The Regulatory Effect Of M2-type Microglia-derived Exosomes On H2O2-induced Apoptosis Of PC12 Cells

Objective:To explore the regulatory effect of M2-type microglia exosomes on H₂O₂-induced apoptosis of PC12 cells,and further explore the related mechanisms affecting apoptosis.Methods:The apoptosis model of PC12 cells in good growth state was established by H₂O₂induction.IL-4 was used to induce the resting microglia to convert into M2-type microglia cells,and M2-type exosomes were isolated by overspeed centrifugation.The exosomes were identified by TEM,NTA and WB.Exosome uptake was measured using confocal microscopy.In order to detect the changes of proteins related to PI3K/AKT signaling pathway,PC12 cells were divided into three groups according to different intervention methods:Control group,H₂O₂group,H₂O₂+Exo group,and WB was used to detect the protein expression of PI3K,p-PI3K,AKT and p-AKT.To observe the related effects of the PI3K inhibitor Ly294002,PC12 cells were divided into four groups:Control group,H₂O₂group,H₂O₂+Exo group,H₂O₂+Exo+Ly294002group,Western blot detection of the protein expression of AKT and p-AKT downstream PI3K.And expression of apoptosis-related protein molecules Bax,Bcl-2,Cleaved caspase-3,Cleaved caspase-9.Flow cytometry was used to detect the apoptosis of PC12 cells.Results:The mRNA expression of M2 microglia markers Arg1 and Ym1 was detected by qRT-PCR to verify that IL-4 was successfully used to induce M2 microglia.The exosomes were identified by TEM,NTA and WB,and it was verified that the exosomes of M2 microglia were extracted successfully.In the detection of PI3K/AKT pathway related protein changes,the expression of p-PI3K/PI3K in H₂O₂group was significantly decreased compared with Control group（P<0.001）;Compared with H₂O₂group,the expression of H₂O₂+Exo group increased（P<0.05）.The expression of p-AKT/AKT in H₂O₂group was significantly decreased compared with Control group（P<0.001）;Compared with H₂O₂group,the expression of H₂O₂+Exo group was significantly increased（P<0.001）.In the detection of the related influence of PI3K inhibitor Ly294002,WB detected the protein expression of AKT and p-AKT downstream of PI3K,as well as apoptosis-related protein molecules Bax,Bcl-2,Cleaved caspase-3,Cleaved caspase-9.Compared with Control group,the expression levels of p-AKT/AKT and Bcl-2 in H₂O₂group were significantly decreased（P<0.001）,Bax,Cleaved caspase-3 and Cleaved caspase-9 expression levels were significantly increased（P<0.001）;Compared with H₂O₂group,the expressions of p-AKT/AKT and Bcl-2 in H₂O₂+Exo group were significantly increased（P<0.001）,Bax,Cleaved caspase-3 and Cleaved caspase-9 expression levels were significantly decreased（P<0.001）;Compared with H₂O₂+Exo group,the expression level of p-AKT/AKT in H₂O₂+Exo+Ly294002group was decreased（P<0.05）,the expression level of Bcl-2 was significantly decreased（P<0.001）,Bax,Cleaved caspase-3 and Cleaved caspase-9 expression levels were up-regulated（P<0.05）.Flow cytometry was used to detect the apoptosis of PC12 cells in each group.Compared with Control group,the apoptosis rate of PC12 cells in H₂O₂group was significantly increased（P<0.001）;Compared with H₂O₂group,the apoptosis rate of H₂O₂+Exo group was significantly decreased（P<0.001）;Compared with H₂O₂+Exo group,apoptosis rate of H₂O₂+Exo+Ly294002group was significantly increased（P<0.05）.Conclusion:In vitro experiments,this study found that M2-type microglia exosome treatment significantly inhibited PC12 cell apoptosis and activated PI3K/AKT signaling pathway,while Ly294002 inhibited PI3K/AKT signaling pathway,showing the opposite effect.The results showed that M2-type microglial exosomes inhibited apoptosis of PC12cells by activating PI3K/AKT pathway.Therefore,the effect of M2-type microglia exosomes on PC12 cells may suggest that M2-type microglia-derived exosomes may be a promising therapeutic agent for nerve injury and provide a feasible treatment for NP.