Bryonolic Acid Transcriptional Control of Anti-inflammatory and Antioxidant Genes in Macrophages in Vitro and in Vivo

Bryonolic acid (BA) (<b>1</b>) 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

Identification of a negative feedback loop in biological oxidant formation fegulated by 4-hydroxy-2-(E)-nonenal

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