The Protective Effect Of Edaravone On PC12 Cells Injuried By CPF And Its Mechanism

Objective: We aim to confirm the impairment of CPF to nerve cells,evaluate the protective effect of Edaravone on PC12 cells injuried by CPF and try to unravel its possible mechanism.Method:(1)Evaluate the ability of PC12 cells treated with CPF and Edaravone by MTT assay;(2)Set up control group,DMSO solvent group,CPF group,Edaravone group and use a fluorescent inverted microscope to observe cell morphology in all groups;(3)Cell apoptosis in different groups were observed by Hoechst33342 stain;(4)The level of reactive oxygen species(ROS),the content of malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)were detected to assess the degree of oxidative stress in all groups;(5)Western blot was used to detect the expression of Nrf-2 in whole cell level and in nucleus level;(6)Screen PC12 cells with Nrf-2 knockdown,expression of Nrf-2 was measured by Western blot to make sure Nrf-2 knockdown is successful;(7)Mark PC12 cells with Nrf-2 knockdown as KD(knockdown),set up KD-DMSO group,KD-CPF group,KD-CPF+Ed group,repeat steps in(1),(2),(3)and(4)to show the effect of Edaravone on KD injuried by CPF under the same condition.Results:(1)MTT find the most appropriate harmful concentration of CPF is 200 ?mol/L,and the most appropriate protective concentration of Edaravone is 25 ?mol/L;PC12 cells are injuried by CPF in a concentration-dependent manner,Edaravone increases the ability of PC12 cells which were injuried by CPF;(2)CPF destroys the integrity of PC12 cell morphology,neurite defect is pretty common in CPF group,cell density gets lower than DMSO group;Edaravone protects PC12 cells away from damage induced by CPF,it makes both cell morphology and cell density be similar to those in DMSO group;(3)PC12 cells in CPF group show apoptotic characteristics,such as karypyknosis,karyorrhexis and cell nucleus appear in strong fluorescence intensity;Edaravone group and DMSO group don't indicate any obvious apoptosis.(4)CPF increases ROS level and MDA content in PC12 cells,meanwhile,it drops SOD activity;but the damage induced by CPF is reversed at last by Edaravone;(5)Compared with DMSO group and CPF group,Western blotshows Edaravone increases the expression of Nrf-2 both in whole cell and nucleus level;(6)Nrf-2 knockdown is effective for our experiment proved by Western blot;(7)The ability of KD is decreased by CPF,but Edaravone doesn't relieve the damage induced by CPF in KD;(8)Serious damage in cell morphology exists in both KD-CPF group and KD-CPF+Ed group,cell integrity is ruined compared with KD-DMSO group;(9)Apoptosis is severe in KD-CPF and KD-CPF+Ed groups,cell nucleus in both groups display a strong fluorescence intensity,karypyknpsis and karyorrhexis are found;KD-DMSO doesn't occur unusual cases;(10)When compared with KD-DMSO group,ROS level and MDA content are higher in KD-CPF group and KD-CPF+Ed group,on the contrary,the activity of SOD gets lower in KD-CPF group and KD-CPF+Ed group.Conlusion: CPF can injury the PC12 cells,both in cell ability and cell morphology.CPF do induce oxidative stress and apoptosis.Edaravone has a protective effect on PC12 cells injuried by CPF.With all results summarized above,we come up with an assumption that Edaravone protects PC12 cells away from damage induced by CPFthrough regulating the expression of Nrf-2 and promoting Nrf-2 protein to transfer to cell nucleus.