inhibitory effect of BMSC-Exos on pyroptosis was comparable to the NLRP3 inhibitor MCC950 and was reversed by pNLRP3 overexpression plasmid.4.PC 12 cells autophagic flux:In the BMSC-Exos treatment group,LC3?/? didn't changed significantly,p-AMPK/AMPK increased(p<0.05),P62 and p-mTOR/mTOR decreased(p<0.05).Both the autophagy inhibitor Cq and AMPK inhibitor compound C reversed the above inhibitory effect on pyroptosis and promting effect on autophagic flux.The results of Co-IP showed that the interaction between NLRP3 and LC3 was increased after the treatment of BMSC-Exos(p<0.05)Conclusions:1.BMSC-Exos could improve PC12 cells viability.2.BMSC-Exos could protect PC 12 cells against OGD/R injury through inhibiting NLRP3 inflammasome-mediated cell pyroptosis.3.BMSC-Exos could protect PC 12 cells against OGD/R injury through attenuating NLRP3 inflammasome-mediated pyroptosis by promoting AMPK-dependent autophagic flux.