7,8-Dihydroxyflavone Protects PC12 Cells Against 6-hydroxyl Dopamine-induced Cytotoxicity Through Nrf2/HO-1 Pathway

7,8-Dihydroxyflavone(7,8-DHF)has been identified as a selective tyrosine kinase receptor B(Trk B)agonist and exerts potent neuroprotective and neurotrophic effects.Our previous study found that 7,8-DHF could protect against 6-OHDA-induced cytotoxicty in PC12 cells,a cell line without Trk B expression.The protective effect of 7,8-DHF seems to be associated with its antioxidant property.However,the antioxidant mechanism of7,8-DHF has not been fully elucidated.In the present study,we further investigated whether Nrf2/HO-1,the most important antioxidant signaling pathway,be involved in the protective effect of 7,8-DHF in 6-OHDA-treated PC12 cells.The results were as follows:1.Preatrement with 7,8-DHF(1,5,25 ?M)for 6 h exerted potent protective effect in6-OHDA(100 ?M for 24 h)-treated PC12 cells.Compared with 6-OHDA group(47.2%),7,8-DHF(25 ?M)elevated the cell viability up to 86.5%(P < 0.01).Thus25 ?M of 7,8-DHF and 100 ?M of 6-OHDA were used in the subsequent studies.2.Exposure to 6-OHDA for 6 h increased the intracellular ROS production by 2.97 fold(P < 0.01).Pretreatment with 7,8-DHF markedly inhibited the ROS level(P < 0.05).3.Exposure to 6-OHDA for 24 h caused 1.86 fold increase in GSSG level(P < 0.01).However,this change could also be inhibited by 7,8-DHF pretreatment(P < 0.01).4.After incubation with 7,8-DHF for different time(1-24 h),the whole cell Nrf2 level kept constant,while HO-1 expression was significantly higher from 6 to 24 h(P <0.01).5.The translocation of Nrf2 from the cytoplasm to the nucleus could induce HO-1expression.7,8-DHF treatment for 3 or 6 h reduced Nrf2 protein level in the cytoplasm(P < 0.01),but it elevated the Nrf2 levels in the nucleus(P < 0.05),indicating that 7,8-DHF might induce Nrf2 translocation.6.Treatment with 7,8-DHF for 3-6 h up-regulated the p-Akt expression(P < 0.05-0.01).In addition,LY294002(10 ?M,a specific PI3 K inhibitor)abrogated DHF-induced HO-1 expression(P < 0.01).This suggests that 7,8-DHF might up-regulate HO-1expression through activating PI3K/Akt signaling pathway.7.Treatment with 7,8-DHF for 3-6 h also up-regulated the p-ERK1/2 expression(P <0.05-0.01).Application of PD98059(20 ?M,a MEK inhibitor)partially blocked DHF-induced HO-1 expression(P < 0.01).This suggests that 7,8-DHF might elevate the HO-1 expression through activating ERK1/2 signaling pathway.8.In MTT experiments,pretreatment with ZnPP(5 ?M,a specific HO-1 inhibitor),LY294002(10 ?M)or PD98059(20 ?M),could partially block the protective effect of 7,8-DHF in 6-OHDA-treated PC12 cells(P < 0.05).These results further support the hypothesis that Nrf2/HO-1,Akt and ERK1/2 signaling pathways might be involved in the protective effect of 7,8-DHF.In summary,7,8-DHF might protect PC12 cells against 6-OHDA-induced cytotoxicity through activating Nrf2/HO-1 pathway.7,8-DHF induced translocation of Nrf2 to the nucleus to up-regulate HO-1 expression.HO-1 was considered as an important antioxidant enzyme by degrading heme to bilirubin,CO and iron.In addition,the activation of Nrf2/HO-1 by 7,8-DHF might be mediated by the upstream PI3K/Akt and ERK1/2 pathways.