Biochemical and Biophysical Research Communications
Carnosic acid, a pro-electrophilic compound, inhibits LPS-induced activation of microglia
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.
Section snippets
Chemicals
The final concentration of DMSO in the culture medium was 0.1%. CA and LPS (L8274, Escherichia coli 026:B6-derived) were obtained from Sigma–Aldrich Chemical Co., Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), bovine calf serum, and penicillin–streptomycin came from Life Technologies (Carlsbad, CA).
Cell culture
MG6 cells [16], obtained from RIKEN Bioresource Center, were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) supplemented with 100 μM
Inhibition of NO production without an effect on cell survival
In order to confirm the absence of cytotoxicity at the concentrations used in the present study, we exposed MG6 cells to different concentrations of CA and LPS (applied alone or in combination). After a 24-h incubation with different CA concentrations (0, 1.0, 3.0, and 10.0 μM), the viability of the cells was determined by performing the standard WST-8 assay. As shown in Fig. 1A, the application of CA for 24 h at the indicated concentrations did not affect the viability of the cells, as indicated
Discussion
As shown in Fig. 3B, we propose the phase 2 induction-dependent CA-mediated inhibition of inflammatory actions of microglial cells. The induction of phase 2 genes greatly activates the redox-regulating mechanism of microglial cells, thus reducing the inflammatory action of these cells [7], [8], [9], [10], [11], [12], [13], [14]. Because CA activated the Keap1/Nrf2 pathway in various biological systems and the activation leads to anti-inflammation [3], [4], [5], [6], neuroprotection [9], [10],
Acknowledgments
The authors thank Dr. Larry D. Frye for editorial help with the manuscript. This work was supported in part by a Grant-in-aid for scientific research (Nos. 22500282; 2011-2013) from JSPS.
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