4-hydroxy-2-nonenal and sulforaphane

Oxidants have been implicated in the pathophysiology of idiopathic pulmonary fibrosis (IPF), especially in myofibroblastic differentiation. We aimed at testing the hypothesis that nuclear factor erythroid 2-related factor 2 (Nrf2), the main regulator of endogenous antioxidant enzymes, is involved in fibrogenesis via myofibroblastic differentiation. Fibroblasts were cultured from the lungs of eight controls and eight IPF patients. Oxidants-antioxidants balance, nuclear Nrf2 expression, and fibroblast phenotype (α-smooth muscle actin and collagen I expression, proliferation, migration, and contraction) were studied under basal conditions and after Nrf2 knockdown or activation by Nrf2 or Keap1 siRNA transfection. The effects of sulforaphane (SFN), an Nrf2 activator, on the fibroblast phenotype were tested under basal and pro-fibrosis conditions (transforming growth factor β [TGF-β]).. Decreased Nrf2 expression was associated with a myofibroblast phenotype in IPF compared with control fibroblasts. Nrf2 knockdown induced oxidative stress and myofibroblastic differentiation in control fibroblasts. Conversely, Nrf2 activation increased antioxidant defences and myofibroblastic dedifferentation in IPF fibroblasts. SFN treatment decreased oxidants, and induced Nrf2 expression, antioxidants, and myofibroblastic dedifferentiation in IPF fibroblasts. SFN inhibited TGF-β profibrotic deleterious effects in IPF and control fibroblasts and restored antioxidant defences. Nrf2 knockdown abolished SFN antifibrosis effects, suggesting that they were Nrf2 mediated.. Our findings confirm that decreased nuclear Nrf2 plays a role in myofibroblastic differentiation and that SFN induces human pulmonary fibroblast dedifferentiation in vitro via Nrf2 activation. Thus, Nrf2 could be a novel therapeutic target in IPF.

Topics: Active Transport, Cell Nucleus; Aldehydes; Animals; Becaplermin; Cell Dedifferentiation; Cell Nucleus; Cells, Cultured; Collagen Type I; Epoxide Hydrolases; Gene Knockdown Techniques; Heme Oxygenase-1; Humans; Idiopathic Pulmonary Fibrosis; Isothiocyanates; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myofibroblasts; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Oxidative Stress; Phenotype; Proto-Oncogene Proteins c-sis; RNA, Small Interfering; Sulfoxides; Thiocyanates; Transforming Growth Factor beta

The transcription factor NF-E2-related factor 2 (Nrf2) mediates transcription of antioxidant/cytoprotective genes by binding to the antioxidant-response element (ARE) within DNA. Upregulation of these genes constitutes a pleiotropic cytoprotective defense pathway, which has been shown to produce neuroprotection in numerous models by decreasing lipid peroxidation (LP) as measured by the neurotoxic LP by-product 4-hydroxynonenal (4-HNE). As neuronal mitochondria have previously been shown to be susceptible to insult-induced LP-mediated oxidative damage, we sought to mechanistically investigate whether Nrf2-ARE activation in vivo could protect mitochondria from subsequent 4-HNE exposure ex vivo. Young adult male CF-1 mice were administered one of two known Nrf2-ARE activators as single intraperitoneal doses-sulforaphane (SFP; 5.0mg/kg) or carnosic acid (CA; 1.0mg/kg)-or their respective vehicles 48 h before Ficoll isolation of rat cerebral cortical mitochondria. Purified mitochondria were then exposed ex vivo to 4-HNE for 15 min at 37 °C, which we showed to cause a concentration-related inhibition of mitochondrial respiration together with covalent binding of 4-HNE to mitochondrial proteins. We chose a 30 μM concentration of 4-HNE, which produced an approximately 50% inhibition of complex I- or complex II-driven respiration, to assess whether prior in vivo Nrf2-ARE-activating compounds would increase the resistance of the isolated cortical mitochondria to 4-HNE's mitotoxic effects. Administration of either compound significantly increased (p < 0.05) expression of heme oxygenase-1 mRNA in cortical tissue 48 h postadministration, verifying that both compounds were capable of inducing the Nrf2-ARE pathway. Moreover, the prior in vivo administration of SFP and CA significantly (p < 0.05) attenuated 4-HNE-induced inhibition of mitochondrial respiration for complex I, but only carnosic acid acted to protect complex II. Furthermore, both CA and SFP significantly (p < 0.05) reduced the amount of 4-HNE bound to mitochondrial proteins as determined by Western blot. These results demonstrate the capability of in vivo Nrf2-ARE induction to protect from 4-HNE toxicity to cortical mitochondria ex vivo. Ongoing studies will determine the therapeutic efficacy of Nrf2-ARE activators to attenuate traumatic brain injury-induced pathophysiology.

Topics: Abietanes; Aldehydes; Animals; Anticarcinogenic Agents; Antioxidant Response Elements; Antioxidants; Cell Respiration; Cysteine Proteinase Inhibitors; Heme Oxygenase-1; Isothiocyanates; Male; Mice; Mitochondria; NF-E2-Related Factor 2; Plant Extracts; Rats; RNA, Messenger; Sulfoxides; Thiocyanates

Recent studies have shown that sulforaphane, a naturally occurring compound that is found in cruciferous vegetables, offers cellular protection in several models of brain injury. When administered following traumatic brain injury (TBI), sulforaphane has been demonstrated to attenuate blood-brain barrier permeability and reduce cerebral edema. These beneficial effects of sulforaphane have been shown to involve induction of a group of cytoprotective, Nrf2-driven genes, whose protein products include free radical scavenging and detoxifying enzymes. However, the influence of sulforaphane on post-injury cognitive deficits has not been examined. In this study, we examined if sulforaphane, when administered following cortical impact injury, can improve the performance of rats tested in hippocampal- and prefrontal cortex-dependent tasks. Our results indicate that sulforaphane treatment improves performance in the Morris water maze task (as indicated by decreased latencies during learning and platform localization during a probe trial) and reduces working memory dysfunction (tested using the delayed match-to-place task). These behavioral improvements were only observed when the treatment was initiated 1h, but not 6h, post-injury. These studies support the use of sulforaphane in the treatment of TBI, and extend the previously observed protective effects to include enhanced cognition.

Topics: Aldehydes; Animals; Anticarcinogenic Agents; Brain Injuries; Cognition Disorders; Disease Models, Animal; Hippocampus; Isothiocyanates; Male; Maze Learning; Memory; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Space Perception; Sulfoxides; Thiocyanates; Time Factors