For the past 40 years or so, oxidative stress has been increasingly recognized as a contributing factor in aging and in various forms of pathophysiology generally associated with aging. Our view of oxidative stress has been largely "superoxide-centric", as we focused on the pathological sources of this oxygen-derived free radical and the types of molecular havoc it can wreak, as well as on the protection provided by the antioxidant enzymes, especially the superoxide dismutases, catalases, and glutathione peroxidases. In the last decade our view of oxidative stress has broadened considerably, and it is now often seen as an imbalance that has its origins in our genes, and the ways in which gene expression is regulated. At the center of this new focus is the transcription factor called nuclear factor (erythroid-derived 2)-like 2, or Nrf2. Nrf2 is referred to as the "master regulator" of the antioxidant response, modulating the expression of hundreds of genes, including not only the familiar antioxidant enzymes, but large numbers of genes that control seemingly disparate processes such as immune and inflammatory responses, tissue remodeling and fibrosis, carcinogenesis and metastasis, and even cognitive dysfunction and addictive behavior. Thus, the dysregulation of Nrf2-regulated genes provides a logical explanation for the connections, both direct and indirect, between observable oxidative stress and perhaps 200 human diseases involving these various physiological processes, each reflecting a network involving many gene products. The evolutionary self-association of these many genes under the common control of Nrf2 suggests that the immune and inflammatory systems may present the largest demand for increased antioxidant protection, apart from constitutive oxidative stress resulting from mitochondrial oxygen consumption for metabolic purposes. Gene expression microarray data on human primary vascular endothelial cells and on the SK-N-MC human neuroblastoma-derived cell line have been obtained in response to the dietary supplement Protandim, a potent composition of highly synergistic phytochemical Nrf2 activators. Pathway analysis of results shows significant modulation by Protandim of pathways involving not only antioxidant enzymes, but of those related to colon cancer, cardiovascular disease, and Alzheimer disease.

Nrf2 is a transcription factor that regulates cellular redox balance and the expression of a wide array of genes involved in immunity and inflammation, including antiviral actions. Nrf2 activity declines with age, making the elderly more susceptible to oxidative stress-mediated diseases, which include type 2 diabetes, chronic inflammation, and viral infections. Published evidence suggests that Nrf2 activity may regulate important mechanisms affecting viral susceptibility and replication. We examined gene expression levels by GeneChip microarray and by RNA-seq assays. We found that the potent Nrf2-activating composition PB125® downregulates ACE2 and TMPRSS2 mRNA expression in human liver-derived HepG2 cells. ACE2 is a surface receptor and TMPRSS2 activates the spike protein for SARS-CoV-2 entry into host cells. Furthermore, in endotoxin-stimulated primary human pulmonary artery endothelial cells, we report the marked downregulation by PB125 of 36 genes encoding cytokines. These include IL-1-beta, IL-6, TNF-α, the cell adhesion molecules ICAM-1, VCAM-1, and E-selectin, and a group of IFN-γ-induced genes. Many of these cytokines have been specifically identified in the “cytokine storm” observed in fatal cases of COVID-19, suggesting that Nrf2 activation may significantly decrease the intensity of the storm.

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Bioactive phytochemicals in Rosmarinus officinalis, Withania somnifera, and Sophora japonica have a long history of human use to promote health. In this study we examined the cellular effects of a combination of extracts from these plant sources based on specified levels of their carnosol/carnosic acid, withaferin A, and luteolin levels, respectively. Individually, these bioactive compounds have previously been shown to activate the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor, which binds to the antioxidant response element (ARE) and regulates the expression of a wide variety of cytoprotective genes. We found that combinations of these three plant extracts act synergistically to activate the Nrf2 pathway, and we identified an optimized combination of the three agents which we named PB125 for use as a dietary supplement. Using microarray, quantitative reverse transcription-PCR, and RNA-seq technologies, we examined the gene expression induced by PB125 in HepG2 (hepatocellular carcinoma) cells, including canonical Nrf2-regulated genes, noncanonical Nrf2-regulated genes, and genes which appear to be regulated by non-Nrf2 mechanisms. Ingenuity Pathway Analysis identified Nrf2 as the primary pathway for gene expression changes by PB125. Pretreatment with PB125 protected cultured HepG2 cells against an oxidative stress challenge caused by cumene hydroperoxide exposure, by both cell viability and cell injury measurements. In summary, PB125 is a phytochemical dietary supplement comprised of extracts of three ingredients, Rosmarinus officinalis, Withania somnifera, and Sophora japonica, with specified levels of carnosol/carnosic acid, withaferin A, and luteolin, respectively. Each ingredient contributes to the activation of the Nrf2 pathway in unique ways, which leads to upregulation of cytoprotective genes and protection of cells against oxidative stress and supports the use of PB125 as a dietary supplement to promote healthy aging.

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A key component of cytoprotective gene regulation is the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), also known as nuclear factor erythroid 2-like 2, from the gene NFE2L2. Under normal conditions, Nrf2 in the cell is targeted for proteasomal degradation by its inhibitor Kelch-like ECH-associated inhibitor 1 (Keap1). When stimulated by oxidative stress, electrophiles, or kinase activation, conformational changes in the Nrf2-Keap1 complex inhibit proteasomal degradation of Nrf2, facilitating an increase in the amount of Nrf2 that binds to antioxidant response element sequences in the promoter regions of a variety of antioxidant, detoxification, and metabolic control genes. Nrf2 activation is mostly associated with beneficial cytoprotective gene regulation, but it can also have deleterious effects. For example, gene mutations in some types of cancers can lead to constitutive activation of Nrf2 and give the tumor cells growth advantages and increased drug resistance. Because cases exist where Nrf2/Keap1/ARE signaling is either too low or too high, there is great interest in the development of both Nrf2 activators and Nrf2 inhibitors as the basis of new therapies.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2; encoded in humans by the NFE2L2 gene) is a transcription factor that regulates the gene expression of a wide variety of cytoprotective phase II detoxification and antioxidant enzymes through a promoter sequence known as the antioxidant-responsive element (ARE). The ARE is a promoter element found in many cytoprotective genes; therefore, Nrf2 plays a pivotal role in the ARE-driven cellular defense system against environmental stresses. Agents that target the ARE/Nrf2 pathway have been tested in a wide variety of disorders, with at least one new Nrf2-activating drug now approved by the US Food and Drug Administration. Examination of in vitro and in vivo experimental results, and taking into account recent human clinical trial results, has led to an opinion that Nrf2-activating strategies – which can include drugs, foods, dietary supplements, and exercise – are likely best targeted at disease prevention, disease recurrence prevention, or slowing of disease progression in early stage illnesses; they may also be useful as an interventional strategy. However, this rubric may be viewed even more conservatively in the pathophysiology of cancer. The activation of the Nrf2 pathway has been widely accepted as offering chemoprevention benefit, but it may be unhelpful or even harmful in the setting of established cancers. For example, Nrf2 activation might interfere with chemotherapies or radiotherapies or otherwise give tumor cells additional growth and survival advantages, unless they already possess mutations that fully activate their Nrf2 pathway constitutively. With all this in mind, the ARE/Nrf2 pathway remains of great interest as a possible target for the pharmacological control of degenerative and immunological diseases, both by activation and by inhibition, and its regulation remains a promising biological target for the development of new therapies.