Effect Of Scutellarin On Apoptosis Of PC12 Cells Under Hypoxia Or Activated Microglia And The Role Of JAK2/STAT3 Signaling Pathway

Objective(s): To investigate the effect of scutellarin on apoptosis and apoptosis-related protein changes in PC12 cells in response to hypoxia or activated microglia,and exploring whether the action of scutellarin is related to the JAK2/STAT3 signaling pathway.Methods: In this experiment,BV-2 microglial cell line and PC12 cell line were used for in vitro experiments.(1)The cultured PC12 cells were treated with oxygen and glucose deprivation(OGD)and divided into three groups: control group,OGD group and OGD+ scutellarin group.CCK-8 cell viability assay was used to determine the optimal concentration of scutellarin in PC12 cells,and Western blot was used to detect the expression of apoptotic protein Cleaved caspase-3.(2)BV-2 microglia were activated with OGD,and PC12 cells were cultured in the medium of activated microglia as conditioned medium(CM).They were divided into three groups: control group,CM group and scutellarin pretreatment group.CCK-8 cell viability assay was used to determine the optimal concentration of scutellarin in BV-2 microglia,TUNEL was used to detect the apoptosis number of PC12 cells in each group,and the expression of apoptosis-related proteins(Caspase-3,Cleaved caspase-3,Bcl-2,Bax)was detected by immunofluorescence double labeling and Western blot.(3)BV-2microglia were activated with OGD,and PC12 cells were cultured in the medium of activated microglia as conditioned medium(CM).They were divided into three groups: control group,CM group and scutellarin pretreatment group.The expression of JAK2/STAT3 signaling pathway(JAK2、p-JAK2、STAT3、p-STAT3)was detected by immunofluorescence double labeling and Western blot.PC12 cells were pretreated with JAK2/STAT3 signaling pathway specific inhibitor(I)AG490 and divided into 6groups: control group,control+I group,CM group,CM+I group,CM+scutellarin group,CM+scutellarin+I group.The expression of apoptosis-related proteins and JAK2/STAT3 signaling pathway were detected by immunofluorescence double labeling and Western blot.Results:(1)After OGD treatment,the viability of PC12 cells decreased significantly(p<0.01),and the expression of apoptotic protein Cleaved caspase-3increased significantly(p<0.01);After scutellarin intervention,there was no significant change in the cell viability and the expression of apoptotic protein Cleaved caspase-3 of PC12 cells in OGD group.(2)BV-2 microglia activated by OGD significantly increased the number of apoptosis of PC12 cells(p<0.01),the expression of apoptotic protein Cleaved caspase-3 and proapoptotic protein Bax increased significantly(p<0.01 or p <0.05),and the expression of anti-apoptotic protein Bcl-2decreased significantly(p<0.01);In the scutellarin pretreatment group,the number of apoptosis of PC12 cells decreased significantly(p <0.01),the expression of apoptotic protein Cleaved caspase-3 and proapoptotic protein Bax decreased significantly(p<0.01 or p<0.05),and the expression of anti-apoptotic protein Bcl-2 increased significantly(p<0.01).(3)BV-2 microglia activated by OGD could significantly inhibit the expression of p-JAK2 and p-STAT3 pathway proteins in PC12 cells(p<0.01 or p<0.05);After scutellarin pretreatment,the expression of p-JAK2 and p-STAT3 pathway proteins in PC12 cells increased significantly(p<0.01 or p<0.05).AG490,a specific inhibitor of JAK2/STAT3 signaling pathway,could significantly increase the number of apoptosis of PC12 cells in the scutellarin treated group(p<0.05),and significantly promote the expression of apoptotic protein Cleaved caspase-3 and pro-apoptotic protein Bax(p<0.05),and significantly inhibit the expression of anti-apoptotic protein Bcl-2 and pathway proteins p-JAK2 and p-STAT3(p<0.01 or p<0.05).Conclusion(s):(1)Scutellarin has no obvious direct effect on the apoptosis of PC12 cells induced by oxygen glucose deprivation(OGD).(2)Scutellarin can inhibit the apoptosis of PC12 cells mediated by microglia activated by OGD.(3)Scutellarin may regulate the apoptosis of PC12 cells mediated by activated BV-2 microglia through JAK2/STAT3 signaling pathway.