Zhi-Hua Chen

4-Hydroxynonenal Induces Adaptive Response and Enhances PC12 Cell Tolerance Primarily through Induction of Thioredoxin Reductase 1 via Activation of Nrf2

4-Hydroxynonenal (4-HNE) is one of the major end products of lipid peroxidation. It has been widely accepted that 4-HNE can induce oxidative stress, implicating into extensive stress-related diseases. In the present study, however, 4-HNE was found to exert adaptive cytoprotective effect at low concentrations, which was primarily through induction of thioredoxin reductase 1 (TR1) via transcriptional activation of NF-E2-related factor 2 (Nrf2). Pretreatment with 4-HNE at sublethal concentrations significantly protected PC12 cells against the subsequent oxidative cell death induced by H2O2 and 6-hydroxydopamine. The cellular antioxidative glutathione system did not show any considerable changes, whereas the TR1 activity as well as the mRNA level was significantly elevated by the 4-HNE treatment. Cells treated with TR1 small interfering RNA exhibited less resistance to oxidative stress, and the adaptive response was completely abolished. The Nrf2 was transcriptionally activated by 4-HNE. Cells treated with Nrf2-small interfering RNA exerted lower constitutive levels of TR1 and exhibited less resistance to oxidative stress, and the 4-HNE-induced TR1 expression and subsequent adaptive response were again abolished in such cells. Treatment with 4-HNE at the adaptive concentration induced transient activation of extracellular signal-regulated protein kinase 1/2 and Akt/protein kinase B. Pharmacological inhibition of both these kinase pathways effectively attenuated 4-HNE-induced TR1 expression and subsequent adaptive protection. The above findings, taken together, suggest that stimulation with 4-HNE at sublethal concentrations induces adaptive response and enhances cell tolerance, primarily through induction of TR1 via transcriptional activation of Nrf2 signaling pathway, thereby protecting cells against the forthcoming oxidative stress.

Induction of Adaptive Response and Enhancement of PC12 Cell Tolerance by 7-Hydroxycholesterol and 15-Deoxy-Δ12,14-Prostaglandin J2 through Up-regulation of Cellular Glutathione via Different Mechanisms

Increasing evidence suggests an adaptive response induced by reactive oxygen species and other physiologically existing oxidative stimuli. We have recently reported that a variety of lipid peroxidation products at sublethal concentrations could induce adaptive response and enhance PC12 cell tolerance, although the detailed underlying molecular mechanisms have not been clearly clarified. In the present study, we found that both 7-hydroxycholesterol (7-OHCh) and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) at sublethal concentrations significantly increased the cellular GSH as well as the enzyme activity of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis. Depletion of cellular GSH by buthionine sulfoximine completely abolished the adaptive response. Interestingly, treatment with 15d-PGJ2 significantly increased the gene expression of both subunits of GCL in an NF-E2-related factor 2 (Nrf2)-dependent manner, whereas neither 7-OHCh induced any considerable changes on the GCL gene expression nor did the Nrf2-small interfering RNA treatment exert any appreciable effects on the GSH elevation and subsequent adaptive response induced by 7-OHCh. These results demonstrate that the adaptive response induced by both 7-OHCh and 15d-PGJ2 is mediated similarly through the up-regulation of GSH but via different mechanisms.

Adaptive response induced by lipid peroxidation products in cell cultures

The adaptive response induced by the lipid peroxidation products, such as phosphatidylcholine hydroperoxide, lysophosphatidylcholine (LysoPC), 15‐deoxy‐Δ12,14‐prostaglandin J2, 4‐hydroxynonenal (4‐HNE), hydroxyoctadecadienoic acid, 7‐hydroxycholesterol, and cholesterol 5β,6β‐epoxide, was investigated in this study. Although these products have been implicated in oxidative stress‐related diseases, pretreatment with such compounds at sublethal concentrations significantly protected PC12 cells against subsequent oxidative stress induced by 6‐hydroxydopamine. Moreover, 4‐HNE and LysoPC also exhibited adaptive protection in human arterial endothelial cells. These findings suggest a general hormetic effect of such compounds in cell cultures and may lead to a reappraisal of the eventual role of reactive oxygen species and lipid peroxidation in organisms.

Adaptation to hydrogen peroxide enhances PC12 cell tolerance against oxidative damage

The adaptive responses to H2O2 and the resulting protective effect against oxidative stress have been investigated using PC12 cells. Pretreatment of sublethal doses of H2O2 significantly protected PC12 cells against the cytotoxicity induced by lethal H2O2. The endogenous antioxidant defense systems, catalase and glutathione peroxidase, were enhanced quickly by the pretreatment of low doses of H2O2. This pretreatment also exerted protective effect against the oxidative insults induced by 6-hydroxydopamine and paraquat, but not against alkyl peroxyl radicals. Our results, taken together, suggest that the stimulation by low dose of H2O2 enriches the cellular antioxidant defense systems, thereby enhancing cell tolerance against the forthcoming oxidative insults induced by H2O2 and related hydroxyl radicals.

Effect of oxygen concentration on free radical-induced cytotoxicity.

Free radicals induce oxidative stress in vivo, leading to various disorders and diseases. In the present study, the effect of oxygen pressure on the cytotoxicity induced by free radicals was studied. It was found that alkyl radicals markedly aggravated Jurkat cell apoptosis under low oxygen pressure and this was ascribed to a hypoxic condition caused by the consumption of oxygen by alkyl radicals giving peroxyl radicals and subsequent lipid peroxidation by a chain mechanism. The intracellular lipid hydroperoxides significantly increased at an early time point even under hypoxia. Cytochrome c was released from the mitochondria, and caspase-9 as well as caspase-3 was activated during apoptosis, indicating that cell death followed by the intrinsic, mitochondrial apoptosis pathway. Pretreatment with VAD-FMK, a caspase inhibitor, attenuated the apoptosis induced by alkyl radicals under hypoxia. Moreover, pretreatment with various antioxidants also significantly rescued the cells from apoptosis. Taken together, the results indicate that free radicals induced hypoxic conditions, which accelerated mitochondria-dependent cell apoptosis.