Anti-inflammatory and antioxidant activities of Nrf2/HO-1 activators: in vitro studies in microglia and retinal cells

Abstract

Inflammation and oxidative stress play essential roles during the progression and resolution of disease states. It is now being recognized that persistent inflammation and oxidative stress underlie several chronic diseases of Western countries. This is relevant in the ageing population, where neurodegenerative diseases and diabetes, among others, are pathologies that require pharmacological interventions targeting multiple pathways involved in these diseases. Microglia cells, the macrophages of the brain, work under normal conditions to protect neurons from infectious agents and by removing dead cells to promote tissue repair. Similarly, retinal pigmented epithelial cells support the eye by protecting photoreceptors and mediating nutrient uptake for retinal tissue. Because of their anatomical position, both cell types participate in the pathophysiology of neurodegenerative diseases and retinal diabetes complications. A highly relevant cellular response to stress is mediated by the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a redox sensitive protein that promotes the up-regulation of cytoprotective enzymes. Among these genes is heme oxygenase-1 (HO-1), which converts heme into iron, carbon monoxide and biliverdin, subsequently reduced to bilirubin by biliverdin reductase. These products exhibit anti-inflammatory and antioxidant actions in models of disease characterized by redox stress and inflammation and lack of HO-1 exacerbates damage in experimental animal models and two human cases reported in the literature. The Nrf2/HO-1 axis is amenable to pharmacological manipulation and molecules that target these pathways are of high interest for the discovery of therapeutic approaches to counteract neuroinflammation and retinal diseases. The work presented herein focuses on characterizing anti-inflammatory and antioxidant activities of Nrf2/HO-1 activators. In the first instance I identified effective Nrf2/HO-1 inducers that modulate the inflammatory response in BV2 murine microglia cells. I searched the literature and selected 56 compounds that activate Nrf2 or HO-1 and analyzed them for HO-1 induction and cytotoxicity in vitro. Approximately 20 compounds increased HO-1 at concentrations of 5 to 20 µM, with carnosol, curcumin, cobalt protoporphyrin-IX and dimethyl fumarate (DMF) exhibiting the best induction/low cytotoxicity profile. HO-1 up-regulation by some compounds resulted in increased bilirubin which correlated with the potency of the inducers to reduce nitrite production after challenge with interferon-ã (INF-ã) or lipopolysaccharide (LPS). The compounds strongly down-regulated inflammation (TNF-á, PGE2 and nitrite) in cells stimulated with INF-ã and LPS; silencing HO-1 or Nrf2 with shRNA partially reversed this effect. I then chose the Nrf2 inducers DMF and carnosol and investigated their effect on antioxidant pathways, oxygen consumption and wound healing in human retinal pigment epithelial cells (ARPE-19) grown in medium containing normal (NG) or high (HG) glucose to mimic hyperglycemia. I found that Nrf2 activation and heme oxygenase increased in ARPE cells treated with DMF or carnosol irrespective of glucose levels. However, in HG retinal cells were more sensitive to regulators of glutathione synthesis and produced lower reactive oxygen species. Culture in HG decreased respiration, ATP levels and damaged mitochondria; treatment with DMF or carnosol did not restore oxygen consumption. Using the scratch assay in vitro I observed that wound closure was faster in HG than NG cells and was accelerated by carnosol. An inhibitor of heme oxygenase reversed this effect, supporting a pro-healing role of HO-1. In summary, Nrf2/HO-1 activators modulate microglia inflammation and exert pharmacological activities in retinal cells cultured under normal or hyperglycemic conditions. Thus, promising chemical scaffolds are identified for the synthesis of potent new derivatives to counteract neuroinflammation and retinal diseases.