Protection Mechanism Study Of Berberine Ameliorates OGD/R-induced Injury Via The Inhibition Of ER Stress And Autophagy In PC12

Objective: To investigate the protective effect and molecular mechanism of Berberine(BBR)on PC12 cell injury induced by Oxygen Glucose Deprivation/Reoxygenation(OGD/R).Methods:(1)Western blot and flow cytometry were used to establish the OGD/R model.(2)The effective working concentration of BBR was screened by CCK-8 kit(Cell Counting Kit-8).(3)Hoechest 33258 staining was used to observe the nucleus morphological changes for each group(Ctrl,OGD/R and OGD/R+BBR).(4)flow cytometry to detect the changes of apoptosis and reactive oxygen species(ROS)in each group(Ctrl,OGD/R and OGD/R + BBR).(5)The endoplasmic reticulum stress and apoptosis-related molecules GRP78,CHOP,Bax,Cleaved caspase3 in each group(Ctrl,OGD/R,OG/R + 4-PBA,and OGD/R + BBR)were detected by western blot and Immunofluorescence staining.(6)The PC12 cells were divided into Ctrl,OGD/R,OGD/R + 3-MA,and OGD/ R + BBR).Autophagy and apoptosis related molecules P-m TOR/m TOR,P62,Beclin-1,LC3-? / LC3-?,BAX and Cleaved caspase3 were detected by western blot.(7)Autophagy double-labeled adenovirus(GFP-RFP-LC3)was used to infected PC12 cells.Each groups' cells were analyzed by confocal microscopy.Results:(1)The OGD/R cell model was successfully established by western blot and flow cytometry.Oxygen-glucose deprivation for 4 hours and then reoxygenation for 18 hours(OGD4+R18)was as to model group for analysis the protective effect of BBR.(2)CCK-8 results showed that in the concentration range of 0.1-50 ?mol/L,BBR can promote the survival rate of PC12 cells.At 100 ?mol/L concentration,the cell survival rate decreased about 50%,which was close to the IC 50 value,and the difference is significant(P <0.05).(3)After OGD for 4 hours,co-culture with different concentrations of BBR for 18 hours on PC12 cells,the CCK-8 results showed that the optimal working concentration of berberine was 5 ?mol/L(P <0.05).(4)Flow cytometry results showed that BBR can significantly reduce the apoptosis and decrease the intracellular ROS level induced by OGD/R(OGD4 + R18)(P <0.05).(5)Hoechest33258 staining experiment showed that compared with the Ctrl group,the nucleus of the OGD/R group showed a bright blue color,indicating a significant increase about the OGD/R group's apoptosis,and the number of bright blue cells in the nucleus of the OGD/R + BBR group decreased,indicating that BBR could be reduced OGD/R-induced apoptosis of PC12 cells.(6)The results of western blot and immunofluorescence experiments showed that compared with the Ctrl group,the expression of endoplasmic reticulum stress and apoptosis-related molecules GRP78,CHOP,Bax,and Cleaved-caspase3 in the OGD/R group were significantly increased,but the agents 4-PBA(ER stress specific inhibitor)and BBR could significantly reduce the expression above-mentioned related endoplasmic reticulum stress and apoptotic molecules(P <0.05).(7)Western blot results showed that compared with the Ctrl group,the autophagy marker molecule LC3-?/LC3-? in the OGD/R group was increased,and the expression of P62 and Beclin-1 increased,the level of phosphorylation of the m TOR was decreased,but the autophagy-specific inhibitors 3-MA and BBR could significantly reduce the ratio of LC3-? / LC3-?(P<0.05),and the expression of P62 and Beclin-1 were reduced(P<0.05).The expression of the phosphorylated m TOR was increased(P <0.05).Conclusions:(1)OGD/R induced endoplasmic reticulum stress' and autophagic apoptosis in PC12 cells.The possible molecular mechanism is that OGD/R induces intracellular ROS levels accumulation.(2)BBR can significantly reduce OGD/R-induced endoplasmic reticulum stress cells Apoptosis,the possible mechanism is to reduce the expression of endoplasmic reticulum stress-related molecules GRP78 and CHOP,and further reduce the expression of apoptosis-related molecules Bax and Cleaved-Caspase3.(3)BBR can significantly reduce OGD/Rinduced autophagic death.The molecular mechanism may be to increase the expression of phosphorylated m TOR,reduce the expression of Beclin-1 and P62,reduce the ratio of LC3-? / LC3-?,improve dysfunctional autophagy flow,and then protect PC12 cells from OGD/R-induced injury.