Carnosic acid, a pro-electrophilic compound, inhibits LPS-induced activation of microglia

Abstract

In the previous studies, we reported that carnosic acid (CA) protects cortical neurons by activating the Keap1/Nrf2 pathway, which activation is initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the “electrophilic” quinone-type CA. Here, we found that the pro-electrophilic CA inhibited the in vitro lipopolysaccharide (LPS)-induced activation of cells of the mouse microglial cell line MG6. LPS induced the expression of IL-1β and IL-6, typical inflammatory cytokines released from microglial cells. CA inhibited the NO production associated with a decrease in the level of inducible NO synthase. Neither CA nor LPS affected cell survival at the concentrations used here. These actions of CA seemed to be mediated by induction of phase 2 genes (gclc, gclm, nqo1 and xct). We propose that an inducer of phase 2 genes may be a critical regulator of microglial activation. Thus, CA is a unique pro-electrophilic compound that provides both a protective effect on neurons and an anti-inflammatory one on microglia through induction of phase 2 genes.

Introduction

Microglia are the resident innate immune cells in the CNS and make up approximately 12% of the total number of cells in the brain [1], [2]. In response to inflammatory triggers such as amyloid-β and lipopolysaccharide (LPS), microglial cells are readily activated and undergo dramatic morphological and physiological transformations [1], [2]. Hyperactivation of microglia may result in deleterious and neurotoxic consequences by excessive production of pro-inflammatory mediators [1], [2]. Reactive oxygen species generated by microglia help to eliminate pathogens in the extracellular milieu but also act on the microglia themselves, altering the intracellular redox balance and functioning as second messengers in the induction of proinflammatory genes [2]. Recent findings indicate that restoration of the proper redox balance may be a determinant in driving microglia back to the resting state [2]. Thus, activation of the transcription factor NF-E2-related factor-2 (Nrf2) [3], [4], a master regulator of cellular redox homeostasis in microglial cells, results in the inhibition of inflammatory responses in the brain [5], [6].

Electrophiles modulate a variety of cellular signaling processes involving several major anti-inflammatory and protective components via activation of the Nrf2/ARE pathway [5], [6], [7], [8], [9]. Electrophilic naturally-occurring products such as curcumin [5], sulforaphane [6], and carnosic acid (CA) [10] can act in this capacity by regulating redox reactions and phase 2 enzymatic activities [7], [8], [9], [10]. In this present study, we focused on carnosic acid (CA) [10], [11], [12], [13]. CA potently activates the Keap1/Nrf2 pathway, which regulates the cellular redox state, in several cells including neuronal, astroglial, and preadipocytic ones [10], [11], [12], [13]. The Keap1/Nrf2 pathway comprises Keap1, a regulator protein, and Nrf2, a transcriptional factor that binds to the ARE [7], [8], [9]. Keap1 is an adapter protein for the ubiquitination of Nrf2 and thus drives the continuous degradation of this transcription factor [7], [8], [9]. When electrophiles such as NEurite outgrowth-Promoting Prostaglandin11 (NEPP11) [14], tert-butyl hyroquinone (TBHQ) [15] or CA [10] react with critical cysteine residues on the Keap1 protein to form an adduct (S-alkylation), they perturb this system and stabilize Nrf2, thus allowing it to be translocated from the cytoplasm into the nucleus, where it binds to AREs and stimulates the expression of phase 2 genes. Here, we examined the effects of CA on microglial activation in vitro and found that CA inhibited microglial activation possibly through induction of phase 2 enzymes.