Induction of adaptive response and enhancement of PC12 cell tolerance by 7-hydroxycholesterol and 15-deoxy-delta(12,14)-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-delta(12,14)-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.     

Peroxynitrite induces HO-1 expression via PI3K/Akt-dependent activation of NF-E2-related factor 2 in PC12 cells

Peroxynitrite is a strong oxidant produced by rapid interaction between superoxide anion and nitric oxide radicals and induces oxidative stress and cell death. Treatment of PC12 cells with 3-morpholinosydnonimine (SIN-1), a generator of peroxynitrite, induced the expression of heme oxygenase-1 (HO-1), an antioxidant cytoprotective enzyme. Inhibition of the HO activity by zinc protoporphyrin IX or knockdown of HO-1 gene expression with siRNA exacerbated the SIN-1-induced apoptosis. After SIN-1 treatment, there was a time-related increase in nuclear localization and subsequent binding of NF-E2-related factor 2 (Nrf2) to the antioxidant-responsive element (ARE). Transfection of PC12 cells with dominant-negative Nrf2 abolished the SIN-1-induced increase in Nrf2-ARE binding and subsequent upregulation of HO-1 expression, leading to enhanced cell death. Upon exposure of PC12 cells to SIN-1, the phosphatidylinositol 3-kinase (PI3K) activity was increased in a time-dependent manner. Pretreatment of cells with LY294002, a pharmacologic inhibitor of PI3K or transfection with the kinase-dead mutant Akt abrogated the SIN-1-induced Nrf2 activation and HO-1 expression. Taken together, these results suggest that peroxynitrite activates Nrf2 via PI3K/Akt signaling and enhances Nrf2-ARE binding, which leads to upregulation of HO-1 expression. The SIN-1-induced HO-1 upregulation may confer the adaptive survival response against nitrosative stress.     

Singlet-oxygen-derived products from linoleate activate Nrf2 signaling in skin cells

Linoleates are required for normal mammalian health and development, but they are also prone to oxidation, resulting in biologically active metabolites such as hydroxyoctadecadienoic acids (HODEs). To investigate the biological activity of 9-EZ-HODE, 10-EZ-HODE, 12-ZE-HODE, and 13-ZE-HODE, the metabolites of singlet-oxygen-derived products from linoleates, we assessed adaptive cytoprotection in HaCaT skin cells. Treating HaCaT cells with sublethal concentrations of 10-EZ-HODE and 12-ZE-HODE, which are singlet-oxygen-mediated specific oxidation metabolites of linoleates, but not 9-EZ-HODE and 13-ZE-HODE, caused resistance to hydrogen peroxide-induced oxidative damage. Microarray analysis of HaCaT cells revealed that 10-EZ-HODE and 12-ZE-HODE induced cellular antioxidant genes that are responsive to nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), such as heme oxygenase-1 and glutathione synthesis enzymes. Although 10-EZ-HODE and 12-ZE-HODE did not induce Nrf2 mRNA, treatment with these metabolites increased the intranuclear expression of Nrf2. These results suggest that 10-EZ-HODE and 12-ZE-HODE initiate adaptive responses that reduce the damage caused by oxidative stress.     

Carbon monoxide produced by heme oxygenase-1 in response to nitrosative stress induces expression of glutamate-cysteine ligase in PC12 cells via activation of phosphatidylinositol 3-kinase and Nrf2 signaling

Induction of heme oxygenase-1 (HO-1) expression has been associated with adaptive cytoprotection against a wide array of toxic insults, but the underlying molecular mechanisms remain largely unresolved. In this study, we investigated the potential role of carbon monoxide (CO), one of the by-products of the HO-1 reaction, in the adaptive survival response to peroxynitrite-induced PC12 cell death. Upon treatment of rat pheochromocytoma (PC12) cells with the peroxynitrite generator 3-morpholinosydnonimine hydrochloride (SIN-1), the cellular GSH level decreased initially, but was gradually restored to the basal level. This was accompanied by increased expression of the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in GSH biosynthesis. The SIN-1-induced GCLC up-regulation was preceded by induction of HO-1 and subsequent CO production. Inhibition of HO activity by zinc protoporphyrin IX or knockdown of HO-1 gene expression by small interfering RNA abrogated the up-regulation of GCLC expression and the subsequent GSH restoration induced by SIN-1. In contrast, additional exposure to the CO-releasing molecule (CO-RM) restored the GSH level previously reduced by inhibition of CO production using zinc protoporphyrin IX. Furthermore, CO-RM treatment up-regulated GCLC expression through activation of Nrf2. The CO-RM-induced activation of Nrf2 was under the control of the phosphatidylinositol 3-kinase/Akt signaling pathway. In conclusion, CO produced by HO-1 rescues PC12 cells from nitrosative stress through induction of GCLC, which is mediated by activation of phosphatidylinositol 3-kinase/Akt and subsequently Nrf2 signaling.     

Selenium deficiency activates mouse liver Nrf2-ARE but vitamin E deficiency does not

Selenium (Se) and vitamin E are antioxidant micronutrients. Se functions through selenoproteins and vitamin E reacts with oxidizing molecules in membranes. The relationship of these micronutrients with the Nrf2-antioxidant response element (ARE) pathway was investigated using ARE-reporter mice and Nrf2-/- mice. Weanling males were fed Se-deficient (0 Se), vitamin E-deficient (0 E), or control diet for 16 or 22 weeks. The ARE reporter was elevated 450-fold in 0 Se liver but was not elevated in 0 E liver. Antioxidant enzymes induced by Nrf2-ARE (glutathione S-transferase (GST), NAD(P)H quinone oxidoreductase (NQOR), and heme oxygenase-1 (HO-1)) were elevated in 0 Se livers but not in 0 E livers. Deletion of Nrf2 had varying effects on the inductions, with GST induction being abolished by it but induction of NQOR and HO-1 still occurring. Thus, Se deficiency, but not vitamin E deficiency, induces a number of enzymes that protect against oxidative stress and modify xenobiotic metabolism through Nrf2-ARE and other stress-response pathways. We conclude that Se deficiency causes cytosolic oxidative stress but that vitamin E deficiency does not. This suggests that the oxidant defense mechanisms in which these antioxidant nutrients function are independent of one another.     

Bisphenol S induces oxidative stress-mediated impairment of testosterone synthesis by inhibiting the Nrf2/HO-1 signaling pathway

Bisphenol S (BPS) is an environmental endocrine disruptor widely used in industrial production. BPS induces oxidative stress and exhibits male reproductive toxicity in mice, but the mechanisms by which BPS impairs steroid hormone synthesis are not fully understood. Nuclear factor erythroid 2-related factor 2(Nrf2)/HO-1 signaling is a key pathway in improving cellular antioxidant defense capacities. Therefore, this study explored the effects of exposure to BPS on testosterone synthesis in adult male mice and its mechanisms with regard to the Nrf2/HO-1 signaling pathway. Adult male C57BL/6 mice were orally exposed to BPS (2, 20, and 200 mg/kg BW) with sesame oil as a vehicle (0.1 ml/10 g BW) per day for 28 consecutive days. The results showed that compared with the control group, serum testosterone levels were substantially reduced in the 20 and 200 mg/kg BPS treatment groups, and testicular testosterone levels were reduced in all BPS treatment groups. These changes were accompanied by a prominent decrease in the expression levels of testosterone synthesis-related enzymes (STAR, CYP11A1, CYP17A1, HSD3B1, and HSD17B3) in the mouse testis. In addition, BPS induced oxidative stress in the testis by upregulating the messenger RNA and protein levels of Keap1 and downregulating the levels of Nrf2, HO-1, and downstream antioxidant enzymes (CAT, SOD1, and Gpx4). In summary, our results indicate that exposure of adult male mice to BPS can inhibit Nrf2/HO-1 signaling and antioxidant enzyme activity, which induces oxidative stress and thereby may impair testosterone synthesis in testicular tissues, leading to reproductive damage.     

Stabilization of Nrf2 protein by D3T provides protection against ethanol-induced apoptosis in PC12 cells

Previous studies have demonstrated that maternal ethanol exposure induces a moderate increase in Nrf2 protein expression in mouse embryos. Pretreatment with the Nrf2 inducer, 3H-1, 2-dithiole-3-thione (D3T), significantly increases the Nrf2 protein levels and prevents apoptosis in ethanol-exposed embryos. The present study, using PC12 cells, was designed to determine whether increased Nrf2 stability is a mechanism by which D3T enhances Nrf2 activation and subsequent antioxidant protection. Ethanol and D3T treatment resulted in a significant accumulation of Nrf2 protein in PC 12 cells. CHX chase analysis has shown that ethanol treatment delayed the degradation of Nrf2 protein in PC12 cells. A significantly greater decrease in Nrf2 protein degradation was observed in the cells treated with D3T alone or with both ethanol and D3T. In addition, D3T treatment significantly reduced ethanol-induced apoptosis. These results demonstrate that the stabilization of Nrf2 protein by D3T confers protection against ethanol-induced apoptosis.     

Gualou Guizhi Granule Protects Against Oxidative Injury by Activating Nrf2/ARE Pathway in Rats and PC12 Cells

Stroke involves numerous pathophysiological processes and oxidative stress is considered as a main cellular event in its pathogenesis. The nuclear factor erythroid-2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway plays a key role in inducing phase II detoxifying enzymes and antioxidant proteins and is now considered as a interesting therapeutic target for the treatment of stroke. The objective of this study is to investigate the protective effect of Gualou Guizhi granule (GLGZG) against oxidative stress and explore the protective mechanism of the Nrf2/ARE pathway. In vivo, administration of GLGZG in a rat model of focal cerebral ischemia significantly suppressed oxidative injury by increasing the activity of superoxide dismutase and glutathione level and decreasing reactive oxygen species and malondialdehyde levels. Western blot analysis showed that GLGZG induced nuclear translocation of Nrf2, and combined with real-time PCR results, which indicated that GLGZG up-regulated the Nrf2/ARE pathway. In addition, in cultured PC12 cells, GLGZG protected against H₂O₂ induced oxidative injury and activated the Nrf2/ARE pathway. All the results demonstrated that GLGZG in the management of cerebral ischemia and H₂O₂ induced oxidative injury may be associated with activation of Nrf2/ARE signaling pathway.