35% Oxygen Preconditioning Protects PC12 Cells Against Death Induced By Hypoxia

Reactive oxygen species, the collective term for superoxide anion (O2.-), hydrogen peroxide (H2O2), hydrol radical (.OH) and others, have been traditionally regarded as cytotoxic, with the potential to cause damage to lipids, protein, and DNA. In contrast to this harmful condition, considerable evidence show that various stimuli can stimulate the enzymatic generation of lower levels of ROS, acting as second messengers in response to these factors.Previous studies have indicated that ischemic preconditioning (IPC) can protect tissues against injury. Now, the concept has been extended to preconditioning triggered by non-ischemic stress, such as generation of reactive oxygen radicals.Extracellular signal-regulated kinase (ERK), a prototype member of the mitogen-activated protein kinase (MAPK) family, is up-regulated by reactive oxygen species production in response to numerous stimuli. Moreover, ERK activation promotes Bcl-2 gene expression through a variety of transcription factors activation. The Bcl-2 protein family has an important role in the regulation of programmed cell death.This article is designed to investigate whether 35% oxygen preconditioning for 3 hours can protect PC12 cells against the death induced by hypoxia (1% O2), and whether 35% oxygen induced adaptive cytoprotection is related to the generation of ROS, ERK signal-transduction pathway, and the overexpression of Bcl-2.MethodsPC12 cells, were maintained in Dulbecco's modified Eagle's medium. PC12 cells were preconditioned with exposure to 35% O2 for 180 min followed by 12 h recovery and subsequent exposure to hypoxia for 72 h. Prior to exposing to 35% O2 and 1% O2, the medium was replaced with serum-free medium. Cells were pre-incubated with related drugs for 60 min before exposing to 35% O2. Viability was measured by MTT method. Intracellular ROS generation was monitored by flow cytometry using peroxide-sensitive fluorescent probe 2',7'-dichlorofluorescein diacetate and dihydroethidium. PC12 cells were transfected using lipofectamine 2000 and ERK SiRNA, as recommended by manufacturer's instructions. ERK mRNA was detected by RT-PCR according to the manufacturer's protocol. Western Blot was performed to estimate the expression of ERK and Bcl-2Results1. cell viability Hypoxia could cause considerable PC12 cells death. But the effect was inhibited by preconditioning with 35% O2 for 3 h followed by 12 h recovery and no efffect was found by 0 h or 24 h recovery, while preconditioning by 50% and 75% O2 just showed the opposite results in MTT assay.2. ROS production PC12 cells in 35% O2 and pyrogallol group produced more O2.? than that in the 21% O2 group and the O2.? in tempol group decreased significantly than that in the 35% O2 group. The production of H2O2 by 35% O2 preconditioning did not increased significantly compared with that of 21% O2 preconditioning. The cell viability increased significantly in pyrogallol and 35% O2 group, and reduced significantly in tempol group.3. ERK expression Thirty-five percent oxygen pretreatment significantly enhanced the cell viability and activated ERK1/2 expression, and these effect was obviously attenuated by PD98059, not by H7 and wortmannin. The activated ERK1/2 overexpression induced by 35% O2 was obviously reduced by treatment with 4-hydroxyl-tempol. Preconditioning with 21% O2 and pyrogallol, could also induced the overexpression of phosphorylated ERK1/2. Contrastively pretreatment with 35% O2 and catalase, or preconditioning with 21% O2 and H2O2 did not change the ERK1/2 expression obviously compared with that of 35% O2 or 21% O2 preconditioning respectively.4. ERK SiRNA RT-PCR analysis showed that ERK mRNA expression was remarkably inhibited by SiRNA. The expression of total and activated ERK1/2 protein in SP group was strongly suppressed. The viability of PC12 cells tranfected with ERK SiRNA was remarkably reduced than that of normal PC12 cells.5. Bcl-2 expression The levels of Bcl-2 and phosphorylated ERK expression increased obviously in 35% O2 group compared with that in other groups and decreased significantly in PD group and SiRNA group compared with that in 21% O2 or 35% O2 group.Conclusion1. 35% O2 preconditioning for 3 h followed by 12 h recovery could protect PC12 cells against death induced by hypoxia.2. PC12 cells preconditioned with 35% O2 could generate O2.?, which played a vital role in protecting PC12 cell against cytotoxicity induced by hypoxia.3. MAPK signal transduction pathway was involved in the cytoprotective effect of 35% O2 preconditioning.4. ERK signal pathway was necessary to the overexpression of the Bcl-2 protein in PC12 cells preconditioned with 35%O2, and Bcl-2 played an important role in this process.