ABSTRACT
The transcription factor NF-E2 p45-related factor 2 (NRF2; encoded by NFE2L2) plays a critical role in the maintenance of cellular redox and metabolic homeostasis, as well as the regulation of inflammation and cellular detoxication pathways. The contribution of the NRF2 pathway to organismal homeostasis is seen in many studies using cell lines and animal models, raising intense attention towards targeting its clinical promise. Over the last three decades, an expanding number of clinical studies have examined NRF2 inducers targeting an ever-widening range of diseases. Full understanding of the pharmacokinetic and pharmacodynamic properties of drug candidates rely partly on the identification, validation, and use of biomarkers to optimize clinical applications. This review focuses on results from clinical trials with four agents known to target NRF2 signaling in preclinical studies (dimethyl fumarate, bardoxolone methyl, oltipraz, and sulforaphane), and evaluates the successes and limitations of biomarkers focused on expression of NRF2 target genes and others, inflammation and oxidative stress biomarkers, carcinogen metabolism and adduct biomarkers in unavoidably exposed populations, and targeted and untargeted metabolomics. While no biomarkers excel at defining pharmacodynamic actions in this setting, it is clear that these four lead clinical compounds do touch the NRF2 pathway in humans.
ABSTRACT
4-Hydroxy-2-(E)-nonenal (4-HNE) is one of the major lipid peroxidation product formed during oxidative stress. At high concentrations, 4-HNE is cytotoxic and exerts deleterious effects that are often associated with the pathology of oxidative stress-driven disease. Alternatively, at low concentrations it functions as a signaling molecule that can activate protective pathways including the antioxidant Nrf2-Keap1 pathway. Although these biphasic signaling properties have been enumerated in many diseases and pathways, it has yet to be addressed whether 4-HNE has the capacity to modulate oxidative stress-driven lipid peroxidation. Here we report an auto-regulatory mechanism of 4-HNE via modulation of the biological oxidant nitric oxide (NO). Utilizing LPS-activated macrophages to induce biological oxidant production, we demonstrate that 4-HNE modulates NO levels via inhibition of iNOS expression. We illustrate a proposed model of control of NO formation whereby at low concentrations of 4-HNE a negative feedback loop maintains a constant level of NO production with an observed inflection at approximately 1 µM, while at higher 4-HNE concentrations positive feedback is observed. Further, we demonstrate that this negative feedback loop of NO production control is dependent on the Nrf2-Keap1 signaling pathway. Taken together, the careful regulation of NO production by 4-HNE argues for a more fundamental role of this lipid peroxidation product in normal physiology.
Subject(s)
Aldehydes/toxicity , Lipid Peroxidation/drug effects , Aldehydes/chemical synthesis , Aldehydes/chemistry , Animals , Cell Line , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , NF-E2-Related Factor 2/deficiency , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Oxidants/chemical synthesis , Oxidants/chemistry , Oxidative Stress/drug effects , Signal Transduction/drug effects , StereoisomerismABSTRACT
Bryonolic acid (BA) (1) is a naturally occurring triterpenoid with pleiotropic properties. This study characterizes the mechanisms mediating the anti-inflammatory and antioxidant activities of BA and validates the utility of BA as a tool to explore the relationships between triterpenoid structure and activity. BA reduces the inflammatory mediator NO by suppressing the expression of the inflammatory enzyme inducible nitric oxide synthase (iNOS) in LPS-activated RAW 264.7 macrophage cells. In addition, BA robustly induces the antioxidant protein heme oxygenase-1 (HO-1) in vitro and in vivo in an Nrf2-dependent manner. Further analyses of Nrf2 target genes reveal selectivity for the timing and level of gene induction by BA in treated macrophages with distinct patterns for Nrf2-regulated antioxidant genes. Additionally, the distinct expression profile of BA on Nrf2 target genes relative to oleanolic acid suggests the importance of the triterpenoid scaffold in dictating the pleiotropic effects exerted by these molecules.
Subject(s)
Antioxidants/pharmacology , Heme Oxygenase-1/drug effects , Macrophages/physiology , Triterpenes/pharmacology , Animals , Anti-Inflammatory Agents/pharmacology , Dose-Response Relationship, Drug , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Mice , Mice, Inbred C57BL , NF-E2-Related Factor 1/drug effects , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type II/analysis , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Oleanolic Acid/chemistry , Oleanolic Acid/pharmacology , Triterpenes/chemistryABSTRACT
Bryonolic acid (BA) is a triterpenoid found in the Cucurbitaceae family of plants. Our interests in the immunomodulatory effects of this class of natural products led us to discover that BA induces a marked increase in the expression of a phase 2 response enzyme, heme oxygenase 1 (HO-1), in a dose-dependent manner. This phenotype has translational implications in malarial disease progression, and consequently we developed a large-scale isolation method for BA that will enable future in vitro and in vivo analyses. We have determined ideal growth conditions and time scale for maximizing BA content in the roots of Cucurbita pepo and analyzed BA production by HPLC. Large-scale extraction yielded 1.34% BA based on dry weight, allowing for the isolation of BA on a multigram scale.
