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Activation of the Keap1/Nrf2 pathway and consequent induction of phase 2 antioxidant enzymes is known to afford neuroprotection. Here, we present a series of novel electrophilic compounds that protect neurons via this pathway. Natural products, such as carnosic acid (CA), are present in high amounts in the herbs rosemary and sage as ortho-dihydroquinones, and have attracted particular attention because they are converted by oxidative stress to their active form (ortho-quinone species) that stimulate the Keap1/Nrf2 transcriptional pathway. Once activated, this pathway leads to the production of a series of antioxidant phase 2 enzymes. Thus, such dihydroquinones function as redox-activated “pro-electrophiles.” Here, we explored the concept that related para-dihydroquinones represent even more effective bioactive pro-electrophiles for the induction of phase 2 enzymes without producing toxic side effects. We synthesized several novel para-hydroquinone-type pro-electrophilic compounds (designated D1 and D2) in order to analyze their protective mechanism. DNA microarray, PCR, and Western blot analyses showed that compound D1 induced expression of heat-shock proteins (HSPs), including HSP70, HSP27 and DnaJ, in addition to phase 2 enzymes such as hemeoxygenase-1 (HO-1), NADP(H) quinine-oxidoreductase1, and the Na+-independent cystine/glutamate exchanger. Treatment with D1 resulted in activation of Nrf2 and HSF-1 transcriptional elements, thus inducing phase 2 enzymes and HSPs, respectively. In this manner, D1 protected neuronal cells from both oxidative and endoplasmic reticulum (ER)-related stress. Additionally, D1 suppressed induction of GRP78, an ER chaperone protein, and inhibited hyperoxidation of peroxiredoxin 2 (PRX2), a molecule that in it reduced state can protect from oxidative stress. These results suggest that D1 is a novel pro-electrophilic compound that activates both the Nrf2 and HSF-1 pathways, and may thus offer protection from oxidative and ER stress.

In our previous studies, we have reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protects cortical neurons by inducing phase 2 enzymes, the induction of which was initiated by activation of the Keap1/Nrf2 pathway , , . In the present study we address the nature of the effector of these neuroprotective effects downstream of the phase 2 enzyme induction. From our results we conclude that activated glutathione (GSH) metabolism may participate in these protective effects. First, we performed cDNA microarray analysis in order to identify the gene(s) responsible for the actions and found that various enzymes involved in the metabolism of GSH (glutathione S-transferase, alpha 4; glutathione S-transferase, alpha 2; and formylglutathione hydrolase) constituted 3 of the top 5 CA-induced genes. The other 2 genes encoded phase 2 enzymes [NAD(P)H-quinone oxidoreductase1and aldehyde dehydrogenase family 3, subfamily A1]. Next, we compared the physiologically-active compounds originating from rosemary (CA, CS, luteolin, genkwanin, rosmarinic acid, caffeic acid, and verbenone) by 3 criteria (enhancement of total glutathione levels, transcriptional activation, neuroprotective effects). By all of these criteria, CA and CS were the most active. In contrast, the other compounds were only weakly active or totally inactive. These results suggest that pro-electrophilic compounds such as CA and CS may protect cortical neurons by causing the following sequential events: S-alkylation --> activation of the Keap1/Nrf2 pathway --> transcriptional activation --> induction of phase 2 enzymes --> activation of GSH metabolism --> neuroprotection.

EGFR/MAPK signaling plays an important role in the inhibitory effect of CA on hepatocyte lipid accumulation.

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